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Molecular Mechanism of Cellulase Production Systems in Penicillium
C H A P T E R
8 Molecular Mechanism of Cellulase Production Systems in Penicillium Ali A. Rastegari Falavarjan Branch, Islamic Azad University, Isfahan, Iran
8.1 INTRODUCTION One of the key technologies in biofuel production is the degradation of complex lignocellulosic biomass to monosaccharides. Lignocellulolytic enzyme systems, mainly produced by filamentous fungi in industry, are widely used in this process (Liu et al., 2013a,b; Merino and Cherry, 2007). Plant biomass-based fuels and chemicals offer an appealing and long-term solution as a replacement to fossil fuels. Enzymatic hydrolysis of biomass to fermentable sugar is a key step in biofuel refineries. However, high-cost cellulases are the major bottlenecks to economically competitive cellulose-to-biofuel conversion. Trichoderma reesei has always been the workhorse in cellulase cocktails, and multiple strategies have been applied to improve its enzyme yields to lower production costs. Fungi from the Penicillium genus have recently attracted a great deal of research attention, and are considered as potential alternatives to T. reesei for second-generation biofuel production (Yao et al., 2015). Degradation of lignocellulosic biomass is carried out primarily by microbial intervention i.e., utilize it as carbon and nutrient/ energy source for their growth. They include species of bacteria (Clostridium, Cellulomonas, Bacillus, Pseudomonas, Fibribacter, Ruminococcus, Butyrivibrio, etc.), fungi (Aspergillus, Rhizopus, Trichoderma, Fusarium, Neurospora, Penicillium, etc.), and actinomycetes (Thermomonospora, Thermoactinomyces, etc.) (Sajith et al., 2016). The aerobic fungal cellulases are usually preferred by the industry, because they are extracellular, adaptive in nature, and usually secreted in large quantities during growth. This is in sharp contrast to many bacterial as well as anaerobic fungal cellulases that exist as tight multienzyme complexes, often membrane bound as cellosomes, from which it is difficult to recover individual active enzyme species and hence makes them less economical. Production of cellulolytic enzyme from aerobic fungi is widespread; among them, species of Aspergillus, Trichoderma, Penicillium, and Sclerotium are found as highly cellulolytic, and are mainly considered for commercial exploitation (Sajith et al., 2016). Different species belonging to the genus Penicillium have shown the ability to produce a complete cellulase system and the Penicillium species generally produces an enzyme mixture with a better ratio between filter paper activity (FPA) and β-glucosidase (BGL) activity than Trichoderma (Syed et al., 2013). This focuses on cellulases from Penicillium species with emphasis on cellulose biodegradation, in the production of biofuels from lignocellulosic biomass. Some species are characterized by enhanced production of cellulases; among them are: P. brasilianum, P. brevicompactum, P. citrinum, P. chrysogenum, P. crustossum, P. decumbens, P. echinulatum, P. expansum, P. funiculosum, P. glabrum, P. griseoroseum, P. janthinellum, P. minioluteum, P. occitanis, P. persicinum, P. pinophilum, P. purpurogenum, and P. verruculosum. Data on the production of cellulases by various Penicillium species are summarized in Table 8.1 (Gusakov and Sinitsyn, 2012). Structurally, fungal cellulases are a simple and modular enzyme with functionally distinct modules or domains. Some of them possess two domains, catalytic domains and carbohydrate-binding domains connected by a serine and threonine-rich polylinker with varying chain length and structure (Fig. 8.1). The carbohydratebinding modules vary in size ranging from 4 to 20 kDa, and are rich in aromatic and often polar amino acid
New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: http://dx.doi.org/10.1016/B978-0-444-63501-3.00008-9
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TABLE 8.1 Cellulase Production By Penicillium Species Organism
Substrate
Fermentation time (h)
Penicillium minioluteum IBT 21486
Solka-Floc cellulose (2%)
230
Protein (g/L)
FPA (FPU/mL)
CMCase (U/mL)
BGL (U/mL) Ref.
0.29
9
1.70
Penicillium pinophilum IBT 10872
0.32
6
2.45
Penicillium verruculosum IBT 18366
0.37
12
0.97
Penicillium brasilianum IBT 20888
0.68
98
1.09
0.35
3.59
1.84
De Castro et al. (2010)
19
3.5
Jorgensen and Olsson (2006)
Penicillium funiculosum ATCC11797
CMC, Avicel, sugarcane bagasse derived materials (7.5 g/L)
81 276
Penicillium brasilianum IBT 20888
Sigmacell 20 cellulose (20 g/L), pretreated spruce (35.8 g/L)
165
0.5
0.59
Penicillium brasilianum IBT 20888
Solka-Floc 200 FCC, oat spelts
170
1.07
0.75
Penicillium pinophilum IBT 4186
xylan, birchwood xylan (40 g/L)
1.08
0.81
2.22
1.7
0.36
0.83
5.4
2.39
0.51
1.96
10.6
1.02
Penicillium persicinum IBT 13226 Penicillium decumbens 114-2
Wheat bran
Penicillium decumbens JU-A10
Krogh et al. (2004)
Jorgensen et al. (2005)
Sun et al. (2008)
Penicillium echinulatum 9A0251
Cellulose plus lactose (1% in total)
168
0.27
1.6
Penicillium citrinum MTCC 6489
Wheat bran
72 168
0.65
1.72
1.89
Penicillium echinumlatum SIM29
Microcrystalline cellulose (1%) plus soy bran (0.2%) plus glucose or sucrose (0.5% 1%)
144
2.0
17
1.5
Dillon et al. (2011)
Penicillium funiculosum
Cellulose (2%) plus cellobiose (0.5 mg/L)
240
2.5
7
8
Rao et al. (1988)
Penicillium echinulatum 9A02S1
Cellulose (1%) plus glucose
192
3.1
2.9
Camassola and Dillon (2007a)
Avicel, Solka-Floc SW44, cellulose or tissue paper (1%) plus wheat bran (2.5%)a
192 or 96
3.49
94.4
3.2
Singhvi et al. (2011)
67.8a
3558a
149a
Penicillium decumbens ML-017
Rice bran
72
5.76a
Penicillium echinulatum 9A02S1
Pretreated sugarcane bagasse, wheat bran
120
33a
282a
59a
Camassola and Dillon (2007b)
Penicillium pinophilum NTG III/6
Solka-Floc BW40, mille barley straw, wheat bran, Avicel PH101 (1% 6%)
72 240
14.5
9.8
175
38
Brown et al. (1987)
Penicillium occitanis Pol6
Avicel PH101 (8%)
187
11.4
12.6
12
17.5
Chaabouni et al. (1994)
Penicillium verruculosum (various strains)
Microcrystalline cellulose, fed-batch mode with glucose
144
47
496
61
Solov’eva et al. (2005)
0.5
Sehnem et al. (2006) Dutta et al. (2008)
(0% 1.5%) plus methylxanthines (1 5 μM) Penicillium janthinellum EU2D-21
a
Liu et al. (2011b)
Activities obtained in solid-state fermentation; expressed in U/g substrate. The highest activity values attained are listed. BGL, -glucosidase; CMC, Caroxymethylcellulose; FPA, Fiter paper activity; FPU, Fiter paper unit.
8.2 CELLULASE PRODUCTION BY PENICILLIUM SPECIES AND ENZYME KINETICS
155
FIGURE 8.1 General structure of cellulase consisting of catalytic domain (CD) and carbohydrate binding domain (CBD) joined via a linker peptide.
residues that immobilize the substrate during catalysis. The active site of the catalytic domain may be topologically tunnel, cleft, or pocket in shape allowing efficient hydrolysis of the substrate. One of the most extensively studied aerobic fungi is T. reesei, which is capable of hydrolyzing native cellulose. T. reesei possesses two genes encoding for exoglucanase, eight for endoglucanases, and seven for glucosidases. In the last few decades, thermophilic fungi have also been studied widely because of the fact that cellulose fibers bulge/swell up at higher temperatures and become easily accessible for hydrolytic enzymes. Talaromyces emersonii is a typical thermophilic fungus capable of producing cellulase, which is active even at 70 C and decomposes the intact cellulose. Two strains of Penicillium have been identified from subtropical soils with the potential for production of cellulase; Chaetomium thermophilum, Sporotrichum thermophile, T. emersonii, and Thermoascus aurantiacus grew well and decomposed cellulose very rapidly, producing thermostable cellulases (Sajith et al., 2016). The lignocellulolytic enzyme activity induced by agricultural waste is significantly higher than that induced by purified cellulose from Penicillium oxalicum GZ-2. However, this feature did not exist in strain T. reesei RUT, suggesting that P. oxalicum GZ-2 possibly has different regulation and induction mechanisms for producing lignocellulolytic enzymes. Understanding how the filamentous fungus P. oxalicum GZ-2 responds to plant biomass and induces an enzyme cocktail to degrade plant polymers may result in new strategies to improve the production of second-generation biofuels. The relationships and roles of each complex plant biomass component (such as cellulose) in inducing and regulating lignocellulose-degrading enzyme gene expression and protein profiles are still poorly understood (Liao et al., 2014). Similarly, a homolog of clr-2/clrB in P. oxalicum was found to be necessary for efficient cellulose production, and its overexpression resulted in a significant increase in cellulases at both transcriptional and protein levels. In addition to the transcriptional activation mechanism, carbon catabolite repression (CCR) triggered by glucose and other easily metabolized carbon sources exists widely in Saccharomyces cerevisiae and filamentous fungi. In cellulolytic fungi, the CCR mechanism is mediated mainly by the transcription factor CreA/Cre1, which suppresses the expression of a majority of cellulase and hemicellulase genes in A. niger, T. reesei, N. crassa, and P. oxalicum. The purpose of this chapter is to investigate how inducible hydrolytic enzymes respond to cellulose and their relationships and roles in protein expression along with the activity of lignocellulolytic enzymes. In recent years, studies based on omics and system biology have provided us with crucial information on the biology of lignocelluloses-degrading enzyme production in cellulolytic fungi. It is widely accepted that the expressions of almost all genes encoding lignocellulose degrading enzymes are triggered by inducer released from complex plant polysaccharides and regulated by transcription factors in a coordinated manner (Yao et al., 2015).
8.2 CELLULASE PRODUCTION BY PENICILLIUM SPECIES AND ENZYME KINETICS In all cases listed in Table 8.1, Penicillium species secreted high levels of BGL. As in the case of T. reesei, when P. pinophilum, P. occitanis, and P. verruculosum were cultured in laboratory fermenters (Table 8.1; last three rows), the cellulase and BGL activities were much higher than those produced by Penicillium species in shake flasks. Up to 14.5 g/L of protein and 9.8 FPU/mL and 38 U/mL of BGL were achieved in submerged culture of P. pinophilum NTG III/6 in a 16 1 stirred-tank fermenter. The cellulase productivity of P. pinophilum NTG III/6 was comparable to that of T. reesei Rut C30 strain (Brown et al., 1987). Similar levels of protein production (11.4 g/L) and FPA (12.6 FPU/mL) were attained for P. occitanis Pol6 grown on 8% microcrystalline cellulose in a 2-l fermenter (Chaabouni et al., 1994). P. verruculosum secreted up to 47 g/L of protein in a 7-l fermenter operating in fed batch mode. The carboxymethyl cellulase (CMCase) and BGL activities attained by P. verruculosum were also
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dramatically higher than those reported for Penicillium species by other researchers (Table 8.1). The protein concentrations obtained in P. verruculosum fermentations are at a level with the highest standards for protein production (Solov’eva et al., 2005). The ratio of BGL: FPA activity was greater than one in many cases or, at least, the activity numbers were comparable in magnitude. A high level of BGL activity is necessary to provide fast and complete conversion of cellobiose to glucose. Cellobiose is the intermediate product of enzymatic cellulose hydrolysis; being inhibitory to exo-cellobiohydrolases (CBHs), this disaccharide reduces the overall conversion of cellulose to the final product. Compared to Penicillia, various strains of T. reesei are usually characterized by essentially lower secretion of BGL (the ratio of BGL: FPA is notably less than one), since most BGLs found in the genome of the latter fungus are intracellular. Thus the T. reesei cellulase preparations have to be supplemented with additional BGL (usually from Aspergillus sp.). However, BGL production by T. reesei has been improved recently due to heterologously expressed BGLs from other microorganisms (Gusakov and Sinitsyn, 2012). The results of another study were superior when than those reported by Castro et al. (2010), who produced cellulase from P. funiculosum using microcrystalline cellulose as substrate. After the optimization of nitrogen and mineral resources, the activities achieved by Castro et al. (2010) in shake flasks were as follows: 0.17 U/mL of FPase, 4.4 5.6 U/mL of CMCase, and 1.3 U/mL of β-glucosidase, and the activities obtained in bioreactor were similar to these values. Furthermore, the production time reported herein was shorter (60 72 hours) than that reported by Castro et al. (2010) (120 hours), resulting in a high productivity value of the cellulase production. Fig. 8.2 shows the enzyme activities for the incubation periods at 37 C and 50 C. The P. funiculosum enzymatic blend displayed good stability over 6 days of incubation considering the three evaluated activities. The high enzymatic preparation stability is of importance because its use in the solid-state fermentation (SSF) process requires good catalytic activity and stability at 50 C (prehydrolysis) and at 37 C (fermentation stage simultaneously to hydrolysis) (Maeda et al., 2013). The maximum velocity (Vmax) and Michaelis Menton constant (Km) are the two constants describing the kinetic characteristics of the enzyme action. The Vmax describes the specific point in an enzymatic reaction at which the rate of the reaction is catalyzed to the maximum, if other factors are optimum; the Vmax is attained only when the substrate concentration is sufficiently available to fill the enzyme’s active site. Km is the substrate concentration required to fill half of the enzyme’s active site. High Km means the enzyme has less affinity toward substrate, whereas low Km indicates high affinity toward substrate and thus higher activity. Liu et al. (2011a) demonstrated the Km and Vmax of two cellulases produced by Aspergillus fumigatus Z5 as 37.8 mg/mL and 437.3 μmol/minutes/mg; and 51.8 mg/mL and 652.7 μmol/minutes/mg, respectively. The Km and Vmax of cellulase produced by A. niger BCRC31494 were found to be 134 mg/mL and 4.6 U/minutes/mg (Li et al., 2012) while Km and Vmax of P. pinophilum were 4.8 mg/mL and 72.5 U/mg, respectively (Pol et al., 2012). Lee et al. (2011) confirmed the Km and Vmax of P. purpurogenum KJS506 as 1.15 mg/mL and 220 U/mg, respectively (Sajith et al., 2016). One of the factors negatively affecting the enzymatic conversion of cellulose to sugars is enzyme inhibition by the reaction products. The most pronounced is the inhibition of CBHs by cellobiose. The inhibition constants for different fungal glycoside hydrolase (GH7) family CBHs have typically been estimated using synthetic chromogenic derivatives of cellobiose or lactose as a substrate. The CBH I from P. occitanis was found to be less FPase/37ºC
CMCase/37ºC
b-glucosidase/37ºC
FPase/50ºC
CMCase/50ºC
b-glucosidase/50ºC
FIGURE 8.2 Stability of the enzyme blend LADEBIO/BR incubated at 37 C and 50 C. 240
160 700 140
400
80 60
300
40
200
20
100 0
0 0
48
96
144 Time (h)
192
240
CMCase (U/mL)
FPase (U/mL)
500
100
160 120 80 40 0
288
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beta-glucosidase (U/mL)
200
600 120
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sensitive to inhibition by cellobiose (Ki 5 2 mM) than most of other characterized enzymes of similar specificity. For a comparison, the reported value of the competitive inhibition constant for T. reesei CBH I is 0.02 mM, while for five CBHs from Phanerochaete chrysosporium, Myceliophthora thermophila (formerly classified as Chrysosporium lucknowense), T. emersonii, and Humicola insolens the Ki value varied in the range of 0.07 0.65 mM. CBHs with reduced inhibition sensitivity to cellobiose, similar to the P. occitanis CBH I, have also been described; they include CBH I, CBH II from Coniophora puteana and CBH IA from T. emersonii with Ki values of 1.2, 2.4, and 2.5 mM, respectively (Gusakov and Sinitsyn, 2012). Structural modeling showed that the reason for the relative insensitivity of P. occitanis CBH I to product inhibition was poor hydrogen bonding and a more open configuration of the active site (Bhiri et al., 2010).
8.3 CELLULOLYTIC SYSTEM INDUCTION AND REGULATION OF PENICILLIUM FUNGI The mechanisms of induction and regulation of cellulose biosynthesis have not been studied as thoroughly in Penicillia as in the Trichoderma and Aspergillus genera. Cellulases are induced in fungi only in the presence of cellulose or other inducers, such as different oligosaccharides (e.g., sophorose, cellobiose, and gentiobiose). Different kinds of pure cellulose or cellulose-containing residues were found to be good inducers and substrates for growth and cellulase production by Penicillium species (Table 8.1) (Rao et al., 1988; Krogh et al., 2004; Jorgensen et al., 2005; Jorgensen and Olsson, 2006). Significant differences were found in the enzyme induction and regulation for Trichoderma and Penicillium fungi. Details on Penicillium cellulases and BGLs, whose complete or partial amino acid sequences are known, are given in Table 8.2. The cellulose degrading multienzyme system of Penicillium species is rather typical for fungi. It consists of a few endoglucanases belonging to the GH families 5, 6, 7, 12, and 45, two or more GH6 and GH7 family cellobiohydrolases (CBHs, including CBH II and CBH I, respectively), and GH1 and GH3 BGLs (Table 8.2). The most abundant Penicillium enzymes in the carbohydrateactive enzyme (CAZy) database are those from the P. chrysogenum Wisconsin 54 1255 strain, for which the full fungal genome has recently been sequenced (Van den Berg et al., 2008). However, their properties have not been studied and the enzymes are not classified according to their substrate specificity (as EG, CBH, or BGL); their affiliation into a particular family is based on amino acid sequences translated from the respective genes. Although Houbraken et al. (2011) recently demonstrated that both Fleming’s penicillin-producing strain and Wisconsin 54 1255 strain are not P. chrysogenum but P. rubens, the name P. chrysogenum is used in this chapter, since it is still present in protein databases and the new P. rubens name is not yet widely accepted. Two families, GH5 represented by endoglucanases and GH7 represented by CBHs, and a few endoglucanases, contain most of Penicillium cellulases, for which the sequence information is available (Table 8.2). Twenty three out of 27 characterized or putative Penicillium BGL belong to the GH3 family. So far, only one endo-1,4-β-glucanase (EG) (from P. decumbens), with unusually high activity toward glucomannan, belongs to the GH45 family that contains exclusively endoglucanases. Four proteins of the GH61 family were found in the P. chrysogenum genome (Van den Berg et al., 2008). The GH61 proteins have formerly been classified as endoglucanases; however, very recent studies revealed that they are metal-dependent oxidative enzymes that cleave cellulose. These enzymes may act as enhancers of the activity of other cellulases. Most Penicillium CBHs and endoglucanases have a modular structure typical for fungal cellulases; that is, they consist of a catalytic module and a cellulose-binding module (CBM) connected with a flexible peptide linker. However, Penicillium cellulases without a CBM, consisting only of a catalytic module, have also been described; for example, the GH12 family EGa from P. brasilianum, the GH5 family EG IIa and EG IIb, as well as GH12 EG III from P. verruculosum. The single-module structure of these enzymes seems to be encoded on a gene level. However, the low-molecular-weight forms of catalytically active cellulases without a CBM may also be formed as a result of partial proteolysis of the intact enzymes possessing the CBM. Such a situation was observed for EG I as well as for CBH I and II of P. verruculosum; high- and low-molecular-weight forms of both CBHs are present in the fungal culture broth in comparable quantities. The overall content of CBHs in the crude P. verruculosum preparation was approximately 70% of total proteins, while the content of endoglucanases and BGL was approximately 15% and 4%, respectively (Gusakov and Sinitsyn, 2012). Based on the sequences of Penicillium GH7 CBHs and other characterized GH7 fungal enzymes of similar specificity retrieved from the CAZy database, a phylogenetic tree was constructed (Fig. 8.3) (Dereeper et al., 2010; Phylogeny.fr). The analyzed enzymes shared 44% of conserved amino acid positions. As can be seen in Fig. 8.1, all Penicillium CBHs fall into enzyme groups located at the bottom part of the figure. They display the highest similarity to CBHs
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TABLE 8.2 Cellulases and β-Glucosidases from Penicillium Species Belonging to Different Glycoside Hydrolase Families and Whose Complete or Partial Amino Acid Sequences are Known GH family
Enzymes
Organism
Accession numbera
1
Three enzymesb
Penicillium chrysogenum
B6GZU4, B6HAS4, B6H4K5
Bgl1
Penicillium funiculosum
ACO82080
Penicillium brasilianum
A5A4M8
Penicillium chrysogenum
See CAZy database for numbersd
Penicillium decumbens
D3JUX2, B3GK87
Penicillium occitanis
A7LKA2
Penicillium pinophilum
NA
Bgl3
Penicillium purpurogenum
C9E9M9
EG
Penicillium brasilianum
B8Q961
Penicillium canescens
NA
Penicillium chrysogenum
See CAZy database for numbersd
Penicillium decumbens
A9Z054, AFG25592
Egl1
Penicillium echinulatum
C5J4L7
Egl2
Penicillium janthinellum
Q12665
Bgl2
Penicillium multicolor
BSMEI8
Eng5
Penicillium pinophilum
C0L2S4
Penicillium purpurogenum
NA
Penicillium verruculosum
NA
One enzyme
Penicillium chrysogenum
B6H8F7
CBH II
Penicillium decumbens
ADX86895
Cellulase
Penicillium funiculosum
B5TMG4
Penicillium verruculosum
NA
Penicillium chrysogenum
Q5S1P9
Two enzymes
Penicillium chrysogenum
B6HE71, B6HC69
CBH I (two versions)
Penicillium decumbens
C9EI49, A3RG86
Egl1 (two versions)
Penicillium decumbens
B0FMT4, B6ZBT2
Xylanase/CBH I (two versions)
Penicillium funiculosum
Q8WZJ4, ADX60067
CBH
Penicillium glabrum
AEL78901
Cbh1
Penicillium janthinellum
Q06886
Cbh1
Penicillium occitanis
Q68HC2
Egl1
Penicillium oxalicum
C5MRS3
Cbh1 (four versions)
Penicillium oxalicum
See CAZy database for numbersd
EG
Penicillium purpurogenum
AEL78899
CBH
Penicillium purpurogenum
AEL78900
EG
Penicillium sp.
AEG74551
Penicillium verruculosum
NA
EGa
Penicillium brasilianum
NA
Egl3
Penicillium canescens
NA
3
Bgl1 17 enzymes
b
Bgl1 (two versions) c
Bgl1 Bgl
5
c
Egl2 13 enzymes
b
Egl2 (two versions) c
c
EG
c
EG IIa and EG IIb 6
b
c
CBH II 7
Cbh1 b
CBH I 12
c
(Continued)
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TABLE 8.2 GH family
(Continued) Organism
Accession numbera
Penicillium chrysogenum
B6H819, B6H7Y5, B6HDE0
Nonclassified
Penicillium decumbens
B5M080
EG V
Penicillium decumbens
B5AKD1
Penicillium chrysogenum
See CAZy database for numbersd
Enzymes Three enzymesb c
45 61
159
b
Four proteins
a
UniProtKB/TrEMBL or GenBank accession numbers are shown, if available. Nonclassified enzymes; data from geneme. Fragments of the amino acid sequence. d Carbohydrate-active enzymes database, www.cazy.org. Bgb, β-glucosidase; CAZy, Carbohydrate-active enzyme; CBH/Cbh, Cellobiohydrolase; EG/Egl/Eng, Endoglucanase; GH, Glycoside hydrolase; NA, Data not available. Data from Carbohydrate-active enzymes database, www.cazy.org. b c
100 99
Melanocarpus albomyces Q8J0K6 Humicola grisea O93780
Chaetomium thermophilium Q4JI68 Acremonium thermophilium A7WNU0 Trichoderma reesei P62694 Trichoderma harzianum Q9P8P3 Claviceps purpurea Q00082 Chaetomium thermophilium Q5G2D5 Humicola grisea P15828 Thie lavia australien is CAD79782 Myceliophthora thermophilia AAQ38146
89 100 74 91
17
90 90
96 22
Neuro spora crassa EAA33262 Acremo nium thermophilium A7WNT9
43 87
Humicola insolens P56680
82 Fusicoccum sp. A7LN91
94 46 90 82
46
86
89 97
67
Volvariella volvacea Q9Y895 Phane rocha ete chrysosporium Q01762 Phanerochaete chrysosporium P13860 Phanero chaete chrysosporium Q09431 Irpex lacteus Q9Y722 Irpex lacteus Q9y723 Cochliobolus carbonum Q00328
Aspergillus niger Q9UVS9 Penicillium chrysogenum B6HC69 Penicillium decumbens A3RG86 Talaromyces emersonii Q8TFL9 90 23 Thermoascus aurantiacus A7WNU2 Penicillium verrucuiosum 100 Penicillium purpurogenum AEL78900 Penicillium funiculosum Q8WZJ4 95 93 Penicillium occitanis Q68HC2 87 24 Penicillium glabrum AEL78901 Aspergillus nidulans EAA66593 88 Aspergillus aculeatus O59843 96 Aspergillus niger O9UVS8 Penicillium janthinellum Q06886 99 98 Penicillium DECUMBENS C9E149 Penicillium oxalicum B3GS64 Penicillium chrysogenum Q5S1P9 91
84
0.4
FIGURE 8.3 Phylogenetic tree for cellobiohydrolases from family 7 of glycoside hydrolases. Distances along the horizontal axis reflect the degree of relatedness of the sequences. Branch support values are shown in percentages. The phylogram was constructed using the Phylogeny.fr web service. IV. APPLICATIONS
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FIGURE 8.4 Progress kinetics of Avicel (5 mg/ mL) hydrolysis by purified cellobiohydrolases from Penicillium verruculosum and Trichoderma reesei. In the presence of purified Aspergillus japonicus β-glucosidase (0.5 U/mL) at 40 C, pH 5.0, protein loading of 0.1 mg/mL. hm, High-molecular-weight enzyme form; lm, Low-molecular-weight enzyme form; Pv, Penicillium verruculosum; Tr, Trichoderma reesei.
from Aspergilli and slightly lower similarity than those from T. emersonii and T. aurantiacus. CBHs from other fungi located overhead, including the CBH I of T. reesei, share lower similarity with Penicillium enzymes. The highest similarity of the Penicillium CBHs to the enzymes from Aspergillus, Talaromyces, and Thermoascus is not surprising, since all four genera listed belong to the same family of fungi (Trichocomaceae) (Gusakov and Sinitsyn, 2012). Extremely high hydrolytic performance has also been reported for the intact high-molecular-weight forms of CBH I and CBH II from P. verruculosum in hydrolysis of Avicel in the presence of purified Aspergillus japonicas BGL (Morozova et al., 2010). Both enzymes are notably more effective than the respective CBHs I and II of T. reesei (Fig. 8.4). It is noteworthy that the performance of the catalytic module of the P. verruculosum CBH II (its lowmolecular-weight form without a CBM) was only slightly worse than that of its intact form but better than the performance of the native T. reesei CBH II. The catalytic modules of cellulases usually display significantly lower activities against crystalline cellulose in comparison with full-size enzymes possessing a CBM (Gusakov and Sinitsyn, 2012; Morozova et al., 2010). The intact CBH I from P. verruculosum demonstrated even higher benefits over the T. reesei CBHs in hydrolysis of lignocellulosic feedstocks, such as pretreated corn residues and sugarcane bagasse. It is interesting to note that the enzymes from P. funiculosum and P. verruculosum, whose extremely high saccharification performance has been documented, share a high degree of identity (Figure 8.3; lines 10 and 12 from the bottom).
8.4 LIGNOCELLULOLYTIC ENZYME PRODUCTION IN PENICILLIUM Production of cellulase and hemicellulase by the wild type P. oxalicum strain is severely repressed in the presence of excessive glucose or glycerol. After multiple rounds of mutagenesis of isolate 114 using ultraviolet irradiation and nitrosoguanidine, a mutant JU1 with the ability to produce cellulose in glucose containing medium was obtained (Fig. 8.5) (Qu et al., 1984). In glucose-free medium (containing 2% holocellulose plus 0.5% wheat bran), JU1 also produced higher cellulose activity (3.1 units of filter paper activity [FPA]/mL) than 114 2 did (0.8 U/mL). Notably, JU1 showed quite different morphology from 114 2, including slower hyphal extension, thicker hyphae, loss of bluish-green conidial pigment, lower amounts of conidia production, and circumscribed pink colonies. JU1 was then adapted repeatedly on spent ammonium sulfite liquor (SASL; one kind of pulp mill effluent) gradient agar plates to improve its tolerance to this toxic liquor. The experimental evolution generated a SASL-tolerant mutant JU-A10. JU-A10 grew faster than JU1 in SASL, synthetic high sulfate medium, and even nontoxic glucose medium. The time of maximum cellulase production by JU-A10 was 20 hours earlier than that of JU1 in SASL-waste fiber medium. Through genome shuffling, three fusants with more than two-fold higher cellulase (FPA) productivities were obtained from JU-A10 (Cheng et al., 2009). In addition, several other cellulase
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FIGURE 8.5 Genealogy of Penicillium oxalicum strains. Strains shown in light gray have been lost over the years. SASL, Spent ammonium sulfite liquor.
high-producing mutants, such as JU-A10 1, JU-140 12, and JU-A10-T, were also derived from JU-A10 by further mutagenesis and screening (Fig. 8.5) (Liu et al., 2013b). Rational strain engineering of P. oxalicum was performed based on the clear genetic background provided by genome sequencing. The assembled genome size of wild type strain 114 2 was 30.19 Mb, and was predicted to encode 10,021 proteins. For mutant JU-A10-T, the genome size was 30.69 Mb, with 10,473 protein-coding genes predicted. To the best of our knowledge, these are the first genome sequences of industrial cellulose-producing Penicillium species. Homologs of most proteins known to affect cellulose production in fungi have been annotated in P. oxalicum, and some have been targeted for the purpose of strain improvement. The gene encoding the ortholog of Neurospora crassa NCU05137 (a secreted protein whose deletion led to increased cellulase expression) in P. oxalicum, PDE_01641, was deleted in both 114 2 and JU-A10-T. Deletion of the PDE_01641 gene increased cellulase (FPA) production and cell growth on cellulose in 114 2, and resulted in a 36% increase in cellulase production at 48 hours of fermentation in JU-A10-T. Deletion of gene bgl2 (encoding major intracellular β-glucosidase) improved the final cellulose activity (after 144 h of fermentation in the medium containing 1% cellulose plus 1% wheat bran) to 0.88 U/mL, which was 3.3-fold that of 114 2. Combined manipulations of some crucial (positive or negative) transcription regulators resulted in an up to 10-fold elevation of cellulase production in 114 2. Engineering strategies are being implemented in industrial hyper-producing mutants to further improve production levels. Strains with specific genotypes have also been developed to facilitate the engineering and functional genomics in P. oxalicum. Strain Dpku70, in which the nonhomologous endjoining pathway was disrupted, was constructed to improve gene-targeting efficiency (Li et al., 2010). The frequency of homologous recombination in Dpku70 reached 100%, much higher than that in the parent wild type 114 2 (20% 90%, depending on genes). A pyrG-auxotrophic mutant M12 was isolated through the spontaneous mutagenesis of 114 2 (Shen et al., 2008). The pyrG marker can be selected bidirectionally (either presence or absence), thus allowing marker recycling for multiple gene manipulations (Liu et al., 2013b; Steiger et al., 2011). Posttranslational modifications such as glycosylation are widely detected for secreted proteins from fungi. Some cellulases purified from strain JU1 had very similar amino acid compositions but different molecular weights, indicating that they might be encoded by the same gene but underwent diverse posttranslational modifications (Qu et al., 1988). On the 2D electrophoresis map of cultured supernatant, 106 secreted protein spots were assigned into a total of 38 protein models, which clearly confirmed the hypothesis of posttranslation modification. The map also showed that most isoforms of one protein had similar molecular weights but differed greatly in isoelectric points. The diversity of N-glycosylation on CBHI (Cel7A-2) from P. oxalicum was studied in detail (Liu et al., 2013b; Gao et al., 2012). Four glycoforms of CBHI with identical amino acid sequences but different N-glycan structures were purified from JU-A10. The four proteins had different specific activities, optimum temperatures, and optimum pHs. In particular, one of the glycoforms, CBHI-A (carrying [Man]3
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[GlcNAc]2 1 GlcNAc at Asn137), showed significant synergism on cellulose degradation with commercial enzyme systems. CBHI-A had no hydrolytic activity on cellulose and p-nitrophenyl-β-D-cellobioside (a commonly used substrate for CBH assays), but could decrease the hydrogen bond intensity and crystalline degree of cotton fibers. It is reasonable to suppose that the properties of other enzyme components are also affected by diverse glycosylations. Regulation of lignocellulolytic enzyme production is mainly accomplished by controlling the transcriptional levels of genes encoding these enzymes. As reported in other fungi, transcriptions of most cellulases and hemicellulases in P. oxalicum are simultaneously induced or repressed (i.e., co-regulated) by carbon sources (Liu et al., 2013b). Three cellulase samples, obtained by cultivation of P. brasilianum on different carbon sources and demonstrating two-fold higher glucose production in 69-hour hydrolysis of steam pretreated spruce than Celluclast 1.5 L, displayed 70% 80% adsorption on this lignin-rich substrate, while the T. reesei enzymes from Celluclast 1.5 L bound to the substrate almost completely (by 94% 98%). Since the degree of adsorption of P. brasilianum enzymes on pure Sigmacell cellulose was almost three-fold lower, the researchers concluded that the enzyme binding to lignin component of the substrate, due to hydrophobic interactions, takes place in the case of pretreated spruce (Gusakov and Sinitsyn, 2012). Orthologs of several transcription factors previously known to be involved in this regulation were annotated in P. oxalicum, some of which were functionally studied. As expected, the CCR factor CreA and deubiquitinating enzyme CreB that stabilizes CreA negatively regulates cellulase production in P. oxalicum under both repressing and inducing conditions. The regulatory roles of transcription factors ACEI (negative regulator of cellulase expression (Aro et al., 2003)), ClrB [activator of cellulase and xylanase expression (Coradetti et al., 2012)], and XlnR [activator of xylanase and cellulase expression (Van Peij et al., 1998)] were also confirmed in P. oxalicum. To get a systematic understanding of the transcription factors regulating cellulase expression, a single-gene deletion strain set covering more than 400 transcription factors was constructed in P. oxalicum. The above mentioned strain, Dpku70 (Fig. 8.5) (Li et al., 2010), was used as the parent strain to facilitate the efficiency of targeted gene deletion. Some transcription factors, whose functions were not reported previously, were shown to strongly affect cellulase production. It appears that we are still far away from a complete understanding of the mechanisms controlling lignocellulolytic enzyme expression in fungi. Genome-wide transcription analysis also provides some other clues about the hyper-producing phenotype in JU-A10-T (Fig. 8.6). First, more genes in JU-A10-T are expressed at low levels than those in 114 2. That is to say, the cell factory of JU-A10-T concentrates on fewer biological processes. Second, the expression levels of genes for the pentose phosphate pathway (providing nicotinamide adenine dinucleotide phosphate (NADPH) and precursors for amino acid biosynthesis), lysine and cysteine synthesis, ribosome, and protein folding are upregulated in JU-A10-T, all facilitating the high-level synthesis of lignocellulolytic enzymes. Third, amino acid degradation and most secondary metabolisms are reduced in JU-A10-T, which may increase the fluxes and energy used for enzyme synthesis. The results provide implications for future rational design of strains for higher production of lignocellulolytic enzymes (Liu et al., 2013b). Another crucial factor affecting the performance of cellulases in lignocelluloses hydrolysis is cellular metabolism, and regulatory network of P. decumbens and P. chrysogenum was also supported by analyzing the top hits of a BLASTp search of P. decumbens proteins in the National Center for Biotechnology Information (NCBI) nonredundant protein database. P. decumbens and P. chrysogenum shared 7,035 orthologous proteins with an average amino acid sequence identity of 69.3%. The protein identity was similar to that among A. nidulans, A. fumigatus, and Aspergillus oryzae (66% 70%), and lower than that among T. reesei, Trichoderma atroviride and Trichoderma virens (70% 78%). Notably, the predicted proteome of P. decumbens was 21.7% smaller than that of P. chrysogenum. Homologous gene family analysis suggested that the difference was mainly due to the higher number of species-specific genes in P. chrysogenum, and to a less extent, due to the expansion (in P. chrysogenum) or contraction (in P. decumbens) of shared gene families between the two species. Comparison of protein gene ontology (GO) classifications shows that P. decumbens had clearly fewer proteins involved in cellular metabolism, biosynthesis, and transport than P. chrysogenum (Fig. 8.7). The result was also confirmed by comparison of the number of some functional proteins, such as short-chain dehydrogenases, cytochrome P450s, secondary metabolism key enzymes, and membrane transporters, between the two species. In addition, P. decumbens had 35.4% fewer protein kinases and 18.5% fewer transcription factors than P. chrysogenum, respectively. On the other hand, P. decumbens was rich with proteins containing carbohydrate-binding domains (Fig. 8.7) and involved in plant cell-wall degradation (Fig. 8.8) compared with P. chrysogenum. When compared with five other fungal species, the higher number of carbohydrate-binding proteins in P. decumbens was also noted (Fig. 8.7) (Liu et al., 2013c).
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FIGURE 8.6 Summary of proteins and cellular processes involved in cellulase production in penicillium oxalicum. Black arrows indicate mass flows and gray arrows indicate regulatory interactions. Genes, proteins or pathways of remarkable expression changes in JU-A10-T compared with those in 114 2 are marked (triangle for upregulation and inverted triangle for downregulation). BGL, β-glucosidase; CDT, Cellodextrin transporter; GT, Glucose transporter; PPP, Pentose phosphate pathway; PT, Pentose transporter.
FIGURE 8.7 Comparison of number of proteins in selected Gene Ontology terms (level 3) involved in carbohydrate utilization and cellular metabolism. The maximum number in each term was set to be 100%. Sc, Saccharomyces cerevisiae; Nc, Neurospora crassa; Tr, Trichoderma reesei; Pd, P. decumbens; Pc, P. chrysogenum; Ag, A. niger; An, A. nidulans.
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FIGURE 8.8 Comparison of numbers of plant cell wall-degrading enzymes among five fungal species. BGLs, β-glucosidases. CBM1 proteins, proteins containing fungal cellulose binding domains. Numbers of proteins (columns) and corresponding CAZyme families (diamonds, only those of cellulases, β-glucosidases, hemicellulases and pectinases) are shown. Tannases, cellobiose dehydrogenases and feruloyl esterases not assigned to CAZy families are not included.
Interestingly, the number of proteins involved in cellular metabolism and regulation in P. decumbens was similar to those in T. reesei. This may mean a relatively simple cellular metabolism network might be more suitable for high-level production of extracellular lignocellulolytic enzymes.
8.5 CONCLUSION Some Penicillium cellulases possess unique properties compared with other known microbial enzymes, such as a higher specific activity, lower sensitivity to the product inhibition, and weaker adsorption on the lignin component of the biomass (reduced inhibition by lignin). These enzymes may be potential candidates for heterologous expression in high productive hosts, together with other cellulases and hemicellulases hydrolyzing the biomass polysaccharides, or act as accessory enzymes to them. Alternatively, Penicillium strains with extraordinary cellulose saccharification ability, high levels of BGL, and overall protein production may become hosts for heterologous expression of useful foreign enzymes/proteins to further increase the hydrolytic potential of these fungi. Currently, relatively little information is known about the full pattern of cellulolytic and hemicellulolytic genes in Penicillia. Although the genome of P. chrysogenum Wisconsin 54 1255 strain has recently been sequenced, properties of the enzymes found in the genome remain unstudied; most of them are not even classified from the point of view of their substrate specificity. Sequencing genomes of other Penicillium species, their annotation, purification, and characterization of encoded enzymes will likely allow finding novel enzymes with interesting properties useful for biofuel production. The effects of different carbon sources on the production level of lignocellulolytic enzymes have been studied, and the related molecular mechanisms have been investigated. When compared with the widely used cellulase producer Trichoderma reesei, some unique features have been found in P. oxalicum, including higher β-glucosidase activity, higher numbers of lignocellulolytic enzyme gene, and different response of cellulase gene expression to some disaccharides. Based on the results from systems biology studies, the production level in P. oxalicum is expected to be further elevated. To achieve this goal, research on the related cellular processes (e.g., signal transduction, transcription regulation, and protein secretion) is being conducted and highly efficient genetic manipulation tools (e.g., multiple gene targeting) are being developed.
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Improving lignocellulolytic enzyme production with Penicillium: from strain screening to systems biology. Biofuels 4 (5), 523 534. Liu, G., Zhang, L., Wei, X., Zou, G., Qin, Y., Ma, L., et al., 2013c. Genomic and secretomic analyses reveal unique features of the lignocellulolytic enzyme system of Penicillium decumbens. PLoS One 8 (2), 1 12. Maeda, R.N., Barcelos, C.A., Santa Anna, L.M., Pereira Jr., N., 2013. Cellulase production by Penicillium funiculosum and its application in the hydrolysis of sugar cane bagasse for second generation ethanol production by fed batch operation. J. Biotechnol. 163, 38 44. Merino, S.T., Cherry, J., 2007. Progress and challenges in enzyme development for biomass utilization. Adv. Biochem. Eng. Biotechnol. 108, 95 120. Morozova, V.V., Gusakov, A.V., Andrianov, R.M., Pravilnikov, A.G., Osipov, D.O., Sinitsyn, A.P., 2010. Cellulases of Penicillium verruculosum. Biotechnol. J. 5 (8), 871 880. Phylogeny.fr. 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Sehnem, N.T., de Bittencourt, L.R., Camassola, M., Dillon, A.J.P., 2006. Cellulase production by Penicillium echinulatum on lactose. Appl. Microbiol. Biotechnol. 72 (1), 163 167. Shen, Y., Zhang, Y., Ma, T., et al., 2008. Simultaneous saccharification and fermentation of acid pretreated corncobs with a recombinant Saccharomyces cerevisiae expressing β-glucosidase. Bioresource Technol. 99 (11), 5099 5103. Singhvi, M.S., Adsul, M.G., Gokhale, D.V., 2011. Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose. Biores. Technol. 102 (11), 6569 6572. Solov’eva, I.V., Okunev, O.N., Vel’kov, V.V., Koshelev, A.V., Bubnova, T.V., Kondrat’eva, E.G., et al., 2005. The selection and properties of Penicillium verruculosum mutants with enhanced production of cellulases and xylanases. Microbiol. 74 (2), 141 146. Steiger, M.G., Vitikainen, M., Uskonen, P., Brunner, K., Adam, G., Pakula, T., et al., 2011. Transformation system for Hypocrea jecorina (Trichoderma reesei) that favors homologous integration and employs reusable bidirectionally selectable markers. Appl. Environ. Microbiol. 77 (1), 114 121. Sun, X., Liu, Z., Zheng, K., Song, X., Qu, Y., 2008. The composition of basal and induced cellulose systems in Penicillium decumbens under induction or repression conditions. Enzyme Microb. Technol. 42 (7), 560 567. Syed, S., Ul-Hassan, S.R., Johri, S., 2013. A novel cellulase from an endophyte, Penicillium sp. NFCCI 2862. American J. Microbiol. Res. 1 (4), 84 91. Van den Berg, M.A., Albang, R., Albermann, K., Badger, J.H., Daran, J.M., Driessen, A.J., et al., 2008. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat. Biotechnol. 26 (10), 1161 1168. Van Peij, N.N., Visser, J., De Graaff, L.H., 1998. Isolation and analysis of xlnR, encoding a transcriptional activator co-ordinating xylanolytic expression in Aspergillus niger. Mol. Microbiol. 27 (1), 131 142. Yao, G., Li, Z., Gao, L., Wu, R., Kan, Q., Liu, G., et al., 2015. Redesigning the regulatory pathway to enhance cellulase production in Penicillium oxalicum. Biotechnol. Biofuels 8, 1 16.
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8 Molecular Mechanism of Cellulase Production Systems in Penicillium Ali A. Rastegari Falavarjan Branch, Islamic Azad University, Isfahan, Iran
8.1 INTRODUCTION One of the key technologies in biofuel production is the degradation of complex lignocellulosic biomass to monosaccharides. Lignocellulolytic enzyme systems, mainly produced by filamentous fungi in industry, are widely used in this process (Liu et al., 2013a,b; Merino and Cherry, 2007). Plant biomass-based fuels and chemicals offer an appealing and long-term solution as a replacement to fossil fuels. Enzymatic hydrolysis of biomass to fermentable sugar is a key step in biofuel refineries. However, high-cost cellulases are the major bottlenecks to economically competitive cellulose-to-biofuel conversion. Trichoderma reesei has always been the workhorse in cellulase cocktails, and multiple strategies have been applied to improve its enzyme yields to lower production costs. Fungi from the Penicillium genus have recently attracted a great deal of research attention, and are considered as potential alternatives to T. reesei for second-generation biofuel production (Yao et al., 2015). Degradation of lignocellulosic biomass is carried out primarily by microbial intervention i.e., utilize it as carbon and nutrient/ energy source for their growth. They include species of bacteria (Clostridium, Cellulomonas, Bacillus, Pseudomonas, Fibribacter, Ruminococcus, Butyrivibrio, etc.), fungi (Aspergillus, Rhizopus, Trichoderma, Fusarium, Neurospora, Penicillium, etc.), and actinomycetes (Thermomonospora, Thermoactinomyces, etc.) (Sajith et al., 2016). The aerobic fungal cellulases are usually preferred by the industry, because they are extracellular, adaptive in nature, and usually secreted in large quantities during growth. This is in sharp contrast to many bacterial as well as anaerobic fungal cellulases that exist as tight multienzyme complexes, often membrane bound as cellosomes, from which it is difficult to recover individual active enzyme species and hence makes them less economical. Production of cellulolytic enzyme from aerobic fungi is widespread; among them, species of Aspergillus, Trichoderma, Penicillium, and Sclerotium are found as highly cellulolytic, and are mainly considered for commercial exploitation (Sajith et al., 2016). Different species belonging to the genus Penicillium have shown the ability to produce a complete cellulase system and the Penicillium species generally produces an enzyme mixture with a better ratio between filter paper activity (FPA) and β-glucosidase (BGL) activity than Trichoderma (Syed et al., 2013). This focuses on cellulases from Penicillium species with emphasis on cellulose biodegradation, in the production of biofuels from lignocellulosic biomass. Some species are characterized by enhanced production of cellulases; among them are: P. brasilianum, P. brevicompactum, P. citrinum, P. chrysogenum, P. crustossum, P. decumbens, P. echinulatum, P. expansum, P. funiculosum, P. glabrum, P. griseoroseum, P. janthinellum, P. minioluteum, P. occitanis, P. persicinum, P. pinophilum, P. purpurogenum, and P. verruculosum. Data on the production of cellulases by various Penicillium species are summarized in Table 8.1 (Gusakov and Sinitsyn, 2012). Structurally, fungal cellulases are a simple and modular enzyme with functionally distinct modules or domains. Some of them possess two domains, catalytic domains and carbohydrate-binding domains connected by a serine and threonine-rich polylinker with varying chain length and structure (Fig. 8.1). The carbohydratebinding modules vary in size ranging from 4 to 20 kDa, and are rich in aromatic and often polar amino acid
New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: http://dx.doi.org/10.1016/B978-0-444-63501-3.00008-9
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TABLE 8.1 Cellulase Production By Penicillium Species Organism
Substrate
Fermentation time (h)
Penicillium minioluteum IBT 21486
Solka-Floc cellulose (2%)
230
Protein (g/L)
FPA (FPU/mL)
CMCase (U/mL)
BGL (U/mL) Ref.
0.29
9
1.70
Penicillium pinophilum IBT 10872
0.32
6
2.45
Penicillium verruculosum IBT 18366
0.37
12
0.97
Penicillium brasilianum IBT 20888
0.68
98
1.09
0.35
3.59
1.84
De Castro et al. (2010)
19
3.5
Jorgensen and Olsson (2006)
Penicillium funiculosum ATCC11797
CMC, Avicel, sugarcane bagasse derived materials (7.5 g/L)
81 276
Penicillium brasilianum IBT 20888
Sigmacell 20 cellulose (20 g/L), pretreated spruce (35.8 g/L)
165
0.5
0.59
Penicillium brasilianum IBT 20888
Solka-Floc 200 FCC, oat spelts
170
1.07
0.75
Penicillium pinophilum IBT 4186
xylan, birchwood xylan (40 g/L)
1.08
0.81
2.22
1.7
0.36
0.83
5.4
2.39
0.51
1.96
10.6
1.02
Penicillium persicinum IBT 13226 Penicillium decumbens 114-2
Wheat bran
Penicillium decumbens JU-A10
Krogh et al. (2004)
Jorgensen et al. (2005)
Sun et al. (2008)
Penicillium echinulatum 9A0251
Cellulose plus lactose (1% in total)
168
0.27
1.6
Penicillium citrinum MTCC 6489
Wheat bran
72 168
0.65
1.72
1.89
Penicillium echinumlatum SIM29
Microcrystalline cellulose (1%) plus soy bran (0.2%) plus glucose or sucrose (0.5% 1%)
144
2.0
17
1.5
Dillon et al. (2011)
Penicillium funiculosum
Cellulose (2%) plus cellobiose (0.5 mg/L)
240
2.5
7
8
Rao et al. (1988)
Penicillium echinulatum 9A02S1
Cellulose (1%) plus glucose
192
3.1
2.9
Camassola and Dillon (2007a)
Avicel, Solka-Floc SW44, cellulose or tissue paper (1%) plus wheat bran (2.5%)a
192 or 96
3.49
94.4
3.2
Singhvi et al. (2011)
67.8a
3558a
149a
Penicillium decumbens ML-017
Rice bran
72
5.76a
Penicillium echinulatum 9A02S1
Pretreated sugarcane bagasse, wheat bran
120
33a
282a
59a
Camassola and Dillon (2007b)
Penicillium pinophilum NTG III/6
Solka-Floc BW40, mille barley straw, wheat bran, Avicel PH101 (1% 6%)
72 240
14.5
9.8
175
38
Brown et al. (1987)
Penicillium occitanis Pol6
Avicel PH101 (8%)
187
11.4
12.6
12
17.5
Chaabouni et al. (1994)
Penicillium verruculosum (various strains)
Microcrystalline cellulose, fed-batch mode with glucose
144
47
496
61
Solov’eva et al. (2005)
0.5
Sehnem et al. (2006) Dutta et al. (2008)
(0% 1.5%) plus methylxanthines (1 5 μM) Penicillium janthinellum EU2D-21
a
Liu et al. (2011b)
Activities obtained in solid-state fermentation; expressed in U/g substrate. The highest activity values attained are listed. BGL, -glucosidase; CMC, Caroxymethylcellulose; FPA, Fiter paper activity; FPU, Fiter paper unit.
8.2 CELLULASE PRODUCTION BY PENICILLIUM SPECIES AND ENZYME KINETICS
155
FIGURE 8.1 General structure of cellulase consisting of catalytic domain (CD) and carbohydrate binding domain (CBD) joined via a linker peptide.
residues that immobilize the substrate during catalysis. The active site of the catalytic domain may be topologically tunnel, cleft, or pocket in shape allowing efficient hydrolysis of the substrate. One of the most extensively studied aerobic fungi is T. reesei, which is capable of hydrolyzing native cellulose. T. reesei possesses two genes encoding for exoglucanase, eight for endoglucanases, and seven for glucosidases. In the last few decades, thermophilic fungi have also been studied widely because of the fact that cellulose fibers bulge/swell up at higher temperatures and become easily accessible for hydrolytic enzymes. Talaromyces emersonii is a typical thermophilic fungus capable of producing cellulase, which is active even at 70 C and decomposes the intact cellulose. Two strains of Penicillium have been identified from subtropical soils with the potential for production of cellulase; Chaetomium thermophilum, Sporotrichum thermophile, T. emersonii, and Thermoascus aurantiacus grew well and decomposed cellulose very rapidly, producing thermostable cellulases (Sajith et al., 2016). The lignocellulolytic enzyme activity induced by agricultural waste is significantly higher than that induced by purified cellulose from Penicillium oxalicum GZ-2. However, this feature did not exist in strain T. reesei RUT, suggesting that P. oxalicum GZ-2 possibly has different regulation and induction mechanisms for producing lignocellulolytic enzymes. Understanding how the filamentous fungus P. oxalicum GZ-2 responds to plant biomass and induces an enzyme cocktail to degrade plant polymers may result in new strategies to improve the production of second-generation biofuels. The relationships and roles of each complex plant biomass component (such as cellulose) in inducing and regulating lignocellulose-degrading enzyme gene expression and protein profiles are still poorly understood (Liao et al., 2014). Similarly, a homolog of clr-2/clrB in P. oxalicum was found to be necessary for efficient cellulose production, and its overexpression resulted in a significant increase in cellulases at both transcriptional and protein levels. In addition to the transcriptional activation mechanism, carbon catabolite repression (CCR) triggered by glucose and other easily metabolized carbon sources exists widely in Saccharomyces cerevisiae and filamentous fungi. In cellulolytic fungi, the CCR mechanism is mediated mainly by the transcription factor CreA/Cre1, which suppresses the expression of a majority of cellulase and hemicellulase genes in A. niger, T. reesei, N. crassa, and P. oxalicum. The purpose of this chapter is to investigate how inducible hydrolytic enzymes respond to cellulose and their relationships and roles in protein expression along with the activity of lignocellulolytic enzymes. In recent years, studies based on omics and system biology have provided us with crucial information on the biology of lignocelluloses-degrading enzyme production in cellulolytic fungi. It is widely accepted that the expressions of almost all genes encoding lignocellulose degrading enzymes are triggered by inducer released from complex plant polysaccharides and regulated by transcription factors in a coordinated manner (Yao et al., 2015).
8.2 CELLULASE PRODUCTION BY PENICILLIUM SPECIES AND ENZYME KINETICS In all cases listed in Table 8.1, Penicillium species secreted high levels of BGL. As in the case of T. reesei, when P. pinophilum, P. occitanis, and P. verruculosum were cultured in laboratory fermenters (Table 8.1; last three rows), the cellulase and BGL activities were much higher than those produced by Penicillium species in shake flasks. Up to 14.5 g/L of protein and 9.8 FPU/mL and 38 U/mL of BGL were achieved in submerged culture of P. pinophilum NTG III/6 in a 16 1 stirred-tank fermenter. The cellulase productivity of P. pinophilum NTG III/6 was comparable to that of T. reesei Rut C30 strain (Brown et al., 1987). Similar levels of protein production (11.4 g/L) and FPA (12.6 FPU/mL) were attained for P. occitanis Pol6 grown on 8% microcrystalline cellulose in a 2-l fermenter (Chaabouni et al., 1994). P. verruculosum secreted up to 47 g/L of protein in a 7-l fermenter operating in fed batch mode. The carboxymethyl cellulase (CMCase) and BGL activities attained by P. verruculosum were also
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dramatically higher than those reported for Penicillium species by other researchers (Table 8.1). The protein concentrations obtained in P. verruculosum fermentations are at a level with the highest standards for protein production (Solov’eva et al., 2005). The ratio of BGL: FPA activity was greater than one in many cases or, at least, the activity numbers were comparable in magnitude. A high level of BGL activity is necessary to provide fast and complete conversion of cellobiose to glucose. Cellobiose is the intermediate product of enzymatic cellulose hydrolysis; being inhibitory to exo-cellobiohydrolases (CBHs), this disaccharide reduces the overall conversion of cellulose to the final product. Compared to Penicillia, various strains of T. reesei are usually characterized by essentially lower secretion of BGL (the ratio of BGL: FPA is notably less than one), since most BGLs found in the genome of the latter fungus are intracellular. Thus the T. reesei cellulase preparations have to be supplemented with additional BGL (usually from Aspergillus sp.). However, BGL production by T. reesei has been improved recently due to heterologously expressed BGLs from other microorganisms (Gusakov and Sinitsyn, 2012). The results of another study were superior when than those reported by Castro et al. (2010), who produced cellulase from P. funiculosum using microcrystalline cellulose as substrate. After the optimization of nitrogen and mineral resources, the activities achieved by Castro et al. (2010) in shake flasks were as follows: 0.17 U/mL of FPase, 4.4 5.6 U/mL of CMCase, and 1.3 U/mL of β-glucosidase, and the activities obtained in bioreactor were similar to these values. Furthermore, the production time reported herein was shorter (60 72 hours) than that reported by Castro et al. (2010) (120 hours), resulting in a high productivity value of the cellulase production. Fig. 8.2 shows the enzyme activities for the incubation periods at 37 C and 50 C. The P. funiculosum enzymatic blend displayed good stability over 6 days of incubation considering the three evaluated activities. The high enzymatic preparation stability is of importance because its use in the solid-state fermentation (SSF) process requires good catalytic activity and stability at 50 C (prehydrolysis) and at 37 C (fermentation stage simultaneously to hydrolysis) (Maeda et al., 2013). The maximum velocity (Vmax) and Michaelis Menton constant (Km) are the two constants describing the kinetic characteristics of the enzyme action. The Vmax describes the specific point in an enzymatic reaction at which the rate of the reaction is catalyzed to the maximum, if other factors are optimum; the Vmax is attained only when the substrate concentration is sufficiently available to fill the enzyme’s active site. Km is the substrate concentration required to fill half of the enzyme’s active site. High Km means the enzyme has less affinity toward substrate, whereas low Km indicates high affinity toward substrate and thus higher activity. Liu et al. (2011a) demonstrated the Km and Vmax of two cellulases produced by Aspergillus fumigatus Z5 as 37.8 mg/mL and 437.3 μmol/minutes/mg; and 51.8 mg/mL and 652.7 μmol/minutes/mg, respectively. The Km and Vmax of cellulase produced by A. niger BCRC31494 were found to be 134 mg/mL and 4.6 U/minutes/mg (Li et al., 2012) while Km and Vmax of P. pinophilum were 4.8 mg/mL and 72.5 U/mg, respectively (Pol et al., 2012). Lee et al. (2011) confirmed the Km and Vmax of P. purpurogenum KJS506 as 1.15 mg/mL and 220 U/mg, respectively (Sajith et al., 2016). One of the factors negatively affecting the enzymatic conversion of cellulose to sugars is enzyme inhibition by the reaction products. The most pronounced is the inhibition of CBHs by cellobiose. The inhibition constants for different fungal glycoside hydrolase (GH7) family CBHs have typically been estimated using synthetic chromogenic derivatives of cellobiose or lactose as a substrate. The CBH I from P. occitanis was found to be less FPase/37ºC
CMCase/37ºC
b-glucosidase/37ºC
FPase/50ºC
CMCase/50ºC
b-glucosidase/50ºC
FIGURE 8.2 Stability of the enzyme blend LADEBIO/BR incubated at 37 C and 50 C. 240
160 700 140
400
80 60
300
40
200
20
100 0
0 0
48
96
144 Time (h)
192
240
CMCase (U/mL)
FPase (U/mL)
500
100
160 120 80 40 0
288
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beta-glucosidase (U/mL)
200
600 120
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sensitive to inhibition by cellobiose (Ki 5 2 mM) than most of other characterized enzymes of similar specificity. For a comparison, the reported value of the competitive inhibition constant for T. reesei CBH I is 0.02 mM, while for five CBHs from Phanerochaete chrysosporium, Myceliophthora thermophila (formerly classified as Chrysosporium lucknowense), T. emersonii, and Humicola insolens the Ki value varied in the range of 0.07 0.65 mM. CBHs with reduced inhibition sensitivity to cellobiose, similar to the P. occitanis CBH I, have also been described; they include CBH I, CBH II from Coniophora puteana and CBH IA from T. emersonii with Ki values of 1.2, 2.4, and 2.5 mM, respectively (Gusakov and Sinitsyn, 2012). Structural modeling showed that the reason for the relative insensitivity of P. occitanis CBH I to product inhibition was poor hydrogen bonding and a more open configuration of the active site (Bhiri et al., 2010).
8.3 CELLULOLYTIC SYSTEM INDUCTION AND REGULATION OF PENICILLIUM FUNGI The mechanisms of induction and regulation of cellulose biosynthesis have not been studied as thoroughly in Penicillia as in the Trichoderma and Aspergillus genera. Cellulases are induced in fungi only in the presence of cellulose or other inducers, such as different oligosaccharides (e.g., sophorose, cellobiose, and gentiobiose). Different kinds of pure cellulose or cellulose-containing residues were found to be good inducers and substrates for growth and cellulase production by Penicillium species (Table 8.1) (Rao et al., 1988; Krogh et al., 2004; Jorgensen et al., 2005; Jorgensen and Olsson, 2006). Significant differences were found in the enzyme induction and regulation for Trichoderma and Penicillium fungi. Details on Penicillium cellulases and BGLs, whose complete or partial amino acid sequences are known, are given in Table 8.2. The cellulose degrading multienzyme system of Penicillium species is rather typical for fungi. It consists of a few endoglucanases belonging to the GH families 5, 6, 7, 12, and 45, two or more GH6 and GH7 family cellobiohydrolases (CBHs, including CBH II and CBH I, respectively), and GH1 and GH3 BGLs (Table 8.2). The most abundant Penicillium enzymes in the carbohydrateactive enzyme (CAZy) database are those from the P. chrysogenum Wisconsin 54 1255 strain, for which the full fungal genome has recently been sequenced (Van den Berg et al., 2008). However, their properties have not been studied and the enzymes are not classified according to their substrate specificity (as EG, CBH, or BGL); their affiliation into a particular family is based on amino acid sequences translated from the respective genes. Although Houbraken et al. (2011) recently demonstrated that both Fleming’s penicillin-producing strain and Wisconsin 54 1255 strain are not P. chrysogenum but P. rubens, the name P. chrysogenum is used in this chapter, since it is still present in protein databases and the new P. rubens name is not yet widely accepted. Two families, GH5 represented by endoglucanases and GH7 represented by CBHs, and a few endoglucanases, contain most of Penicillium cellulases, for which the sequence information is available (Table 8.2). Twenty three out of 27 characterized or putative Penicillium BGL belong to the GH3 family. So far, only one endo-1,4-β-glucanase (EG) (from P. decumbens), with unusually high activity toward glucomannan, belongs to the GH45 family that contains exclusively endoglucanases. Four proteins of the GH61 family were found in the P. chrysogenum genome (Van den Berg et al., 2008). The GH61 proteins have formerly been classified as endoglucanases; however, very recent studies revealed that they are metal-dependent oxidative enzymes that cleave cellulose. These enzymes may act as enhancers of the activity of other cellulases. Most Penicillium CBHs and endoglucanases have a modular structure typical for fungal cellulases; that is, they consist of a catalytic module and a cellulose-binding module (CBM) connected with a flexible peptide linker. However, Penicillium cellulases without a CBM, consisting only of a catalytic module, have also been described; for example, the GH12 family EGa from P. brasilianum, the GH5 family EG IIa and EG IIb, as well as GH12 EG III from P. verruculosum. The single-module structure of these enzymes seems to be encoded on a gene level. However, the low-molecular-weight forms of catalytically active cellulases without a CBM may also be formed as a result of partial proteolysis of the intact enzymes possessing the CBM. Such a situation was observed for EG I as well as for CBH I and II of P. verruculosum; high- and low-molecular-weight forms of both CBHs are present in the fungal culture broth in comparable quantities. The overall content of CBHs in the crude P. verruculosum preparation was approximately 70% of total proteins, while the content of endoglucanases and BGL was approximately 15% and 4%, respectively (Gusakov and Sinitsyn, 2012). Based on the sequences of Penicillium GH7 CBHs and other characterized GH7 fungal enzymes of similar specificity retrieved from the CAZy database, a phylogenetic tree was constructed (Fig. 8.3) (Dereeper et al., 2010; Phylogeny.fr). The analyzed enzymes shared 44% of conserved amino acid positions. As can be seen in Fig. 8.1, all Penicillium CBHs fall into enzyme groups located at the bottom part of the figure. They display the highest similarity to CBHs
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TABLE 8.2 Cellulases and β-Glucosidases from Penicillium Species Belonging to Different Glycoside Hydrolase Families and Whose Complete or Partial Amino Acid Sequences are Known GH family
Enzymes
Organism
Accession numbera
1
Three enzymesb
Penicillium chrysogenum
B6GZU4, B6HAS4, B6H4K5
Bgl1
Penicillium funiculosum
ACO82080
Penicillium brasilianum
A5A4M8
Penicillium chrysogenum
See CAZy database for numbersd
Penicillium decumbens
D3JUX2, B3GK87
Penicillium occitanis
A7LKA2
Penicillium pinophilum
NA
Bgl3
Penicillium purpurogenum
C9E9M9
EG
Penicillium brasilianum
B8Q961
Penicillium canescens
NA
Penicillium chrysogenum
See CAZy database for numbersd
Penicillium decumbens
A9Z054, AFG25592
Egl1
Penicillium echinulatum
C5J4L7
Egl2
Penicillium janthinellum
Q12665
Bgl2
Penicillium multicolor
BSMEI8
Eng5
Penicillium pinophilum
C0L2S4
Penicillium purpurogenum
NA
Penicillium verruculosum
NA
One enzyme
Penicillium chrysogenum
B6H8F7
CBH II
Penicillium decumbens
ADX86895
Cellulase
Penicillium funiculosum
B5TMG4
Penicillium verruculosum
NA
Penicillium chrysogenum
Q5S1P9
Two enzymes
Penicillium chrysogenum
B6HE71, B6HC69
CBH I (two versions)
Penicillium decumbens
C9EI49, A3RG86
Egl1 (two versions)
Penicillium decumbens
B0FMT4, B6ZBT2
Xylanase/CBH I (two versions)
Penicillium funiculosum
Q8WZJ4, ADX60067
CBH
Penicillium glabrum
AEL78901
Cbh1
Penicillium janthinellum
Q06886
Cbh1
Penicillium occitanis
Q68HC2
Egl1
Penicillium oxalicum
C5MRS3
Cbh1 (four versions)
Penicillium oxalicum
See CAZy database for numbersd
EG
Penicillium purpurogenum
AEL78899
CBH
Penicillium purpurogenum
AEL78900
EG
Penicillium sp.
AEG74551
Penicillium verruculosum
NA
EGa
Penicillium brasilianum
NA
Egl3
Penicillium canescens
NA
3
Bgl1 17 enzymes
b
Bgl1 (two versions) c
Bgl1 Bgl
5
c
Egl2 13 enzymes
b
Egl2 (two versions) c
c
EG
c
EG IIa and EG IIb 6
b
c
CBH II 7
Cbh1 b
CBH I 12
c
(Continued)
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TABLE 8.2 GH family
(Continued) Organism
Accession numbera
Penicillium chrysogenum
B6H819, B6H7Y5, B6HDE0
Nonclassified
Penicillium decumbens
B5M080
EG V
Penicillium decumbens
B5AKD1
Penicillium chrysogenum
See CAZy database for numbersd
Enzymes Three enzymesb c
45 61
159
b
Four proteins
a
UniProtKB/TrEMBL or GenBank accession numbers are shown, if available. Nonclassified enzymes; data from geneme. Fragments of the amino acid sequence. d Carbohydrate-active enzymes database, www.cazy.org. Bgb, β-glucosidase; CAZy, Carbohydrate-active enzyme; CBH/Cbh, Cellobiohydrolase; EG/Egl/Eng, Endoglucanase; GH, Glycoside hydrolase; NA, Data not available. Data from Carbohydrate-active enzymes database, www.cazy.org. b c
100 99
Melanocarpus albomyces Q8J0K6 Humicola grisea O93780
Chaetomium thermophilium Q4JI68 Acremonium thermophilium A7WNU0 Trichoderma reesei P62694 Trichoderma harzianum Q9P8P3 Claviceps purpurea Q00082 Chaetomium thermophilium Q5G2D5 Humicola grisea P15828 Thie lavia australien is CAD79782 Myceliophthora thermophilia AAQ38146
89 100 74 91
17
90 90
96 22
Neuro spora crassa EAA33262 Acremo nium thermophilium A7WNT9
43 87
Humicola insolens P56680
82 Fusicoccum sp. A7LN91
94 46 90 82
46
86
89 97
67
Volvariella volvacea Q9Y895 Phane rocha ete chrysosporium Q01762 Phanerochaete chrysosporium P13860 Phanero chaete chrysosporium Q09431 Irpex lacteus Q9Y722 Irpex lacteus Q9y723 Cochliobolus carbonum Q00328
Aspergillus niger Q9UVS9 Penicillium chrysogenum B6HC69 Penicillium decumbens A3RG86 Talaromyces emersonii Q8TFL9 90 23 Thermoascus aurantiacus A7WNU2 Penicillium verrucuiosum 100 Penicillium purpurogenum AEL78900 Penicillium funiculosum Q8WZJ4 95 93 Penicillium occitanis Q68HC2 87 24 Penicillium glabrum AEL78901 Aspergillus nidulans EAA66593 88 Aspergillus aculeatus O59843 96 Aspergillus niger O9UVS8 Penicillium janthinellum Q06886 99 98 Penicillium DECUMBENS C9E149 Penicillium oxalicum B3GS64 Penicillium chrysogenum Q5S1P9 91
84
0.4
FIGURE 8.3 Phylogenetic tree for cellobiohydrolases from family 7 of glycoside hydrolases. Distances along the horizontal axis reflect the degree of relatedness of the sequences. Branch support values are shown in percentages. The phylogram was constructed using the Phylogeny.fr web service. IV. APPLICATIONS
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FIGURE 8.4 Progress kinetics of Avicel (5 mg/ mL) hydrolysis by purified cellobiohydrolases from Penicillium verruculosum and Trichoderma reesei. In the presence of purified Aspergillus japonicus β-glucosidase (0.5 U/mL) at 40 C, pH 5.0, protein loading of 0.1 mg/mL. hm, High-molecular-weight enzyme form; lm, Low-molecular-weight enzyme form; Pv, Penicillium verruculosum; Tr, Trichoderma reesei.
from Aspergilli and slightly lower similarity than those from T. emersonii and T. aurantiacus. CBHs from other fungi located overhead, including the CBH I of T. reesei, share lower similarity with Penicillium enzymes. The highest similarity of the Penicillium CBHs to the enzymes from Aspergillus, Talaromyces, and Thermoascus is not surprising, since all four genera listed belong to the same family of fungi (Trichocomaceae) (Gusakov and Sinitsyn, 2012). Extremely high hydrolytic performance has also been reported for the intact high-molecular-weight forms of CBH I and CBH II from P. verruculosum in hydrolysis of Avicel in the presence of purified Aspergillus japonicas BGL (Morozova et al., 2010). Both enzymes are notably more effective than the respective CBHs I and II of T. reesei (Fig. 8.4). It is noteworthy that the performance of the catalytic module of the P. verruculosum CBH II (its lowmolecular-weight form without a CBM) was only slightly worse than that of its intact form but better than the performance of the native T. reesei CBH II. The catalytic modules of cellulases usually display significantly lower activities against crystalline cellulose in comparison with full-size enzymes possessing a CBM (Gusakov and Sinitsyn, 2012; Morozova et al., 2010). The intact CBH I from P. verruculosum demonstrated even higher benefits over the T. reesei CBHs in hydrolysis of lignocellulosic feedstocks, such as pretreated corn residues and sugarcane bagasse. It is interesting to note that the enzymes from P. funiculosum and P. verruculosum, whose extremely high saccharification performance has been documented, share a high degree of identity (Figure 8.3; lines 10 and 12 from the bottom).
8.4 LIGNOCELLULOLYTIC ENZYME PRODUCTION IN PENICILLIUM Production of cellulase and hemicellulase by the wild type P. oxalicum strain is severely repressed in the presence of excessive glucose or glycerol. After multiple rounds of mutagenesis of isolate 114 using ultraviolet irradiation and nitrosoguanidine, a mutant JU1 with the ability to produce cellulose in glucose containing medium was obtained (Fig. 8.5) (Qu et al., 1984). In glucose-free medium (containing 2% holocellulose plus 0.5% wheat bran), JU1 also produced higher cellulose activity (3.1 units of filter paper activity [FPA]/mL) than 114 2 did (0.8 U/mL). Notably, JU1 showed quite different morphology from 114 2, including slower hyphal extension, thicker hyphae, loss of bluish-green conidial pigment, lower amounts of conidia production, and circumscribed pink colonies. JU1 was then adapted repeatedly on spent ammonium sulfite liquor (SASL; one kind of pulp mill effluent) gradient agar plates to improve its tolerance to this toxic liquor. The experimental evolution generated a SASL-tolerant mutant JU-A10. JU-A10 grew faster than JU1 in SASL, synthetic high sulfate medium, and even nontoxic glucose medium. The time of maximum cellulase production by JU-A10 was 20 hours earlier than that of JU1 in SASL-waste fiber medium. Through genome shuffling, three fusants with more than two-fold higher cellulase (FPA) productivities were obtained from JU-A10 (Cheng et al., 2009). In addition, several other cellulase
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FIGURE 8.5 Genealogy of Penicillium oxalicum strains. Strains shown in light gray have been lost over the years. SASL, Spent ammonium sulfite liquor.
high-producing mutants, such as JU-A10 1, JU-140 12, and JU-A10-T, were also derived from JU-A10 by further mutagenesis and screening (Fig. 8.5) (Liu et al., 2013b). Rational strain engineering of P. oxalicum was performed based on the clear genetic background provided by genome sequencing. The assembled genome size of wild type strain 114 2 was 30.19 Mb, and was predicted to encode 10,021 proteins. For mutant JU-A10-T, the genome size was 30.69 Mb, with 10,473 protein-coding genes predicted. To the best of our knowledge, these are the first genome sequences of industrial cellulose-producing Penicillium species. Homologs of most proteins known to affect cellulose production in fungi have been annotated in P. oxalicum, and some have been targeted for the purpose of strain improvement. The gene encoding the ortholog of Neurospora crassa NCU05137 (a secreted protein whose deletion led to increased cellulase expression) in P. oxalicum, PDE_01641, was deleted in both 114 2 and JU-A10-T. Deletion of the PDE_01641 gene increased cellulase (FPA) production and cell growth on cellulose in 114 2, and resulted in a 36% increase in cellulase production at 48 hours of fermentation in JU-A10-T. Deletion of gene bgl2 (encoding major intracellular β-glucosidase) improved the final cellulose activity (after 144 h of fermentation in the medium containing 1% cellulose plus 1% wheat bran) to 0.88 U/mL, which was 3.3-fold that of 114 2. Combined manipulations of some crucial (positive or negative) transcription regulators resulted in an up to 10-fold elevation of cellulase production in 114 2. Engineering strategies are being implemented in industrial hyper-producing mutants to further improve production levels. Strains with specific genotypes have also been developed to facilitate the engineering and functional genomics in P. oxalicum. Strain Dpku70, in which the nonhomologous endjoining pathway was disrupted, was constructed to improve gene-targeting efficiency (Li et al., 2010). The frequency of homologous recombination in Dpku70 reached 100%, much higher than that in the parent wild type 114 2 (20% 90%, depending on genes). A pyrG-auxotrophic mutant M12 was isolated through the spontaneous mutagenesis of 114 2 (Shen et al., 2008). The pyrG marker can be selected bidirectionally (either presence or absence), thus allowing marker recycling for multiple gene manipulations (Liu et al., 2013b; Steiger et al., 2011). Posttranslational modifications such as glycosylation are widely detected for secreted proteins from fungi. Some cellulases purified from strain JU1 had very similar amino acid compositions but different molecular weights, indicating that they might be encoded by the same gene but underwent diverse posttranslational modifications (Qu et al., 1988). On the 2D electrophoresis map of cultured supernatant, 106 secreted protein spots were assigned into a total of 38 protein models, which clearly confirmed the hypothesis of posttranslation modification. The map also showed that most isoforms of one protein had similar molecular weights but differed greatly in isoelectric points. The diversity of N-glycosylation on CBHI (Cel7A-2) from P. oxalicum was studied in detail (Liu et al., 2013b; Gao et al., 2012). Four glycoforms of CBHI with identical amino acid sequences but different N-glycan structures were purified from JU-A10. The four proteins had different specific activities, optimum temperatures, and optimum pHs. In particular, one of the glycoforms, CBHI-A (carrying [Man]3
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[GlcNAc]2 1 GlcNAc at Asn137), showed significant synergism on cellulose degradation with commercial enzyme systems. CBHI-A had no hydrolytic activity on cellulose and p-nitrophenyl-β-D-cellobioside (a commonly used substrate for CBH assays), but could decrease the hydrogen bond intensity and crystalline degree of cotton fibers. It is reasonable to suppose that the properties of other enzyme components are also affected by diverse glycosylations. Regulation of lignocellulolytic enzyme production is mainly accomplished by controlling the transcriptional levels of genes encoding these enzymes. As reported in other fungi, transcriptions of most cellulases and hemicellulases in P. oxalicum are simultaneously induced or repressed (i.e., co-regulated) by carbon sources (Liu et al., 2013b). Three cellulase samples, obtained by cultivation of P. brasilianum on different carbon sources and demonstrating two-fold higher glucose production in 69-hour hydrolysis of steam pretreated spruce than Celluclast 1.5 L, displayed 70% 80% adsorption on this lignin-rich substrate, while the T. reesei enzymes from Celluclast 1.5 L bound to the substrate almost completely (by 94% 98%). Since the degree of adsorption of P. brasilianum enzymes on pure Sigmacell cellulose was almost three-fold lower, the researchers concluded that the enzyme binding to lignin component of the substrate, due to hydrophobic interactions, takes place in the case of pretreated spruce (Gusakov and Sinitsyn, 2012). Orthologs of several transcription factors previously known to be involved in this regulation were annotated in P. oxalicum, some of which were functionally studied. As expected, the CCR factor CreA and deubiquitinating enzyme CreB that stabilizes CreA negatively regulates cellulase production in P. oxalicum under both repressing and inducing conditions. The regulatory roles of transcription factors ACEI (negative regulator of cellulase expression (Aro et al., 2003)), ClrB [activator of cellulase and xylanase expression (Coradetti et al., 2012)], and XlnR [activator of xylanase and cellulase expression (Van Peij et al., 1998)] were also confirmed in P. oxalicum. To get a systematic understanding of the transcription factors regulating cellulase expression, a single-gene deletion strain set covering more than 400 transcription factors was constructed in P. oxalicum. The above mentioned strain, Dpku70 (Fig. 8.5) (Li et al., 2010), was used as the parent strain to facilitate the efficiency of targeted gene deletion. Some transcription factors, whose functions were not reported previously, were shown to strongly affect cellulase production. It appears that we are still far away from a complete understanding of the mechanisms controlling lignocellulolytic enzyme expression in fungi. Genome-wide transcription analysis also provides some other clues about the hyper-producing phenotype in JU-A10-T (Fig. 8.6). First, more genes in JU-A10-T are expressed at low levels than those in 114 2. That is to say, the cell factory of JU-A10-T concentrates on fewer biological processes. Second, the expression levels of genes for the pentose phosphate pathway (providing nicotinamide adenine dinucleotide phosphate (NADPH) and precursors for amino acid biosynthesis), lysine and cysteine synthesis, ribosome, and protein folding are upregulated in JU-A10-T, all facilitating the high-level synthesis of lignocellulolytic enzymes. Third, amino acid degradation and most secondary metabolisms are reduced in JU-A10-T, which may increase the fluxes and energy used for enzyme synthesis. The results provide implications for future rational design of strains for higher production of lignocellulolytic enzymes (Liu et al., 2013b). Another crucial factor affecting the performance of cellulases in lignocelluloses hydrolysis is cellular metabolism, and regulatory network of P. decumbens and P. chrysogenum was also supported by analyzing the top hits of a BLASTp search of P. decumbens proteins in the National Center for Biotechnology Information (NCBI) nonredundant protein database. P. decumbens and P. chrysogenum shared 7,035 orthologous proteins with an average amino acid sequence identity of 69.3%. The protein identity was similar to that among A. nidulans, A. fumigatus, and Aspergillus oryzae (66% 70%), and lower than that among T. reesei, Trichoderma atroviride and Trichoderma virens (70% 78%). Notably, the predicted proteome of P. decumbens was 21.7% smaller than that of P. chrysogenum. Homologous gene family analysis suggested that the difference was mainly due to the higher number of species-specific genes in P. chrysogenum, and to a less extent, due to the expansion (in P. chrysogenum) or contraction (in P. decumbens) of shared gene families between the two species. Comparison of protein gene ontology (GO) classifications shows that P. decumbens had clearly fewer proteins involved in cellular metabolism, biosynthesis, and transport than P. chrysogenum (Fig. 8.7). The result was also confirmed by comparison of the number of some functional proteins, such as short-chain dehydrogenases, cytochrome P450s, secondary metabolism key enzymes, and membrane transporters, between the two species. In addition, P. decumbens had 35.4% fewer protein kinases and 18.5% fewer transcription factors than P. chrysogenum, respectively. On the other hand, P. decumbens was rich with proteins containing carbohydrate-binding domains (Fig. 8.7) and involved in plant cell-wall degradation (Fig. 8.8) compared with P. chrysogenum. When compared with five other fungal species, the higher number of carbohydrate-binding proteins in P. decumbens was also noted (Fig. 8.7) (Liu et al., 2013c).
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FIGURE 8.6 Summary of proteins and cellular processes involved in cellulase production in penicillium oxalicum. Black arrows indicate mass flows and gray arrows indicate regulatory interactions. Genes, proteins or pathways of remarkable expression changes in JU-A10-T compared with those in 114 2 are marked (triangle for upregulation and inverted triangle for downregulation). BGL, β-glucosidase; CDT, Cellodextrin transporter; GT, Glucose transporter; PPP, Pentose phosphate pathway; PT, Pentose transporter.
FIGURE 8.7 Comparison of number of proteins in selected Gene Ontology terms (level 3) involved in carbohydrate utilization and cellular metabolism. The maximum number in each term was set to be 100%. Sc, Saccharomyces cerevisiae; Nc, Neurospora crassa; Tr, Trichoderma reesei; Pd, P. decumbens; Pc, P. chrysogenum; Ag, A. niger; An, A. nidulans.
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FIGURE 8.8 Comparison of numbers of plant cell wall-degrading enzymes among five fungal species. BGLs, β-glucosidases. CBM1 proteins, proteins containing fungal cellulose binding domains. Numbers of proteins (columns) and corresponding CAZyme families (diamonds, only those of cellulases, β-glucosidases, hemicellulases and pectinases) are shown. Tannases, cellobiose dehydrogenases and feruloyl esterases not assigned to CAZy families are not included.
Interestingly, the number of proteins involved in cellular metabolism and regulation in P. decumbens was similar to those in T. reesei. This may mean a relatively simple cellular metabolism network might be more suitable for high-level production of extracellular lignocellulolytic enzymes.
8.5 CONCLUSION Some Penicillium cellulases possess unique properties compared with other known microbial enzymes, such as a higher specific activity, lower sensitivity to the product inhibition, and weaker adsorption on the lignin component of the biomass (reduced inhibition by lignin). These enzymes may be potential candidates for heterologous expression in high productive hosts, together with other cellulases and hemicellulases hydrolyzing the biomass polysaccharides, or act as accessory enzymes to them. Alternatively, Penicillium strains with extraordinary cellulose saccharification ability, high levels of BGL, and overall protein production may become hosts for heterologous expression of useful foreign enzymes/proteins to further increase the hydrolytic potential of these fungi. Currently, relatively little information is known about the full pattern of cellulolytic and hemicellulolytic genes in Penicillia. Although the genome of P. chrysogenum Wisconsin 54 1255 strain has recently been sequenced, properties of the enzymes found in the genome remain unstudied; most of them are not even classified from the point of view of their substrate specificity. Sequencing genomes of other Penicillium species, their annotation, purification, and characterization of encoded enzymes will likely allow finding novel enzymes with interesting properties useful for biofuel production. The effects of different carbon sources on the production level of lignocellulolytic enzymes have been studied, and the related molecular mechanisms have been investigated. When compared with the widely used cellulase producer Trichoderma reesei, some unique features have been found in P. oxalicum, including higher β-glucosidase activity, higher numbers of lignocellulolytic enzyme gene, and different response of cellulase gene expression to some disaccharides. Based on the results from systems biology studies, the production level in P. oxalicum is expected to be further elevated. To achieve this goal, research on the related cellular processes (e.g., signal transduction, transcription regulation, and protein secretion) is being conducted and highly efficient genetic manipulation tools (e.g., multiple gene targeting) are being developed.
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