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Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw
b i o m a s s a n d b i o e n e r g y 7 8 ( 2 0 1 5 ) 9 9 e1 0 9
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Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw Hao Fang, Liming Xia* Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
article info
abstract
Article history:
The cellobiohydrolase II (CBH II) gene cbh2 from Trichoderma reesei was cloned and its co-
Received 18 April 2014
dons were optimized in accordance with the codon usage frequencies of the host Pichia
Received in revised form
pastoris. The AOX1 strong promoter inducible by methanol was employed to efficiently
8 April 2015
express the foreign gene cbh2 in P. pastoris. It was found that 5.84 ± 0.42 U cm3 CBH II was
Accepted 16 April 2015
obtained at 96 h using the synthetic cbh2 gene whose codons were optimized, 2.02-fold
Available online
higher than using the native cbh2 gene (2.89 ± 0.32 U cm3), indicating that the codon optimization strategy was an effective approach to enhance the heterologous expression of
Keywords:
CBH II in P. pastoris. The product of recombinant P. pastoris CBH II had an approximate
Trichoderma reesei cellobiohydrolase
molecular weight 58 kDa. Its optimal pH and temperature were 5.0 and 50 C, respectively.
II
The recombinant CBH II was used to enhance the yields of the enzymatic hydrolysis of the
Pichia pastoris
corn stover and rice straw pretreated with sodium hydroxide by improving the exo-exo-
Heterologous expression
synergism between CBH II and CBH I in T. reesei cellulase. The yields 94.7% and 83.3%
Codon optimization
were achieved in the enzymatic hydrolysis of corn stover and rice straw pretreated by
Exo-exo-synergism
sodium hydroxide, respectively.
Enzymatic hydrolysis
1.
Introduction
During the bioprocesses of converting lignocelluloses which are one of the most abundant renewable resources on the earth to reducing sugars that could be used as feedstock for a variety of value-added chemicals such as fuels, cellulase with a complete composition plays a very important role, presenting a hopeful alternative for conventional chemical processes. Trichoderma reesei, a mesophilic soft-rot ascomycete fungus
* Corresponding author. Tel./fax: þ86 571 87951840. E-mail address: [email protected] (L. Xia). http://dx.doi.org/10.1016/j.biombioe.2015.04.014 0961-9534/© 2015 Published by Elsevier Ltd.
© 2015 Published by Elsevier Ltd.
that is widely used as the biotechnological workhorse of the genus, is a household name in both academic and industrydriven studies into lignocelluloses degrading enzymes and their applications [1e3]. A complete mixture of cellulase from T. reesei is composed of cellobiohydrolases (CBH: EC 3.2.1.91), exoglucanases which release cellobiose as main product from crystalline cellulose; endoglucanases (EG: EC 3.2.1.4), preferably attack amorphous cellulose and some short chain oligomers; and b-glucosidases (EC 3.2.1.21), which hydrolyze cellooligosaccharides and cellobiose into glucose [4e6].
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Synergisms exist between the components of T. reesei cellulase. The best known is the synergism between b-glucosidases and cellobiohydrolases. Many studies pivoted the b-glucosidases to enhance that synergism, diminishing the inhibitory effect of cellobiose accumulation in enzymatic hydrolysis on cellobiohydrolases (and endoglucanases) caused by the deficiency of T. reesei cellulase in the bglucosidase content [5e8]. Nonetheless the exo-exosynergism between cellobiohydrolase I (CBH I) coded by cbh1 gene and the cellobiohydrolase II (CBH II) coded by cbh2 gene in T. reesei cellulase is of importance because CBH I mainly acts on reducing ends and CBH II preferably acts on non-reducing ends, preparing a more readily hydrolysable substrate for each other [4,9]. In T. reesei, CBH II has higher specific activity toward microcrystalline cellulose than CBH I, but the latter one has 50e60% of the total secreted protein of T. reesei due to the fact that the promoter of cbh1 gene is regarded as a strong promoter [10]. In contrast, CBH II only accounts for 10e15% of total protein [10], thus rendering it deficient in CBH II that is essential to an excellent exo-exosynergism. So improving CBH II is important to a great performance of T. reesei cellulase in enzymatic hydrolysis of lignocelluloses and the deficiency of CBH II is one of the limiting factors of T. reesei cellulase needed to be overcome using different strategies. Producing T. reesei CBH II in Pichia pastoris is a desirable approach because P. pastoris is an excellent expression system for expressing heterologous protein in secreted forms with many advantages among which the most important one is its perfect protein processing mechanism including signal peptide cleavage, protein folding, and post-translational modification inside the cell, and secretion ability into medium with normal function [11]. Owing to the large difference of codon usage preference in different species and some other reasons such as unstable mRNA of exotic gene, heterologous gene expressions always result in low extracellular proteins production, especially for those proteins with active functions. Codon optimization in accordance with the codon usage preference of host cell, therefore, is a promising and effective technique to increase heterologous protein expression level. This has been proven by some reports [11e13], although they had differentiated outcomes. Corn stover and rice straw, main agricultural residues in northern and southern China respectively, could be made use of as renewable feedstock for biorefinery. A great amount of corn stover and rice straw is always set on fire every year in China, giving rise to severe environmental problems. Producing lignocellulosic ethanol or other value-added products from corn stover or other agricultural residues, therefore, is feasible and has many advantages in China [14]. Thus corn stover and rice straw were used as substrate of enzymatic hydrolysis to compare the performance of the cellulolytic enzyme mixture. Pretreatment is required to smash the recalcitrant structure of lignocelluloses naturally formed before enzymatic hydrolysis in order to increase the enzymatic digestibility and to facilitate subsequent conversion. Sodium hydroxide pretreatment, carried out at lower temperature and pressure than acid hydrolysis and steam explosion, can substantially increase the lignin removal and enhance the accessibility and digestibility of cellulose [7,15].
So it was used in this work for the pretreatment of corn stover and rice straw. In this work, the codons of T. reesei cbh2 gene were optimized according to the codon usage preference of P. pastoris. Then the synthetic cbh2 gene with codons optimized and the native T. reesei cbh2 gene were put into the P. pastoris expression vector pPIC9K containing the a-Factor secretion signal, respectively. The former one was used for the efficient expression in P. pastoris GS115 and the latter one was used as control. The enzymatic properties of recombinant CBH II were investigated. And the recombinant CBH II was added to the cellulolytic enzymes mixture so as to enhance exo-exo-synergism between CBH I and CBII, thereby improving the enzymatic hydrolysis yields of corn stover and rice straw.
2.
Material and methods
2.1.
Microorganisms, plasmids and mediums
Escherichia coli strain DH5a was used for plasmid manipulation and propagation throughout the work as described by Sambrook et al. [16]. T. reesei ZU-02 [4,8,17] provided total mRNA containing the mRNA of the cbh2 gene used as the template of reverse transcriptase polymerase chain reaction (RT-PCR) for producing cDNA, which was subsequently used as the template of native cbh2 gene cloning. P. pastoris GS115 was the host of cbh2 gene expression. All the microorganisms above were from the strain collection of Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University. The pMD18-T cloning vector (Takara, Otsu, Japan) was used for TA cloning. The plasmid pPIC9K, harboring a-factor signal peptide gene and the methanol-inducible alcohol oxidase gene (AOX1) that is a strong promoter controlling recombinant gene expression, was employed as expression vector of cbh2 gene in P. pastoris GS115 [18]. LuriaeBertani (LB) medium for E. coli cultivation had the following composition: 10 kg m3 tryptone, 5 kg m3 yeast extract, 10 kg m3 NaCl, pH 7.0. The potato dextrose agar (PDA) slants used for short term T. reesei ZU-02 culture preservation were composed of 200 kg m3 potato, 20 kg m3 dextrose and 20 kg m3 agar. The seed medium for T. reesei was as follows: 15 kg m3 glucose, 20 kg m3 yeast extract, 2.5 kg m3 (NH4)2SO4, kg m3 KH2PO4, 0.8 kg m3 MgSO4, kg m3 CaC12, 0.005 kg m3 FeSO4$7H2O, 0.0016 kg m3 MnSO4$H2O, 0.0014 kg m3 ZnSO4$7H2O, 0.0037 kg m3 CoCl2$6H2O. The ingredients of YPD medium for P. pastoris GS115 cultivation were as follows: 10 kg m3 yeast extract, 20 kg m3 peptone, 20 kg m3 glucose. MD medium used for screening transformation consisted of 3.4 kg m3 yeast nitrogen base (YNB) without amino acids, 4 104 kg m3 biotin, and 20 kg m3 glucose. The composition of BMGY medium for seed preparation of P. pastoris transformants was as follows: 10 kg m3 glycerol, 10 kg m3 yeast extract, 20 kg m3 peptone, 3.4 kg m3 YNB, 10 kg m3 (NH4)2SO4 and 4 104 kg m3 biotin in pH 6.0 0.1 mol L1 potassium phosphate buffer. The BMMY medium for recombinant gene induction and fermentation was comprised of 5 kg m3
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methanol, 10 kg m3 yeast extract, 20 kg m3 peptone, 3.4 kg m3 YNB, 10 kg m3 (NH4)2SO4 and 4 104 kg m3 biotin in pH 6.0100 mmol L1 potassium phosphate buffer. When solid agar plates were required, 15e20 kg m3 agar powder was added before sterilization.
2.2. Cloning of native cbh2 gene and synthesis of codon optimized cbh2 gene T. reesei ZU-02 spores were inoculated into the seed medium (containing microcrystalline cellulose instead of glucose) and incubated at 30 C and 2.83 Hz for 2 d for total RNA preparation. Five cubic centimeters mycelium was frozen with liquid nitrogen and ground to fine powder with mortar and pestle. Total RNA was extracted from the fine powder by the TRNzol reagent (Takara, Dalian, China). The cDNA of T. reesei cbh2 gene was produced through reverse transcriptase reaction using the total RNA as the template with the reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Takara, Dalian, China). DNA amplification was carried out with a forward primer (50 -ATAGAATTCCTCAAGCTTG CTCAAGCGTCT-30 , containing EcoR I site) and a reverse primer (50 -CGCGCGGCCGCTTACAGGAACGATGGGTTTGC-30 , containing Not I site) from the cDNA as template. PCR conditions included 94 C for 10 min, followed by 30 (94 C for 1 min, 50 C for 1 min, 72 C for 2 min), and 72 C for 10 min. The resultant gene product was cloned into the pMD18-T cloning vector and sent to Sangon Biotech (Shanghai, China) to be sequenced. The amino acid sequence of T. reesei CBH II (GenBank accession: AAA34210.1) was reverse-translated into the preferentially used codons in P. pastoris. The codon usage detail of P. pastoris, in the form of codon usage frequency, is shown in Table 1, according to which the codons were optimized. Codons with low usage percentage (<15%) were replaced by higher frequently used ones [11]. Then the codon optimized cbh2 gene containing the restriction sites, EcoR I in the upstream and Not I in the downstream for the subsequent gene manipulation, was synthesized by Sangon Biotech (Shanghai, China).
2.3.
Construction of expression plasmid
The pMD18-T-cbh2 harboring T. reesei cbh2 gene was double digested by the restriction enzymes EcoR I and Not I that were purchased from Takara Biotech (Dalian, China), releasing the cbh2 gene fragment which was subsequently linked to the pPIC9K fragment produced from the double digestion with the same restriction enzymes. As a result, the final plasmid pPIC9K-cbh2 for the heterologous expression in P. pastoris was obtained. As for the synthetic T. reesei cbh2 gene from Sangon Biotech (Shanghai, China), it was contained in pUC18 plasmid with EcoR I and Not I restriction sites. The ligation of pPIC9K fragment and the synthetic cbh2 gene fragment was the same as the procedure described above. These two pPIC9K-cbh2 plasmids containing native and synthetic cbh2 gene fragment were linearized with restriction enzyme Bgl II prior to being used for transformation of P. pastoris.
Table 1 e Comparison of codon usage of native and synthetic cbh2 genes with that of the host strain Pichia pastoris. Amino acids
Ala
Arg
Asn Asp Cys Gln Glu Gly
His Ile
Leu
Lys Met Phe Pro
Ser
Stop
Thr
Trp
Codons
GCT GCC GCA GCG AGA AGG CGT CGA CGC CGG AAC AAT GAT GAC TGT TGC CAA CAG GAA GAG GGT GGA GGC GGG CAT CAC ATT ATC ATA TTG TTA CTT CTG CTA CTC AAG AAA ATG TTC TTT CCA CCT CCC CCG TCT TCC TCA AGT AGC TCG TAA TAG TGA ACT ACC ACA ACG TGG
Codon usage frequency Pichia pastoris
Native cbh2
0.45 0.26 0.23 0.06 0.48 0.16 0.16 0.10 0.05 0.05 0.52 0.48 0.58 0.42 0.64 0.36 0.61 0.39 0.57 0.43 0.41 0.32 0.14 0.13 0.57 0.43 0.50 0.31 0.19 0.32 0.17 0.17 0.15 0.11 0.08 0.53 0.47 1.00 0.54 0.46 0.41 0.35 0.15 0.09 0.30 0.20 0.18 0.15 0.09 0.08 0.53 0.32 0.15 0.40 0.25 0.24 0.11 1.00
0.30 (18) 0.38 (23) 0.22 (13) 0.10 (6) 0.13 (2) 0.00 (0) 0.13 (2) 0.20 (3) 0.40 (6) 0.13 (2) 0.63 (19) 0.37 (11) 0.38 (8) 0.62 (13) 0.50 (6) 0.50 (6) 0.52 (11) 0.48 (10) 0.30 (3) 0.70 (7) 0.23 (9) 0.30 (12) 0.40 (16) 0.08 (3) 0.25 (1) 0.75 (3) 0.56 (10) 0.44 (8) 0.00 (0) 0.19 (7) 0.00 (0) 0.30 (11) 0.22 (8) 0.08 (3) 0.22 (8) 0.90 (9) 0.10 (1) 1.00 (5) 0.33 (4) 0.67 (8) 0.32 (10) 0.35 (11) 0.13 (4) 0.19 (6) 0.15 (7) 0.23 (11) 0.09 (4) 0.04 (2) 0.23 (11) 0.26 (12) 1.00 (1) 0.00 (0) 0.00 (0) 0.30 (11) 0.38 (14) 0.14 (5) 0.19 (7) 1.00 (12)
Synthetic cbh2 0.87 0.08 0.05 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.9 0.10 0.62 0.38 0.83 0.17 0.81 0.19 0.70 0.30 0.78 0.23 0.00 0.00 0.75 0.25 0.78 0.22 0.00 0.97 0.00 0.03 0.00 0.00 0.00 1.00 0.00 1.00 0.92 0.08 0.77 0.23 0.00 0.00 0.81 0.17 0.02 0.00 0.00 0.00 1.00 0.00 0.00 0.70 0.24 0.05 0.00 1.00
(52) (5) (3) (0) (15) (0) (0) (0) (0) (0) (27) (3) (13) (8) (10) (2) (17) (4) (7) (3) (31) (9) (0) (0) (3) (1) (14) (4) (0) (36) (0) (1) (0) (0) (0) (10) (0) (5) (11) (1) (24) (7) (0) (0) (38) (8) (1) (0) (0) (0) (1) (0) (0) (26) (9) (2) (0) (12)
(continued on next page)
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Table 1 e (continued ) Amino acids
Tyr Val
Codons
TAC TAT GTT GTC GTG GTA
Codon usage frequency Pichia pastoris
Native cbh2
0.55 0.45 0.43 0.23 0.19 0.15
0.57 (12) 0.43 (9) 0.21 (6) 0.46 (13) 0.25 (7) 0.07 (2)
Synthetic cbh2 0.90 0.10 0.89 0.11 0.00 0.00
(19) (2) (25) (3) (0) (0)
The information on the codon usage of P. pastoris is from the codon usage database (http://www.kazusa.or.jp/codon).
1.0. Subsequently, the 100 cm3 BMMY mediums in 500 cm3 Erlenmeyer flasks inoculated P. pastoris transformants were incubated at 30 C and 4.17 Hz for 120 h. Methanol was added to a concentration of 10 kg m3 at every 24 h to keep the durative induction and expression of the cbh2 gene in P. pastoris transformants driven by the AOX1 promoter intensively inducible in the presence of an appropriate concentration of methanol. Sampling was implemented periodically for analysis. At least three parallel experiments and samples were adopted to abate the detrimental effects of possible errors on experimental results.
2.6. 2.4. Transformation of P. pastoris and screening of transformants P. pastoris GS115 stored on YPD slant was inoculated into 5 cm3 YPD medium and cultured at 30 C and 4.17 Hz for 12 h. Then it was collected by centrifugation (3000 g, 5 min) and grown in 50 cm3 YPD medium at 30 C and 4.17 Hz until OD600 ¼ 1.3e1.5. The competent cells of P. pastoris GS115 were prepared, the transformation was carried out, and the screening of P. pastoris transformants was conducted, according to the Invitrogen pPIC9K Expression Manual. The linearized pPIC9Kcbh2 was transformed into the competent cells of P. pastoris GS115 by electroporation using SCIENTZ-2B Genepulser (Ningbo, China) under the conditions: 1.25 kV, 25 uF, 200 U. Afterwards, potential transformants were selected on MD and YPD agar plates using the antibiotic G418 at five concentrations: 1.0, 2.0, 3.0, 4.0 and 5.0 mg cm3. In theory, the transformants appearing on the plates containing higher G418 concentration often have a larger number of insertions or integrations of recombinant DNA fragments into yeast genome. Hence the P. pastoris transformants growing on the plates containing 5.0 or 4.0 mg cm3 G418 were chosen for further research.
2.5.
Verification of P. pastoris transformants by PCR
The chromosomal DNA of P. pastoris was produced using Ezup Column Yeast Genomic DNA Purification Kit purchased from Sangon Biotech (Shanghai, China). Then PCR of the chromosomal DNA of P. pastoris was carried out to verify the insertion of recombinant DNA into yeast genome using AOX1 primes 50 and 50 -GGCAAATGGGACTGGTTCCAATTGACAAGC-30 CATTCTGACATCCT-30 . PCR conditions were 94 C for 10 min, followed by 30 (94 C for 1 min, 60 C for 1 min, 72 C for 2 min), and 72 C for 10 min.
Cellobiohydrolase activity (CBHA) assay, which was modified from the standard method recommended by International Union of Pure and Applied Chemistry (IUPAC) [19], was implemented in 10 cm3 cuvettes using microcrystalline cellulose PH101 (SigmaeAldrich, St. Louis, MO, USA) as the substrate. Each cuvette was added with 0.02 g microcrystalline cellulose and 0.5 cm3 proper diluted enzyme. Then 1 cm3 50 mmol L1 citrate buffer was supplemented to form a 1.5 cm3 reaction mixture and have pH value controlled at 4.8. Having been blended thoroughly, the mixture was incubated in water bath at 50 C with a shaking of 2.33 Hz for 30 min. Thereafter, the reaction was stopped in ice bath and the reducing sugars in the supernatant were determined by DNS method. One Unit (1 U) of CBHA was defined as the amount of enzyme required for generating 1 mg reducing sugars in 1 h [4].
2.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The supernatant of the fermentation broth sampled from fermentation process was determined by SDS-PAGE with 0.5 kg m3 stacking gel and 1.2 kg m3 separating gel. Approximately 15e20 mm3 of the sample was loaded into each well of the staking gel. After electrophoresis, the gels were stained in the Coomassie brilliant blue solution (3 kg m3 methanol, 1 kg m3 acetic acid and 0.01 kg m3 Coomassie brilliant blue R-250) for 40 min and then destained for overnight in the destaining solution comprised of 3 kg m3 methanol and 1 kg m3 acetic acid. The molecular mass of the recombinant protein was estimated using the protein molecular weight marker purchased from Takara (Dalian, China) as a standard.
2.8. 2.5.1.
Cellobiohydrolase activity assay
Determination of soluble protein concentration
Induction of recombinant P. pastoris and fermentation
The colonies of P. pastoris transformants were inoculated into 5 cm3 BMGY medium and incubated at 30 C and 4.17 Hz for 12 h. Then the cells were collected by centrifugation (3000 g, 5 min) and cultured in 50 cm3 BMGY medium for 16e18 h. At this point, the cultures had OD600 values between 2 and 6 (logarithmic growth phase). The cells were collected by centrifugation (3000 g, 5 min) and the pellets were resuspended in 100 cm3 BMMY medium with a starting OD600 value
Soluble protein concentration in fermentation broth was determined using Bradford reagent (SigmaeAldrich, St. Louis, MO, USA). Bovine serum albumin (BSA) was used as the standard protein samples at the gradual concentrations with proper range to prepare a standard curve for the quantitative determination of soluble protein concentration. All the samples blended with Bradford reagent were measured spectrophotometrically at a wavelength of 595 nm.
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2.9. Enzymatic properties of CBH II expressed by P. pastoris
column). The enzymatic hydrolysis yield was calculated according to the following equation [4,20]:
The pH optimum of CBH II was measured between pH 3.0 and pH 8.0 at 50 C using the highest CBHA as the 100% standard. The pH range of 3.0e6.0 was adjusted with 50 mmol L1 citrate buffer solution and the pH range of 6.0e8.0 was adjusted with 50 mmol L1 sodium phosphate buffer. The optimal temperature was determined at a temperature gradient between 30 C and 70 C with 5 intervals using 50 mmol L1 citrate buffer solution, pH 5.0. The highest CBHA was also used as the 100% standard. The temperature was controlled in a thermostat water bath.
2.10.
Yield (%) ¼ reducing sugars (g) 0.9 100/ (cellulose þ hemicellulose) in substrate (g)
(1)
0.9 is the conversion factor used for deleting the interfering effect of the molecular weight changes of sugars before and after hydrolysis on the accuracy of calculation results.
3.
Results and discussion
3.1. Construction of expression vector, transformation of P. pastoris and transformants screening
Enzymatic hydrolysis of corn stover and rice straw
The corn stover (Zea mays Longyu 98 aged 101 d) and rice straw (Oryza sativa Qianyou 817 aged 124 d) were harvested and collected from Nanyang area (E112 310 , N33 010 ) of Henan Province and Hangzhou area (E120 090 , N30 140 ) of Zhejiang Province in October 2012, respectively. The definition of corn stover and rice straw in this work included stalk, husk and leaves. They were air dried and then stored at room temperature. They must be used within 12 months after being harvested. Before pretreatment, the stored corn stover and rice straw were pulverized with a laboratory hammer mill and screened to obtain the particles less than 2 mm in size using a sieve shaker. Subsequently, both of them were pretreated with sodium hydroxide pretreatment method proposed by Chen et al. [7]. Thereafter, the solid cellulosic residues were collected and washed with tap water to neutral pH. And then they were kept at 4 C in refrigerator for water content balance. The information about the sodium hydroxide pretreated corn stover (SHPCS), the sodium hydroxide pretreated rice straw (SHPRS), and the raw materials are shown in Table 2. Enzymatic hydrolysis of SHPCS or SHPRS was carried out in 250 cm3 Erlenmeyer flasks with a 50 cm3 mixture of 2.5 cm3 1 mol L1 citrate buffer solution (pH 4.8), 100 kg m3 substrate, enzymes, and a supplemented amount of water. The enzymatic hydrolysis was implemented at 50 C in water bath with a shaking of 2.33 Hz. Sampling was conducted periodically during the enzymatic hydrolysis for subsequent analysis.
2.11. Measurement of monomeric sugars and calculation of yield Glucose and xylose produced from the enzymatic hydrolysis were analyzed and quantified by higher performance liquid chromatography (HPLC) (Waters HPX-87P ion exclusion
From the online database (http://www.kazusa.or.jp/codon), it was found that some codons of T. reesei native cbh2 gene are rarely used in P. pastoris (usage frequency less than 15%). They are GCG (Ala), CGA/C/G (Arg), GGC/G (Gly), CTA/C (Leu), CCG (Pro), AGC (Ser), TCG (Ser), and ACG (Thr). All of them were replaced with those codons with the highest usage frequency for the specific amino acid. And some codons with the usage frequencies higher than 15% but lower than the most highly used codons were also replaced to further increase the expression efficiency of cbh2 gene due to the larger availabilities of the tRNAs having the anticodons with the higher usage frequencies in P. pastoris. The detailed changes from native cbh2 gene to synthetic codon-optimized cbh2 gene are listed in Table 1. The synthetic cbh2 gene from Sangon Biotech (Shanghai, China) was in the vector pUC18 with EcoR I and Not I restriction sites for subsequent DNA manipulation. The native cbh2 gene was cloned from T. reesei ZU-02 using the procedures as described in Materials and Methods. Then these two genes and the pPIC9K were double digested with EcoR I and Not I restriction enzymes and the cbh2 genes and the pPIC9K fragment were separated via agarose gel electrophoresis. Afterwards, the DNAs in collected gels were extracted and purified. Then the DNA fragments were ligated to form the final expression vector harboring native cbh2 gene or synthetic cbh2 gene that is shown in Fig. 1. And the agarose electrophoresis result of pPIC9K-cbh2 is presented in Fig. 2A. Because both native cbh2 gene and synthetic cbh2 gene have the same molecular weight, they are in the exactly same band in the agarose electrophoresis. And due to the super helix structure of the closed circle plasmid, the band of pPIC9K-cbh2 exhibits the molecular weight smaller than its actual molecular weight that is given in Fig. 1, approximately 9.5 kb.
Table 2 e Information about the pretreated and raw materials of corn stover and rice straw. Quantities are mass fractions of dry material (%). SHPCS, sodium hydroxide pretreated corn stover; SHPRS, sodium hydroxide pretreated rice straw. Cellulose Raw corn stover SHPCS Raw rice straw SHPRS
38.5 63.6 38.6 59.7
± 0.4 ± 0.9 ± 0.3 ± 0.7
Hemicellulose 21.8 ± 24.3 ± 20.9 ± 21.1 ±
0.5 0.6 1.5 1.7
Lignin 19.0 8.9 15.6 9.3
± 0.3 ± 0.3 ± 2.4 ± 0.5
Others 20.7 3.2 24.9 9.9
± 0.4 ± 0.1 ± 1.5 ± 1.3
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tenth generation corresponding P. pastoris transformants. This demonstrated the good stability of integrated cbh2 genes that is required to be tested because of the possibility of losing the genetic traits newly conferred through genetic engineering from generation to the next.
3.2. Induction of recombinant P. pastoris and fermentation
Fig. 1 e Recombinant plasmid pPIC9K-cbh2 with PAOX1cbh2-TAOX1 expression cassette used for transformation of Pichia patoris GS115. The native Trichoderma reesei cbh2 gene (or synthetic cbh2 gene whose codons were optimized) was linked between the AXO1 promoter and the AOX1 terminator, the strong promoter is inducible by methanol. The selective marker (HIS4) and the resistance markers, Kan (R) and Amp (R), in the plasmid were used for yeast and bacterial transformants selection, respectively. The image of plasmid structure was pictured using WinPlas 2.7 software (Rich Goldstein).
The constructed expression vectors were transformed into E. coli DH5a for plasmid amplification. The E. coli DH5a transformants containing pPIC9K-cbh2 were verified by colony PCR (Fig. 2B) and thereafter they were cultured for plasmid preparation. The recombinant plasmids linearized with restriction enzyme Bgl II were used for the transformation of P. pastoris by electroporation. The aim of plasmid linearization is to improve the efficiency of P. pastoris transformation by electroporation and different restriction enzymes would generate Mutþ or MutS recombinants. The former one is the phenotype of utilizing methanol normally and the latter one is the phenotype incapable of metabolize methanol efficiently because it cannot produce alcohol oxidase, which could be readily distinguished by patching on MD versus MM plates [11]. After P. pastoris transformation and transformants screening as described in Material and Methods, 24 positive transformants with the highest resistance to the geneticin G418 were selected and subjected to genomic DNA preparation for PCR verification (Fig. 2C). The PCR verification used the P. pastoris transformants containing blank plasmids and original strain as control (lane 5e7 in Fig. 2C). The bands with molecular weight about 1.3 kb (lane 1e4 in Fig. 2C) indicated the existence of native cbh2 gene or synthetic cbh2 gene in P. pastoris genomic DNA, confirming that all the transformants' genomic DNAs were integrated by exotic cbh2 gene. In Fig. 2C, lane 1 and 3 were the PCR products from the genomic DNAs of the first generation P. pastoris transformants encompassing native cbh2 gene or synthetic cbh2 gene, respectively. And lane 2 and 4 were the PCR products from the genomic DNAs of the
P. pastoris PC1 and PC2 were cultured in 50 cm3 BMMY medium in 250 cm3 Erlenmeyer flasks for fermentation. At every 24 h point during the fermentation process, 10 kg m3 methanol was added to induce the strong expression of AOX1 promoter. According to the previous research [18], 10 kg m3 methanol was the most suitable concentration for recombinant P. pastoris to produce heterologous enzyme. The concentrations of methanol lower than 10 kg m3 were not adequate for sufficient cell growth rate, resulting in lower yields of recombinant proteins [21,22]. Yet the methanol concentrations higher than 10 kg m3 had negative effect on cell growth and inhibitory influence on the induction of AOX1 promoter due to toxic products of alcohol oxidation [23]. The time course of CBH II production by P. pastoris PC1 and PC2 was shown in Fig. 3. As shown in Fig. 3A, the CBHAs of the transformants PC1 and PC2 reached the crest values at 96 h during the induction and fermentation processes, 2.89 ± 0.32 U cm3 for the transformant PC1 harboring native cbh2 gene and 5.84 ± 0.42 U cm3 for PC2 harboring synthetic cbh2 gene whose codons were optimized according to preferential codon usage of the host P. pastoris for the improved heterologous expression and production of CBH II, and thereafter declined probably due to the degradation of CBH II caused by the proteases released by the aged P. pastoris [24]. The CBHA of the enzyme expressed by the synthetic cbh2 gene was 2.02 times higher than that of the native cbh2 gene. As presented in Fig. 3B, the soluble protein excreted by the transformant PC2 was also significantly higher than that of PC1. PC2 had 0.63 ± 0.06 mg cm3 soluble protein at 96 h, 1.66 times higher than PC1 that is 0.38 ± 0.04 mg cm3. The results of CBHA and soluble protein comparisons demonstrate that codon optimization strategy used in this work was efficacious and capable of increasing the expression level of heterologous genes, especially for those host strains with large difference in codon usage patterns from the original strains with the native genes. However, the codon optimization had differentiated outcomes in the literature reports [11e13], ranging from 1.24 to more than 10-fold increase in enzyme production performance. It depends on how well the codon optimization strategy suits the host strain for exotic gene expression. The supernatants of fermentation broths from the P. pastoris transformants PC1 and PC2, as well as the original P. pastoris strain, were collected at 96 h and analyzed through SDS-PAGE to estimate the molecular weight of the product from recombinant P. pastoris. Fig. 4 presents the result that lane 1 and lane 2 are the CBH II bands with the identical molecular weight approximately 58 kDa produced by PC1 and PC2, respectively, and lane 0 for the original P. pastoris has no CBH II band, demonstrating that both the native cbh2 gene and the synthetic cbh2 gene were successfully expressed and secreted by recombinant P. pastoris without any change in
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Fig. 2 e Results from agarose gel electrophoresis. (A) the final expression vector pPIC9K-cbh2, (B) Colony PCR the Escherichia coli transformants containing the plasmid with native cbh2 gene (lane 1) and the plasmid with synthetic cbh2 gene (lane 2), respectively, (C) PCR results of Pichia pastoris transformants harboring the expression cassettes with native cbh2 gene and synthetic cbh2 gene, respectively. Lane 1: first generation of P. patoris transformant with native cbh2 gene, Lane 2: tenth generation of P. patoris transformant with native cbh2 gene, Lane 3: first generation of P. patoris transformant with synthetic cbh2 gene, Lane 4: tenth generation of P. patoris transformant with synthetic cbh2 gene, Lane 5e7: controls.
amino acid sequence of the CBH II protein structure. The actual molecular weights of recombinant CBH II proteins expressed by P. pastoris 58 kDa are higher than that of the recombinant CBH II protein expressed by E. coli 53 kDa (data not shown) which is close to its putative molecular weight calculated theoretically from the amino acids. The reason for this phenomenon is that N-glycosylation makes the recombinant enzyme ~10 kDa larger than the native nonglycosylated enzyme [11,12]. The approximately 5 kDa enlarged molecular weight of recombinant protein in this work indicates that the overglycosylation problem for foreign gene expression in P. pastoris was not very serious.
3.3. Enzymatic properties of CBH II expressed by P. pastoris The effects of pH and temperature on the activity of CBH II produced by recombinant P. pastoris toward microcrystalline cellulose PH101 (SigmaeAldrich, St. Louis, MO, USA) were
investigated to determine the optimal pH value and temperature. The results are shown in Fig. 5A and B, the former one indicating that the optimal pH value for CBH II was 5.0 and the latter one demonstrating that the optimal temperature for CBH II was 50 C. Because of the amino acids of the CBH II proteins encoded by the native cbh2 gene and the synthetic cbh2 gene whose codons were optimized are exactly the same, nothing is different between the two CBH II proteins and they are treated as the same CBH II. It was found in Fig. 5 that CBH II had lower stability in the interval of higher pH (5e8) and in the interval of higher temperature (50e70 C) than in the interval of lower pH (3e5) and in the interval of lower temperature (30e50 C), respectively. The reason for former one may be that CBH is more unstable under the low hydrogen ion concentration condition caused by high pH than under the high ion concentration condition caused by low pH. So perhaps hydrogen ion concentration is important for CBH II to maintain its active spatial conformation and enzymatic activity. The explanation for latter one is that high temperature not
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Fig. 4 e SDS-PAGE analysis of the culture supernatant of recombinant P. pastoris. Lane 1: PC1 containing native cbh2 gene, Lane 2: PC2 containing synthetic cbh2 gene, Lane 0: control. All the samples are collected at 24 h during fermentation.
Fig. 3 e Cellobiohydrolase activities (A) and soluble protein concentrations (B) in the fermentation broth obtained as a function of time from Pichia patoris transformant PC1 containing native cbh2 gene and PC2 containing synthetic cbh2 gene grown in the 100 cm3 BMMY mediums in 500 cm3 Erlenmeyer flasks incubated in incubatory shakers at 30 C and a shaking of 4.17 Hz. The initial pH and OD600 were 6.0 and 1.0, respectively. Methanol was added to a concentration of 10 kg m¡3 at every 24 h to keep the durative induction and expression of the cbh2 gene in P. pastoris transformants. Data shown are average values of triplicate samples and error bars are standard deviations.
only negatively influences the CBHA but also causes CBH II deactivation. Low temperature, nonetheless, has just negative effect on CBHA but no deactivation effect on CBH II. P. pastoris is an ideal host strain for the heterologous expression of cbh2 gene and an excellent producer of CBH II because it has a number of advantages over bacteria and fungi. For example, AOX1 promoter that is one of the strongest promoters can be employed to efficiently express recombinant genes and the simple composition of extracellular proteins facilitates downstream processing. And the codon optimization is necessary to overcome the codon usage bias between different species, which was also proven to be effective in improving heterologous protein production in this study. Fungi have many their own merits such as great ability of producing extracellular proteins but have obvious demerits, the difficulty of genetic engineering manipulation and the
Fig. 5 e Effects of pH (A) and temperature (B) on CBH II activity from recombinant P. pastoris. (A) the temperature was set at 50 C, (B) the pH was adjust to 5.0 with 50 mmol L¡1 citrate buffer solution. Fractional activity (%) was percentage of the residual activity of CBH II at nonoptimal condition to that at optimal condition. Data shown are average values of triplicate samples and error bars are standard deviations.
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complexity of extracellular protein making downstream processing not easy. But if enzyme separation and purification from enzyme mixture is not taken into consideration or not essential, fungi would be a perfect hosts given that many molecular biology techniques of fungi is maturing, especially for some model organisms such as T. reesei and Aspergillus niger [25e28]. Moreover, the genes of the components of cellulase had been successfully cloned and expressed in T. reesei to enhance both the cellulase production and its performance in enzymatic hydrolysis of lignocelluloses [4,8,29]. In addition, it was found that T. reesei cbh2 gene could be expressed in E. coli but its product was exclusively localized to the outer membrane of E. coli and unable to be excreted, although E. coli has several advantages over eukaryotic hosts including shorter time scales, a well-developed genetic toolbox, and less complex high-throughput screening through directed evolution [30]. P. pastoris, therefore, is an excellent alternative for producing CBH II or other enzymes with simple composition easy for separation and purification.
3.4.
Enzymatic hydrolysis of SHPCS and SHPRS
The recombinant CBH II was added to T. reesei cellulase to enhance the exo-exo-synergism for the enhancement of the enzymatic hydrolysis of lignocelluloses. Excessive amounts of A. niger cellobiase and T. reesei xylanase were supplemented in order to exclude the disturbing effects of cellobiase or xylanase deficiency on the result and enhance the enzymatic hydrolysis yields of SHPCS and SHPRS. As shown in Fig. 6, when the cellobiase and xylanase added one after one, the enzymatic hydrolysis yields of both the cellulolytic enzyme mixtures with and without recombinant CBH II were obviously improved. This observation indicates that T. reesei cellulase lacks of cellobiase and xylanase. It is well known that T. reesei is deficient in cellobiase, which can be solved by fermentation or genetic engineering techniques [5,6,8]. In all cases in Fig. 6, the cellulolytic enzyme mixture with recombinant CBH II performed better than that without recombinant CBH II, demonstrating that the deficiency in cellobiase and xylanase did not disturb the result and that the enhancement of enzymatic hydrolysis of SHPCS was caused by the exo-exo-synergism improved by adding recombinant CBH II. When 20 FPIU g1 dry substrate cellulase, 10 IU g1 dry substrate cellobiase, 300 IU g1 dry substrate xylanase and 20 U g1 dry substrate recombinant CBH II were used, a high enzymatic hydrolysis yield of SHPCS (94.7%) was obtained. This result not only shows the hydrolyzing efficiency of cellulolytic enzyme mixture but also demonstrates that the pretreatment method is suitable for corn stover. Same situation took place in the enzymatic hydrolysis of SHPRS, shown in Fig. 7. Exo-exo-synergism played the same positive role in all cases shown in Fig. 7AeC. This phenomenon proves that exo-exo-synergism functions authentically in the enzymatic hydrolysis regardless of what substrate being used. When 20 FPIU g1 dry substrate cellulase, 10 IU g1 dry substrate cellobiase, 300 IU g1 dry substrate xylanase and
given in Section 2.10. Data shown are average values of triplicate samples.
Fig. 6 e Time courses of enzymatic hydrolysis of SHPCS carried out in 250 cm3 Erlenmeyer flasks with a volume of 50 cm3. (A) The dosages of cellulase and recombinant CBH II were 20 FPIU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. (B) The dosages of cellulase, cellobiase and recombinant CBH II were 20 FPIU g¡1 dry substrate, 10 IU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. (C) The dosages of cellulase, cellobiase, xylanase and recombinant CBH II were 20 FPIU g¡1 dry substrate, 10 IU g¡1 dry substrate, 300 IU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. The temperature, agitation, and initial pH were 50 C, 1.67 Hz, and 4.8, respectively. The enzymatic hydrolysis yields were calculated in accordance with Eq. (1)
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20 U g1 dry substrate recombinant CBH II were used, the enzymatic hydrolysis yield of SHPRS was 83.3%, which is good although lower than that of SHPRS. This may be due to some reasons. For instance, the different properties of the substrates vary the results because different substrate has differences in chemical composition and structure. In general rice straw is not as good as corn stover when used as substrate for production of reducing sugars because of higher ash content. Another probable reason is that the pretreatment method best for corn stover did not suit rice straw because pretreatment methods and related conditions differ from one type of lignocellulosic material to another. The reason why the same pretreatment method was used although it was not most optimal for rice straw is that the different pretreatment method may be the interfering factor on the comparison. It is proven in this work that exo-exo-synergism played an important role in enzymatic hydrolysis of lignocelluloses, which always pales in comparison with the fame of the synergism between cellobiase and cellobiohydrolases/endoglucanases. No further enhancement of enzymatic hydrolysis could be observed when more than 20 U g1 substrate recombinant CBH II was added because at that point exo-exosynergism was no longer the limiting factor of the enzymatic hydrolysis [31]. It demonstrates that the strategies employed in this work to enhance heterologous expression of T. reesei cbh2 gene in P. pastoris and improve the exo-exosynergism in cellulase to increase the enzymatic hydrolysis yield by adding recombinant CBH II worked well and is a good paradigm for others. Corn stover and rice straw are main agricultural residues in the north and south of China, respectively. The abundance of these agricultural residues and improper disposal such as combustion cause serious environmental problems in today's China. Therefore, the utilization and conversion of the agricultural residues to valuable chemicals or products is drawing more and more attention in both science community and industry sectors. The good paradigm introduced in this work (94.7% conversion of corn stover and 83.3% conversion of rice straw to monomeric sugars) gives us a possible solution to produce fermentable sugars from lignocellulosic biomass inexpensively, renewably, and sustainably. Those fermentable sugars from lignocelluloses are ideal alternatives to starch sugar for production of biofuels, chemicals or others, which is profoundly meaningful to the countries including China who have increasingly enormous demand for food but limited food resources. Fig. 7 e Time courses of enzymatic hydrolysis of SHPRS carried out in 250 cm3 Erlenmeyer flasks with a volume of 50 cm3. (A) The dosages of cellulase and recombinant CBH II were 20 FPIU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. (B) The dosages of cellulase, cellobiase and recombinant CBH II were 20 FPIU g¡1 dry substrate, 10 IU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. (C) The dosages of cellulase, cellobiase, xylanase and recombinant CBH II were 20 FPIU g¡1 dry substrate, 10 IU g¡1 dry substrate, 300 IU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. The temperature, agitation, and initial pH were 50 C, 1.67 Hz, and 4.8, respectively. The enzymatic
4.
Conclusions
The native cbh2 gene cloned from T. reesei ZU-02 and the synthetic cbh2 gene whose codons were optimized according to the codon usage frequencies of the host strain P. pastoris were introduced into P. pastoris GS115, respectively. Through the comparison in fermentation, it was found that codon
hydrolysis yields were calculated in accordance with Eq. (1) given in Section 2.10. Data shown are average values of triplicate samples.
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optimization strategy could significantly enhance the heterologous expression of T. reesei CBH II in P. pastoris. The product of the recombinant P. pastoris was analyzed and its enzymatic properties were investigated. High enzymatic hydrolysis yields of SHPCS and SHPRS were obtained by adding recombinant CBH II to enhance exo-exo-synergism in cellulase.
Acknowledgments This work was financially supported by the National High Technology Research and Development Program (2007AA05Z401) and by the Program for Zhejiang Leading Team of Science and Technology Innovation (2011R50002) of China.
references
[1] Peterson R, Nevalainen H. Trichoderma reesei RUT-C30-thirty years of strain improvement. Microbiology 2012;158(1):58e68. [2] Schuster A, Schmoll M. Biology and biotechnology of Trichoderma. Appl Microbiol Biotechnol 2010;87(3):787e99. [3] Yu L, Chao Y, Wensel P, Chen S. Hydrodynamic and kinetic study of cellulase production by Trichoderma reesei with pellet morphology. Biotechnol Bioeng 2012;109(7):1755e68. [4] Fang H, Xia L. High activity cellulase production by recombinant Trichoderma reesei ZU-02 with the enhanced cellobiohydrolase production. Bioresour Technol 2013;144:693e7. [5] Fang H, Zhao C, Song X-Y. Optimization of enzymatic hydrolysis of steam-exploded corn stover by two approaches: response surface methodology or using cellulase from mixed cultures of Trichoderma reesei RUT-C30 and Aspergillus niger NL02. Bioresour Technol 2010;101(11):4111e9. [6] Fang H, Zhao C, Song X-Y, Chen M, Chang Z, Chu J. Enhanced cellulolytic enzyme production by the synergism between Trichoderma reesei RUT-C30 and Aspergillus niger NL02 and by the addition of surfactants. Biotechnol Bioprocess Eng 2013;18(2):390e8. [7] Chen M, Zhao J, Xia L. Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars. Carbohydr Polym 2008;71(3):411e5. [8] Wang B, Xia L. High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei. Bioresour Technol 2011;102(6):4568e72. [9] Nidetzky B, Steiner W, Hayn M, Claeyssens M. Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction. Biochem J 1994;298(3):705e10. [10] Maras M, De Bruyn A, Schraml J, Herdewijn P, Claeyssens M, Fiers W, et al. Structural characterization of N-linked oligosaccharides from cellobiohydrolase I secreted by the filamentous fungus Trichoderma reesei RUTC 30. Eur J Biochem 1997;245(3):617e25. [11] Teng D, Fan Y, Yang YL, Tian ZG, Luo J, Wang JH. Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Appl Microbiol Biotechnol 2007;74(5):1074e83.
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[12] Akcapinar GB, Gul O, Sezerman U. Effect of codon optimization on the expression of Trichoderma reesei endoglucanase 1 in Pichia pastoris. Biotechnol Prog 2011;27(5):1257e63. [13] Te'o VS, Cziferszky AE, Bergquist PL, Nevalainen KM. Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei. FEMS Microbiol Lett 2000;190(1):13e9. [14] Fang X, Shen Y, Zhao J, Bao X, Qu Y. Status and prospect of lignocellulosic bioethanol production in China. Bioresour Technol 2010;101(13):4814e9. [15] Galbe M, Zacchi G. Pretreatment: the key to efficient utilization of lignocellulosic materials. Biomass Bioenergy 2012;46:70e8. [16] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Laboratory Press; 1989. [17] Liming X, Xueliang S. High-yield cellulase production by Trichoderma reesei ZU-02 on corn cob residue. Bioresour Technol 2004;91(3):259e62. [18] Jin X, Meng N, Xia LM. Expression of an Endo-beta-1,4glucanase gene from Orpinomyces PC-2 in Pichia pastoris. Int J Mol Sci 2011;12(5):3366e80. [19] Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59:257e68. [20] Fang H, Xia L. Cellulase production by recombinant Trichoderma reesei and its application in enzymatic hydrolysis of agricultural residues. Fuel 2015;143:211e6. [21] Cregg JM, Cereghino JL, Shi J, Higgins DR. Recombinant protein expression in Pichia pastoris. Mol Biotechnol 2000;16(1):23e52. [22] Zhang W, Inan M, Meagher MM. Fermentation strategies for recombinant protein expression in the methylotrophic yeast Pichia pastoris. Biotechnol Bioprocess Eng 2000;5(4):275e87. [23] Cos O, Ramon R, Montesinos JL, Valero F. Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: a review. Microb Cell Fact 2006;5:17. [24] Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, et al. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 1997;190(1):55e62. [25] Meyer V. Genetic engineering of filamentous fungieprogress, obstacles and future trends. Biotechnol Adv 2008;26(2):177e85. [26] Michielse CB, Hooykaas PJ, van den Hondel CA, Ram AF. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc 2008;3(10):1671e8. [27] Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ. Agrobacterium -mediated transformation as a tool for functional genomics in fungi. Curr Genet 2005;48(1):1e17. [28] Nevalainen KM, Te'o VS, Bergquist PL. Heterologous protein expression in filamentous fungi. Trends Biotechnol 2005;23(9):468e74. [29] Gu B, Xia L. High expression of a neutral endo-betaglucanase gene from Humicola insolens in Trichoderma reesei. J Ind Microbiol Biotechnol 2013;40(7):773e9. [30] Abdeljabbar DM, Song HJ, Link AJ. Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli. Biotechnol Lett 2012;34(1):91e6. [31] Zhou J, Wang Y-H, Chu J, Luo L-Z, Zhuang Y-P, Zhang S-L. Optimization of cellulase mixture for efficient hydrolysis of steam-exploded corn stover by statistically designed experiments. Bioresour Technol 2009;100(2):819e25.
Available online at www.sciencedirect.com
ScienceDirect http://www.elsevier.com/locate/biombioe
Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw Hao Fang, Liming Xia* Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
article info
abstract
Article history:
The cellobiohydrolase II (CBH II) gene cbh2 from Trichoderma reesei was cloned and its co-
Received 18 April 2014
dons were optimized in accordance with the codon usage frequencies of the host Pichia
Received in revised form
pastoris. The AOX1 strong promoter inducible by methanol was employed to efficiently
8 April 2015
express the foreign gene cbh2 in P. pastoris. It was found that 5.84 ± 0.42 U cm3 CBH II was
Accepted 16 April 2015
obtained at 96 h using the synthetic cbh2 gene whose codons were optimized, 2.02-fold
Available online
higher than using the native cbh2 gene (2.89 ± 0.32 U cm3), indicating that the codon optimization strategy was an effective approach to enhance the heterologous expression of
Keywords:
CBH II in P. pastoris. The product of recombinant P. pastoris CBH II had an approximate
Trichoderma reesei cellobiohydrolase
molecular weight 58 kDa. Its optimal pH and temperature were 5.0 and 50 C, respectively.
II
The recombinant CBH II was used to enhance the yields of the enzymatic hydrolysis of the
Pichia pastoris
corn stover and rice straw pretreated with sodium hydroxide by improving the exo-exo-
Heterologous expression
synergism between CBH II and CBH I in T. reesei cellulase. The yields 94.7% and 83.3%
Codon optimization
were achieved in the enzymatic hydrolysis of corn stover and rice straw pretreated by
Exo-exo-synergism
sodium hydroxide, respectively.
Enzymatic hydrolysis
1.
Introduction
During the bioprocesses of converting lignocelluloses which are one of the most abundant renewable resources on the earth to reducing sugars that could be used as feedstock for a variety of value-added chemicals such as fuels, cellulase with a complete composition plays a very important role, presenting a hopeful alternative for conventional chemical processes. Trichoderma reesei, a mesophilic soft-rot ascomycete fungus
* Corresponding author. Tel./fax: þ86 571 87951840. E-mail address: [email protected] (L. Xia). http://dx.doi.org/10.1016/j.biombioe.2015.04.014 0961-9534/© 2015 Published by Elsevier Ltd.
© 2015 Published by Elsevier Ltd.
that is widely used as the biotechnological workhorse of the genus, is a household name in both academic and industrydriven studies into lignocelluloses degrading enzymes and their applications [1e3]. A complete mixture of cellulase from T. reesei is composed of cellobiohydrolases (CBH: EC 3.2.1.91), exoglucanases which release cellobiose as main product from crystalline cellulose; endoglucanases (EG: EC 3.2.1.4), preferably attack amorphous cellulose and some short chain oligomers; and b-glucosidases (EC 3.2.1.21), which hydrolyze cellooligosaccharides and cellobiose into glucose [4e6].
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Synergisms exist between the components of T. reesei cellulase. The best known is the synergism between b-glucosidases and cellobiohydrolases. Many studies pivoted the b-glucosidases to enhance that synergism, diminishing the inhibitory effect of cellobiose accumulation in enzymatic hydrolysis on cellobiohydrolases (and endoglucanases) caused by the deficiency of T. reesei cellulase in the bglucosidase content [5e8]. Nonetheless the exo-exosynergism between cellobiohydrolase I (CBH I) coded by cbh1 gene and the cellobiohydrolase II (CBH II) coded by cbh2 gene in T. reesei cellulase is of importance because CBH I mainly acts on reducing ends and CBH II preferably acts on non-reducing ends, preparing a more readily hydrolysable substrate for each other [4,9]. In T. reesei, CBH II has higher specific activity toward microcrystalline cellulose than CBH I, but the latter one has 50e60% of the total secreted protein of T. reesei due to the fact that the promoter of cbh1 gene is regarded as a strong promoter [10]. In contrast, CBH II only accounts for 10e15% of total protein [10], thus rendering it deficient in CBH II that is essential to an excellent exo-exosynergism. So improving CBH II is important to a great performance of T. reesei cellulase in enzymatic hydrolysis of lignocelluloses and the deficiency of CBH II is one of the limiting factors of T. reesei cellulase needed to be overcome using different strategies. Producing T. reesei CBH II in Pichia pastoris is a desirable approach because P. pastoris is an excellent expression system for expressing heterologous protein in secreted forms with many advantages among which the most important one is its perfect protein processing mechanism including signal peptide cleavage, protein folding, and post-translational modification inside the cell, and secretion ability into medium with normal function [11]. Owing to the large difference of codon usage preference in different species and some other reasons such as unstable mRNA of exotic gene, heterologous gene expressions always result in low extracellular proteins production, especially for those proteins with active functions. Codon optimization in accordance with the codon usage preference of host cell, therefore, is a promising and effective technique to increase heterologous protein expression level. This has been proven by some reports [11e13], although they had differentiated outcomes. Corn stover and rice straw, main agricultural residues in northern and southern China respectively, could be made use of as renewable feedstock for biorefinery. A great amount of corn stover and rice straw is always set on fire every year in China, giving rise to severe environmental problems. Producing lignocellulosic ethanol or other value-added products from corn stover or other agricultural residues, therefore, is feasible and has many advantages in China [14]. Thus corn stover and rice straw were used as substrate of enzymatic hydrolysis to compare the performance of the cellulolytic enzyme mixture. Pretreatment is required to smash the recalcitrant structure of lignocelluloses naturally formed before enzymatic hydrolysis in order to increase the enzymatic digestibility and to facilitate subsequent conversion. Sodium hydroxide pretreatment, carried out at lower temperature and pressure than acid hydrolysis and steam explosion, can substantially increase the lignin removal and enhance the accessibility and digestibility of cellulose [7,15].
So it was used in this work for the pretreatment of corn stover and rice straw. In this work, the codons of T. reesei cbh2 gene were optimized according to the codon usage preference of P. pastoris. Then the synthetic cbh2 gene with codons optimized and the native T. reesei cbh2 gene were put into the P. pastoris expression vector pPIC9K containing the a-Factor secretion signal, respectively. The former one was used for the efficient expression in P. pastoris GS115 and the latter one was used as control. The enzymatic properties of recombinant CBH II were investigated. And the recombinant CBH II was added to the cellulolytic enzymes mixture so as to enhance exo-exo-synergism between CBH I and CBII, thereby improving the enzymatic hydrolysis yields of corn stover and rice straw.
2.
Material and methods
2.1.
Microorganisms, plasmids and mediums
Escherichia coli strain DH5a was used for plasmid manipulation and propagation throughout the work as described by Sambrook et al. [16]. T. reesei ZU-02 [4,8,17] provided total mRNA containing the mRNA of the cbh2 gene used as the template of reverse transcriptase polymerase chain reaction (RT-PCR) for producing cDNA, which was subsequently used as the template of native cbh2 gene cloning. P. pastoris GS115 was the host of cbh2 gene expression. All the microorganisms above were from the strain collection of Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University. The pMD18-T cloning vector (Takara, Otsu, Japan) was used for TA cloning. The plasmid pPIC9K, harboring a-factor signal peptide gene and the methanol-inducible alcohol oxidase gene (AOX1) that is a strong promoter controlling recombinant gene expression, was employed as expression vector of cbh2 gene in P. pastoris GS115 [18]. LuriaeBertani (LB) medium for E. coli cultivation had the following composition: 10 kg m3 tryptone, 5 kg m3 yeast extract, 10 kg m3 NaCl, pH 7.0. The potato dextrose agar (PDA) slants used for short term T. reesei ZU-02 culture preservation were composed of 200 kg m3 potato, 20 kg m3 dextrose and 20 kg m3 agar. The seed medium for T. reesei was as follows: 15 kg m3 glucose, 20 kg m3 yeast extract, 2.5 kg m3 (NH4)2SO4, kg m3 KH2PO4, 0.8 kg m3 MgSO4, kg m3 CaC12, 0.005 kg m3 FeSO4$7H2O, 0.0016 kg m3 MnSO4$H2O, 0.0014 kg m3 ZnSO4$7H2O, 0.0037 kg m3 CoCl2$6H2O. The ingredients of YPD medium for P. pastoris GS115 cultivation were as follows: 10 kg m3 yeast extract, 20 kg m3 peptone, 20 kg m3 glucose. MD medium used for screening transformation consisted of 3.4 kg m3 yeast nitrogen base (YNB) without amino acids, 4 104 kg m3 biotin, and 20 kg m3 glucose. The composition of BMGY medium for seed preparation of P. pastoris transformants was as follows: 10 kg m3 glycerol, 10 kg m3 yeast extract, 20 kg m3 peptone, 3.4 kg m3 YNB, 10 kg m3 (NH4)2SO4 and 4 104 kg m3 biotin in pH 6.0 0.1 mol L1 potassium phosphate buffer. The BMMY medium for recombinant gene induction and fermentation was comprised of 5 kg m3
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methanol, 10 kg m3 yeast extract, 20 kg m3 peptone, 3.4 kg m3 YNB, 10 kg m3 (NH4)2SO4 and 4 104 kg m3 biotin in pH 6.0100 mmol L1 potassium phosphate buffer. When solid agar plates were required, 15e20 kg m3 agar powder was added before sterilization.
2.2. Cloning of native cbh2 gene and synthesis of codon optimized cbh2 gene T. reesei ZU-02 spores were inoculated into the seed medium (containing microcrystalline cellulose instead of glucose) and incubated at 30 C and 2.83 Hz for 2 d for total RNA preparation. Five cubic centimeters mycelium was frozen with liquid nitrogen and ground to fine powder with mortar and pestle. Total RNA was extracted from the fine powder by the TRNzol reagent (Takara, Dalian, China). The cDNA of T. reesei cbh2 gene was produced through reverse transcriptase reaction using the total RNA as the template with the reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Takara, Dalian, China). DNA amplification was carried out with a forward primer (50 -ATAGAATTCCTCAAGCTTG CTCAAGCGTCT-30 , containing EcoR I site) and a reverse primer (50 -CGCGCGGCCGCTTACAGGAACGATGGGTTTGC-30 , containing Not I site) from the cDNA as template. PCR conditions included 94 C for 10 min, followed by 30 (94 C for 1 min, 50 C for 1 min, 72 C for 2 min), and 72 C for 10 min. The resultant gene product was cloned into the pMD18-T cloning vector and sent to Sangon Biotech (Shanghai, China) to be sequenced. The amino acid sequence of T. reesei CBH II (GenBank accession: AAA34210.1) was reverse-translated into the preferentially used codons in P. pastoris. The codon usage detail of P. pastoris, in the form of codon usage frequency, is shown in Table 1, according to which the codons were optimized. Codons with low usage percentage (<15%) were replaced by higher frequently used ones [11]. Then the codon optimized cbh2 gene containing the restriction sites, EcoR I in the upstream and Not I in the downstream for the subsequent gene manipulation, was synthesized by Sangon Biotech (Shanghai, China).
2.3.
Construction of expression plasmid
The pMD18-T-cbh2 harboring T. reesei cbh2 gene was double digested by the restriction enzymes EcoR I and Not I that were purchased from Takara Biotech (Dalian, China), releasing the cbh2 gene fragment which was subsequently linked to the pPIC9K fragment produced from the double digestion with the same restriction enzymes. As a result, the final plasmid pPIC9K-cbh2 for the heterologous expression in P. pastoris was obtained. As for the synthetic T. reesei cbh2 gene from Sangon Biotech (Shanghai, China), it was contained in pUC18 plasmid with EcoR I and Not I restriction sites. The ligation of pPIC9K fragment and the synthetic cbh2 gene fragment was the same as the procedure described above. These two pPIC9K-cbh2 plasmids containing native and synthetic cbh2 gene fragment were linearized with restriction enzyme Bgl II prior to being used for transformation of P. pastoris.
Table 1 e Comparison of codon usage of native and synthetic cbh2 genes with that of the host strain Pichia pastoris. Amino acids
Ala
Arg
Asn Asp Cys Gln Glu Gly
His Ile
Leu
Lys Met Phe Pro
Ser
Stop
Thr
Trp
Codons
GCT GCC GCA GCG AGA AGG CGT CGA CGC CGG AAC AAT GAT GAC TGT TGC CAA CAG GAA GAG GGT GGA GGC GGG CAT CAC ATT ATC ATA TTG TTA CTT CTG CTA CTC AAG AAA ATG TTC TTT CCA CCT CCC CCG TCT TCC TCA AGT AGC TCG TAA TAG TGA ACT ACC ACA ACG TGG
Codon usage frequency Pichia pastoris
Native cbh2
0.45 0.26 0.23 0.06 0.48 0.16 0.16 0.10 0.05 0.05 0.52 0.48 0.58 0.42 0.64 0.36 0.61 0.39 0.57 0.43 0.41 0.32 0.14 0.13 0.57 0.43 0.50 0.31 0.19 0.32 0.17 0.17 0.15 0.11 0.08 0.53 0.47 1.00 0.54 0.46 0.41 0.35 0.15 0.09 0.30 0.20 0.18 0.15 0.09 0.08 0.53 0.32 0.15 0.40 0.25 0.24 0.11 1.00
0.30 (18) 0.38 (23) 0.22 (13) 0.10 (6) 0.13 (2) 0.00 (0) 0.13 (2) 0.20 (3) 0.40 (6) 0.13 (2) 0.63 (19) 0.37 (11) 0.38 (8) 0.62 (13) 0.50 (6) 0.50 (6) 0.52 (11) 0.48 (10) 0.30 (3) 0.70 (7) 0.23 (9) 0.30 (12) 0.40 (16) 0.08 (3) 0.25 (1) 0.75 (3) 0.56 (10) 0.44 (8) 0.00 (0) 0.19 (7) 0.00 (0) 0.30 (11) 0.22 (8) 0.08 (3) 0.22 (8) 0.90 (9) 0.10 (1) 1.00 (5) 0.33 (4) 0.67 (8) 0.32 (10) 0.35 (11) 0.13 (4) 0.19 (6) 0.15 (7) 0.23 (11) 0.09 (4) 0.04 (2) 0.23 (11) 0.26 (12) 1.00 (1) 0.00 (0) 0.00 (0) 0.30 (11) 0.38 (14) 0.14 (5) 0.19 (7) 1.00 (12)
Synthetic cbh2 0.87 0.08 0.05 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.9 0.10 0.62 0.38 0.83 0.17 0.81 0.19 0.70 0.30 0.78 0.23 0.00 0.00 0.75 0.25 0.78 0.22 0.00 0.97 0.00 0.03 0.00 0.00 0.00 1.00 0.00 1.00 0.92 0.08 0.77 0.23 0.00 0.00 0.81 0.17 0.02 0.00 0.00 0.00 1.00 0.00 0.00 0.70 0.24 0.05 0.00 1.00
(52) (5) (3) (0) (15) (0) (0) (0) (0) (0) (27) (3) (13) (8) (10) (2) (17) (4) (7) (3) (31) (9) (0) (0) (3) (1) (14) (4) (0) (36) (0) (1) (0) (0) (0) (10) (0) (5) (11) (1) (24) (7) (0) (0) (38) (8) (1) (0) (0) (0) (1) (0) (0) (26) (9) (2) (0) (12)
(continued on next page)
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Table 1 e (continued ) Amino acids
Tyr Val
Codons
TAC TAT GTT GTC GTG GTA
Codon usage frequency Pichia pastoris
Native cbh2
0.55 0.45 0.43 0.23 0.19 0.15
0.57 (12) 0.43 (9) 0.21 (6) 0.46 (13) 0.25 (7) 0.07 (2)
Synthetic cbh2 0.90 0.10 0.89 0.11 0.00 0.00
(19) (2) (25) (3) (0) (0)
The information on the codon usage of P. pastoris is from the codon usage database (http://www.kazusa.or.jp/codon).
1.0. Subsequently, the 100 cm3 BMMY mediums in 500 cm3 Erlenmeyer flasks inoculated P. pastoris transformants were incubated at 30 C and 4.17 Hz for 120 h. Methanol was added to a concentration of 10 kg m3 at every 24 h to keep the durative induction and expression of the cbh2 gene in P. pastoris transformants driven by the AOX1 promoter intensively inducible in the presence of an appropriate concentration of methanol. Sampling was implemented periodically for analysis. At least three parallel experiments and samples were adopted to abate the detrimental effects of possible errors on experimental results.
2.6. 2.4. Transformation of P. pastoris and screening of transformants P. pastoris GS115 stored on YPD slant was inoculated into 5 cm3 YPD medium and cultured at 30 C and 4.17 Hz for 12 h. Then it was collected by centrifugation (3000 g, 5 min) and grown in 50 cm3 YPD medium at 30 C and 4.17 Hz until OD600 ¼ 1.3e1.5. The competent cells of P. pastoris GS115 were prepared, the transformation was carried out, and the screening of P. pastoris transformants was conducted, according to the Invitrogen pPIC9K Expression Manual. The linearized pPIC9Kcbh2 was transformed into the competent cells of P. pastoris GS115 by electroporation using SCIENTZ-2B Genepulser (Ningbo, China) under the conditions: 1.25 kV, 25 uF, 200 U. Afterwards, potential transformants were selected on MD and YPD agar plates using the antibiotic G418 at five concentrations: 1.0, 2.0, 3.0, 4.0 and 5.0 mg cm3. In theory, the transformants appearing on the plates containing higher G418 concentration often have a larger number of insertions or integrations of recombinant DNA fragments into yeast genome. Hence the P. pastoris transformants growing on the plates containing 5.0 or 4.0 mg cm3 G418 were chosen for further research.
2.5.
Verification of P. pastoris transformants by PCR
The chromosomal DNA of P. pastoris was produced using Ezup Column Yeast Genomic DNA Purification Kit purchased from Sangon Biotech (Shanghai, China). Then PCR of the chromosomal DNA of P. pastoris was carried out to verify the insertion of recombinant DNA into yeast genome using AOX1 primes 50 and 50 -GGCAAATGGGACTGGTTCCAATTGACAAGC-30 CATTCTGACATCCT-30 . PCR conditions were 94 C for 10 min, followed by 30 (94 C for 1 min, 60 C for 1 min, 72 C for 2 min), and 72 C for 10 min.
Cellobiohydrolase activity (CBHA) assay, which was modified from the standard method recommended by International Union of Pure and Applied Chemistry (IUPAC) [19], was implemented in 10 cm3 cuvettes using microcrystalline cellulose PH101 (SigmaeAldrich, St. Louis, MO, USA) as the substrate. Each cuvette was added with 0.02 g microcrystalline cellulose and 0.5 cm3 proper diluted enzyme. Then 1 cm3 50 mmol L1 citrate buffer was supplemented to form a 1.5 cm3 reaction mixture and have pH value controlled at 4.8. Having been blended thoroughly, the mixture was incubated in water bath at 50 C with a shaking of 2.33 Hz for 30 min. Thereafter, the reaction was stopped in ice bath and the reducing sugars in the supernatant were determined by DNS method. One Unit (1 U) of CBHA was defined as the amount of enzyme required for generating 1 mg reducing sugars in 1 h [4].
2.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The supernatant of the fermentation broth sampled from fermentation process was determined by SDS-PAGE with 0.5 kg m3 stacking gel and 1.2 kg m3 separating gel. Approximately 15e20 mm3 of the sample was loaded into each well of the staking gel. After electrophoresis, the gels were stained in the Coomassie brilliant blue solution (3 kg m3 methanol, 1 kg m3 acetic acid and 0.01 kg m3 Coomassie brilliant blue R-250) for 40 min and then destained for overnight in the destaining solution comprised of 3 kg m3 methanol and 1 kg m3 acetic acid. The molecular mass of the recombinant protein was estimated using the protein molecular weight marker purchased from Takara (Dalian, China) as a standard.
2.8. 2.5.1.
Cellobiohydrolase activity assay
Determination of soluble protein concentration
Induction of recombinant P. pastoris and fermentation
The colonies of P. pastoris transformants were inoculated into 5 cm3 BMGY medium and incubated at 30 C and 4.17 Hz for 12 h. Then the cells were collected by centrifugation (3000 g, 5 min) and cultured in 50 cm3 BMGY medium for 16e18 h. At this point, the cultures had OD600 values between 2 and 6 (logarithmic growth phase). The cells were collected by centrifugation (3000 g, 5 min) and the pellets were resuspended in 100 cm3 BMMY medium with a starting OD600 value
Soluble protein concentration in fermentation broth was determined using Bradford reagent (SigmaeAldrich, St. Louis, MO, USA). Bovine serum albumin (BSA) was used as the standard protein samples at the gradual concentrations with proper range to prepare a standard curve for the quantitative determination of soluble protein concentration. All the samples blended with Bradford reagent were measured spectrophotometrically at a wavelength of 595 nm.
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2.9. Enzymatic properties of CBH II expressed by P. pastoris
column). The enzymatic hydrolysis yield was calculated according to the following equation [4,20]:
The pH optimum of CBH II was measured between pH 3.0 and pH 8.0 at 50 C using the highest CBHA as the 100% standard. The pH range of 3.0e6.0 was adjusted with 50 mmol L1 citrate buffer solution and the pH range of 6.0e8.0 was adjusted with 50 mmol L1 sodium phosphate buffer. The optimal temperature was determined at a temperature gradient between 30 C and 70 C with 5 intervals using 50 mmol L1 citrate buffer solution, pH 5.0. The highest CBHA was also used as the 100% standard. The temperature was controlled in a thermostat water bath.
2.10.
Yield (%) ¼ reducing sugars (g) 0.9 100/ (cellulose þ hemicellulose) in substrate (g)
(1)
0.9 is the conversion factor used for deleting the interfering effect of the molecular weight changes of sugars before and after hydrolysis on the accuracy of calculation results.
3.
Results and discussion
3.1. Construction of expression vector, transformation of P. pastoris and transformants screening
Enzymatic hydrolysis of corn stover and rice straw
The corn stover (Zea mays Longyu 98 aged 101 d) and rice straw (Oryza sativa Qianyou 817 aged 124 d) were harvested and collected from Nanyang area (E112 310 , N33 010 ) of Henan Province and Hangzhou area (E120 090 , N30 140 ) of Zhejiang Province in October 2012, respectively. The definition of corn stover and rice straw in this work included stalk, husk and leaves. They were air dried and then stored at room temperature. They must be used within 12 months after being harvested. Before pretreatment, the stored corn stover and rice straw were pulverized with a laboratory hammer mill and screened to obtain the particles less than 2 mm in size using a sieve shaker. Subsequently, both of them were pretreated with sodium hydroxide pretreatment method proposed by Chen et al. [7]. Thereafter, the solid cellulosic residues were collected and washed with tap water to neutral pH. And then they were kept at 4 C in refrigerator for water content balance. The information about the sodium hydroxide pretreated corn stover (SHPCS), the sodium hydroxide pretreated rice straw (SHPRS), and the raw materials are shown in Table 2. Enzymatic hydrolysis of SHPCS or SHPRS was carried out in 250 cm3 Erlenmeyer flasks with a 50 cm3 mixture of 2.5 cm3 1 mol L1 citrate buffer solution (pH 4.8), 100 kg m3 substrate, enzymes, and a supplemented amount of water. The enzymatic hydrolysis was implemented at 50 C in water bath with a shaking of 2.33 Hz. Sampling was conducted periodically during the enzymatic hydrolysis for subsequent analysis.
2.11. Measurement of monomeric sugars and calculation of yield Glucose and xylose produced from the enzymatic hydrolysis were analyzed and quantified by higher performance liquid chromatography (HPLC) (Waters HPX-87P ion exclusion
From the online database (http://www.kazusa.or.jp/codon), it was found that some codons of T. reesei native cbh2 gene are rarely used in P. pastoris (usage frequency less than 15%). They are GCG (Ala), CGA/C/G (Arg), GGC/G (Gly), CTA/C (Leu), CCG (Pro), AGC (Ser), TCG (Ser), and ACG (Thr). All of them were replaced with those codons with the highest usage frequency for the specific amino acid. And some codons with the usage frequencies higher than 15% but lower than the most highly used codons were also replaced to further increase the expression efficiency of cbh2 gene due to the larger availabilities of the tRNAs having the anticodons with the higher usage frequencies in P. pastoris. The detailed changes from native cbh2 gene to synthetic codon-optimized cbh2 gene are listed in Table 1. The synthetic cbh2 gene from Sangon Biotech (Shanghai, China) was in the vector pUC18 with EcoR I and Not I restriction sites for subsequent DNA manipulation. The native cbh2 gene was cloned from T. reesei ZU-02 using the procedures as described in Materials and Methods. Then these two genes and the pPIC9K were double digested with EcoR I and Not I restriction enzymes and the cbh2 genes and the pPIC9K fragment were separated via agarose gel electrophoresis. Afterwards, the DNAs in collected gels were extracted and purified. Then the DNA fragments were ligated to form the final expression vector harboring native cbh2 gene or synthetic cbh2 gene that is shown in Fig. 1. And the agarose electrophoresis result of pPIC9K-cbh2 is presented in Fig. 2A. Because both native cbh2 gene and synthetic cbh2 gene have the same molecular weight, they are in the exactly same band in the agarose electrophoresis. And due to the super helix structure of the closed circle plasmid, the band of pPIC9K-cbh2 exhibits the molecular weight smaller than its actual molecular weight that is given in Fig. 1, approximately 9.5 kb.
Table 2 e Information about the pretreated and raw materials of corn stover and rice straw. Quantities are mass fractions of dry material (%). SHPCS, sodium hydroxide pretreated corn stover; SHPRS, sodium hydroxide pretreated rice straw. Cellulose Raw corn stover SHPCS Raw rice straw SHPRS
38.5 63.6 38.6 59.7
± 0.4 ± 0.9 ± 0.3 ± 0.7
Hemicellulose 21.8 ± 24.3 ± 20.9 ± 21.1 ±
0.5 0.6 1.5 1.7
Lignin 19.0 8.9 15.6 9.3
± 0.3 ± 0.3 ± 2.4 ± 0.5
Others 20.7 3.2 24.9 9.9
± 0.4 ± 0.1 ± 1.5 ± 1.3
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tenth generation corresponding P. pastoris transformants. This demonstrated the good stability of integrated cbh2 genes that is required to be tested because of the possibility of losing the genetic traits newly conferred through genetic engineering from generation to the next.
3.2. Induction of recombinant P. pastoris and fermentation
Fig. 1 e Recombinant plasmid pPIC9K-cbh2 with PAOX1cbh2-TAOX1 expression cassette used for transformation of Pichia patoris GS115. The native Trichoderma reesei cbh2 gene (or synthetic cbh2 gene whose codons were optimized) was linked between the AXO1 promoter and the AOX1 terminator, the strong promoter is inducible by methanol. The selective marker (HIS4) and the resistance markers, Kan (R) and Amp (R), in the plasmid were used for yeast and bacterial transformants selection, respectively. The image of plasmid structure was pictured using WinPlas 2.7 software (Rich Goldstein).
The constructed expression vectors were transformed into E. coli DH5a for plasmid amplification. The E. coli DH5a transformants containing pPIC9K-cbh2 were verified by colony PCR (Fig. 2B) and thereafter they were cultured for plasmid preparation. The recombinant plasmids linearized with restriction enzyme Bgl II were used for the transformation of P. pastoris by electroporation. The aim of plasmid linearization is to improve the efficiency of P. pastoris transformation by electroporation and different restriction enzymes would generate Mutþ or MutS recombinants. The former one is the phenotype of utilizing methanol normally and the latter one is the phenotype incapable of metabolize methanol efficiently because it cannot produce alcohol oxidase, which could be readily distinguished by patching on MD versus MM plates [11]. After P. pastoris transformation and transformants screening as described in Material and Methods, 24 positive transformants with the highest resistance to the geneticin G418 were selected and subjected to genomic DNA preparation for PCR verification (Fig. 2C). The PCR verification used the P. pastoris transformants containing blank plasmids and original strain as control (lane 5e7 in Fig. 2C). The bands with molecular weight about 1.3 kb (lane 1e4 in Fig. 2C) indicated the existence of native cbh2 gene or synthetic cbh2 gene in P. pastoris genomic DNA, confirming that all the transformants' genomic DNAs were integrated by exotic cbh2 gene. In Fig. 2C, lane 1 and 3 were the PCR products from the genomic DNAs of the first generation P. pastoris transformants encompassing native cbh2 gene or synthetic cbh2 gene, respectively. And lane 2 and 4 were the PCR products from the genomic DNAs of the
P. pastoris PC1 and PC2 were cultured in 50 cm3 BMMY medium in 250 cm3 Erlenmeyer flasks for fermentation. At every 24 h point during the fermentation process, 10 kg m3 methanol was added to induce the strong expression of AOX1 promoter. According to the previous research [18], 10 kg m3 methanol was the most suitable concentration for recombinant P. pastoris to produce heterologous enzyme. The concentrations of methanol lower than 10 kg m3 were not adequate for sufficient cell growth rate, resulting in lower yields of recombinant proteins [21,22]. Yet the methanol concentrations higher than 10 kg m3 had negative effect on cell growth and inhibitory influence on the induction of AOX1 promoter due to toxic products of alcohol oxidation [23]. The time course of CBH II production by P. pastoris PC1 and PC2 was shown in Fig. 3. As shown in Fig. 3A, the CBHAs of the transformants PC1 and PC2 reached the crest values at 96 h during the induction and fermentation processes, 2.89 ± 0.32 U cm3 for the transformant PC1 harboring native cbh2 gene and 5.84 ± 0.42 U cm3 for PC2 harboring synthetic cbh2 gene whose codons were optimized according to preferential codon usage of the host P. pastoris for the improved heterologous expression and production of CBH II, and thereafter declined probably due to the degradation of CBH II caused by the proteases released by the aged P. pastoris [24]. The CBHA of the enzyme expressed by the synthetic cbh2 gene was 2.02 times higher than that of the native cbh2 gene. As presented in Fig. 3B, the soluble protein excreted by the transformant PC2 was also significantly higher than that of PC1. PC2 had 0.63 ± 0.06 mg cm3 soluble protein at 96 h, 1.66 times higher than PC1 that is 0.38 ± 0.04 mg cm3. The results of CBHA and soluble protein comparisons demonstrate that codon optimization strategy used in this work was efficacious and capable of increasing the expression level of heterologous genes, especially for those host strains with large difference in codon usage patterns from the original strains with the native genes. However, the codon optimization had differentiated outcomes in the literature reports [11e13], ranging from 1.24 to more than 10-fold increase in enzyme production performance. It depends on how well the codon optimization strategy suits the host strain for exotic gene expression. The supernatants of fermentation broths from the P. pastoris transformants PC1 and PC2, as well as the original P. pastoris strain, were collected at 96 h and analyzed through SDS-PAGE to estimate the molecular weight of the product from recombinant P. pastoris. Fig. 4 presents the result that lane 1 and lane 2 are the CBH II bands with the identical molecular weight approximately 58 kDa produced by PC1 and PC2, respectively, and lane 0 for the original P. pastoris has no CBH II band, demonstrating that both the native cbh2 gene and the synthetic cbh2 gene were successfully expressed and secreted by recombinant P. pastoris without any change in
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Fig. 2 e Results from agarose gel electrophoresis. (A) the final expression vector pPIC9K-cbh2, (B) Colony PCR the Escherichia coli transformants containing the plasmid with native cbh2 gene (lane 1) and the plasmid with synthetic cbh2 gene (lane 2), respectively, (C) PCR results of Pichia pastoris transformants harboring the expression cassettes with native cbh2 gene and synthetic cbh2 gene, respectively. Lane 1: first generation of P. patoris transformant with native cbh2 gene, Lane 2: tenth generation of P. patoris transformant with native cbh2 gene, Lane 3: first generation of P. patoris transformant with synthetic cbh2 gene, Lane 4: tenth generation of P. patoris transformant with synthetic cbh2 gene, Lane 5e7: controls.
amino acid sequence of the CBH II protein structure. The actual molecular weights of recombinant CBH II proteins expressed by P. pastoris 58 kDa are higher than that of the recombinant CBH II protein expressed by E. coli 53 kDa (data not shown) which is close to its putative molecular weight calculated theoretically from the amino acids. The reason for this phenomenon is that N-glycosylation makes the recombinant enzyme ~10 kDa larger than the native nonglycosylated enzyme [11,12]. The approximately 5 kDa enlarged molecular weight of recombinant protein in this work indicates that the overglycosylation problem for foreign gene expression in P. pastoris was not very serious.
3.3. Enzymatic properties of CBH II expressed by P. pastoris The effects of pH and temperature on the activity of CBH II produced by recombinant P. pastoris toward microcrystalline cellulose PH101 (SigmaeAldrich, St. Louis, MO, USA) were
investigated to determine the optimal pH value and temperature. The results are shown in Fig. 5A and B, the former one indicating that the optimal pH value for CBH II was 5.0 and the latter one demonstrating that the optimal temperature for CBH II was 50 C. Because of the amino acids of the CBH II proteins encoded by the native cbh2 gene and the synthetic cbh2 gene whose codons were optimized are exactly the same, nothing is different between the two CBH II proteins and they are treated as the same CBH II. It was found in Fig. 5 that CBH II had lower stability in the interval of higher pH (5e8) and in the interval of higher temperature (50e70 C) than in the interval of lower pH (3e5) and in the interval of lower temperature (30e50 C), respectively. The reason for former one may be that CBH is more unstable under the low hydrogen ion concentration condition caused by high pH than under the high ion concentration condition caused by low pH. So perhaps hydrogen ion concentration is important for CBH II to maintain its active spatial conformation and enzymatic activity. The explanation for latter one is that high temperature not
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Fig. 4 e SDS-PAGE analysis of the culture supernatant of recombinant P. pastoris. Lane 1: PC1 containing native cbh2 gene, Lane 2: PC2 containing synthetic cbh2 gene, Lane 0: control. All the samples are collected at 24 h during fermentation.
Fig. 3 e Cellobiohydrolase activities (A) and soluble protein concentrations (B) in the fermentation broth obtained as a function of time from Pichia patoris transformant PC1 containing native cbh2 gene and PC2 containing synthetic cbh2 gene grown in the 100 cm3 BMMY mediums in 500 cm3 Erlenmeyer flasks incubated in incubatory shakers at 30 C and a shaking of 4.17 Hz. The initial pH and OD600 were 6.0 and 1.0, respectively. Methanol was added to a concentration of 10 kg m¡3 at every 24 h to keep the durative induction and expression of the cbh2 gene in P. pastoris transformants. Data shown are average values of triplicate samples and error bars are standard deviations.
only negatively influences the CBHA but also causes CBH II deactivation. Low temperature, nonetheless, has just negative effect on CBHA but no deactivation effect on CBH II. P. pastoris is an ideal host strain for the heterologous expression of cbh2 gene and an excellent producer of CBH II because it has a number of advantages over bacteria and fungi. For example, AOX1 promoter that is one of the strongest promoters can be employed to efficiently express recombinant genes and the simple composition of extracellular proteins facilitates downstream processing. And the codon optimization is necessary to overcome the codon usage bias between different species, which was also proven to be effective in improving heterologous protein production in this study. Fungi have many their own merits such as great ability of producing extracellular proteins but have obvious demerits, the difficulty of genetic engineering manipulation and the
Fig. 5 e Effects of pH (A) and temperature (B) on CBH II activity from recombinant P. pastoris. (A) the temperature was set at 50 C, (B) the pH was adjust to 5.0 with 50 mmol L¡1 citrate buffer solution. Fractional activity (%) was percentage of the residual activity of CBH II at nonoptimal condition to that at optimal condition. Data shown are average values of triplicate samples and error bars are standard deviations.
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complexity of extracellular protein making downstream processing not easy. But if enzyme separation and purification from enzyme mixture is not taken into consideration or not essential, fungi would be a perfect hosts given that many molecular biology techniques of fungi is maturing, especially for some model organisms such as T. reesei and Aspergillus niger [25e28]. Moreover, the genes of the components of cellulase had been successfully cloned and expressed in T. reesei to enhance both the cellulase production and its performance in enzymatic hydrolysis of lignocelluloses [4,8,29]. In addition, it was found that T. reesei cbh2 gene could be expressed in E. coli but its product was exclusively localized to the outer membrane of E. coli and unable to be excreted, although E. coli has several advantages over eukaryotic hosts including shorter time scales, a well-developed genetic toolbox, and less complex high-throughput screening through directed evolution [30]. P. pastoris, therefore, is an excellent alternative for producing CBH II or other enzymes with simple composition easy for separation and purification.
3.4.
Enzymatic hydrolysis of SHPCS and SHPRS
The recombinant CBH II was added to T. reesei cellulase to enhance the exo-exo-synergism for the enhancement of the enzymatic hydrolysis of lignocelluloses. Excessive amounts of A. niger cellobiase and T. reesei xylanase were supplemented in order to exclude the disturbing effects of cellobiase or xylanase deficiency on the result and enhance the enzymatic hydrolysis yields of SHPCS and SHPRS. As shown in Fig. 6, when the cellobiase and xylanase added one after one, the enzymatic hydrolysis yields of both the cellulolytic enzyme mixtures with and without recombinant CBH II were obviously improved. This observation indicates that T. reesei cellulase lacks of cellobiase and xylanase. It is well known that T. reesei is deficient in cellobiase, which can be solved by fermentation or genetic engineering techniques [5,6,8]. In all cases in Fig. 6, the cellulolytic enzyme mixture with recombinant CBH II performed better than that without recombinant CBH II, demonstrating that the deficiency in cellobiase and xylanase did not disturb the result and that the enhancement of enzymatic hydrolysis of SHPCS was caused by the exo-exo-synergism improved by adding recombinant CBH II. When 20 FPIU g1 dry substrate cellulase, 10 IU g1 dry substrate cellobiase, 300 IU g1 dry substrate xylanase and 20 U g1 dry substrate recombinant CBH II were used, a high enzymatic hydrolysis yield of SHPCS (94.7%) was obtained. This result not only shows the hydrolyzing efficiency of cellulolytic enzyme mixture but also demonstrates that the pretreatment method is suitable for corn stover. Same situation took place in the enzymatic hydrolysis of SHPRS, shown in Fig. 7. Exo-exo-synergism played the same positive role in all cases shown in Fig. 7AeC. This phenomenon proves that exo-exo-synergism functions authentically in the enzymatic hydrolysis regardless of what substrate being used. When 20 FPIU g1 dry substrate cellulase, 10 IU g1 dry substrate cellobiase, 300 IU g1 dry substrate xylanase and
given in Section 2.10. Data shown are average values of triplicate samples.
Fig. 6 e Time courses of enzymatic hydrolysis of SHPCS carried out in 250 cm3 Erlenmeyer flasks with a volume of 50 cm3. (A) The dosages of cellulase and recombinant CBH II were 20 FPIU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. (B) The dosages of cellulase, cellobiase and recombinant CBH II were 20 FPIU g¡1 dry substrate, 10 IU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. (C) The dosages of cellulase, cellobiase, xylanase and recombinant CBH II were 20 FPIU g¡1 dry substrate, 10 IU g¡1 dry substrate, 300 IU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. The temperature, agitation, and initial pH were 50 C, 1.67 Hz, and 4.8, respectively. The enzymatic hydrolysis yields were calculated in accordance with Eq. (1)
108
b i o m a s s a n d b i o e n e r g y 7 8 ( 2 0 1 5 ) 9 9 e1 0 9
20 U g1 dry substrate recombinant CBH II were used, the enzymatic hydrolysis yield of SHPRS was 83.3%, which is good although lower than that of SHPRS. This may be due to some reasons. For instance, the different properties of the substrates vary the results because different substrate has differences in chemical composition and structure. In general rice straw is not as good as corn stover when used as substrate for production of reducing sugars because of higher ash content. Another probable reason is that the pretreatment method best for corn stover did not suit rice straw because pretreatment methods and related conditions differ from one type of lignocellulosic material to another. The reason why the same pretreatment method was used although it was not most optimal for rice straw is that the different pretreatment method may be the interfering factor on the comparison. It is proven in this work that exo-exo-synergism played an important role in enzymatic hydrolysis of lignocelluloses, which always pales in comparison with the fame of the synergism between cellobiase and cellobiohydrolases/endoglucanases. No further enhancement of enzymatic hydrolysis could be observed when more than 20 U g1 substrate recombinant CBH II was added because at that point exo-exosynergism was no longer the limiting factor of the enzymatic hydrolysis [31]. It demonstrates that the strategies employed in this work to enhance heterologous expression of T. reesei cbh2 gene in P. pastoris and improve the exo-exosynergism in cellulase to increase the enzymatic hydrolysis yield by adding recombinant CBH II worked well and is a good paradigm for others. Corn stover and rice straw are main agricultural residues in the north and south of China, respectively. The abundance of these agricultural residues and improper disposal such as combustion cause serious environmental problems in today's China. Therefore, the utilization and conversion of the agricultural residues to valuable chemicals or products is drawing more and more attention in both science community and industry sectors. The good paradigm introduced in this work (94.7% conversion of corn stover and 83.3% conversion of rice straw to monomeric sugars) gives us a possible solution to produce fermentable sugars from lignocellulosic biomass inexpensively, renewably, and sustainably. Those fermentable sugars from lignocelluloses are ideal alternatives to starch sugar for production of biofuels, chemicals or others, which is profoundly meaningful to the countries including China who have increasingly enormous demand for food but limited food resources. Fig. 7 e Time courses of enzymatic hydrolysis of SHPRS carried out in 250 cm3 Erlenmeyer flasks with a volume of 50 cm3. (A) The dosages of cellulase and recombinant CBH II were 20 FPIU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. (B) The dosages of cellulase, cellobiase and recombinant CBH II were 20 FPIU g¡1 dry substrate, 10 IU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. (C) The dosages of cellulase, cellobiase, xylanase and recombinant CBH II were 20 FPIU g¡1 dry substrate, 10 IU g¡1 dry substrate, 300 IU g¡1 dry substrate and 20 U g¡1 dry substrate, respectively. The temperature, agitation, and initial pH were 50 C, 1.67 Hz, and 4.8, respectively. The enzymatic
4.
Conclusions
The native cbh2 gene cloned from T. reesei ZU-02 and the synthetic cbh2 gene whose codons were optimized according to the codon usage frequencies of the host strain P. pastoris were introduced into P. pastoris GS115, respectively. Through the comparison in fermentation, it was found that codon
hydrolysis yields were calculated in accordance with Eq. (1) given in Section 2.10. Data shown are average values of triplicate samples.
b i o m a s s a n d b i o e n e r g y 7 8 ( 2 0 1 5 ) 9 9 e1 0 9
optimization strategy could significantly enhance the heterologous expression of T. reesei CBH II in P. pastoris. The product of the recombinant P. pastoris was analyzed and its enzymatic properties were investigated. High enzymatic hydrolysis yields of SHPCS and SHPRS were obtained by adding recombinant CBH II to enhance exo-exo-synergism in cellulase.
Acknowledgments This work was financially supported by the National High Technology Research and Development Program (2007AA05Z401) and by the Program for Zhejiang Leading Team of Science and Technology Innovation (2011R50002) of China.
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