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Biological process for coproduction of hydrogen and thermophilic enzymes during CO fermentation
Bioresource Technology 305 (2020) 123067
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Case Study
Biological process for coproduction of hydrogen and thermophilic enzymes during CO fermentation Seong Hyuk Leea,1, Sung-Mok Leea,1, Jung-Hyun Leea,b, Hyun Sook Leea,b, Sung Gyun Kanga,b, a b
T
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Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Busan 49111, Republic of Korea Department of Marine Biotechnology, University of Science and Technology, Daejeon 34113, Republic of Korea
ARTICLE INFO
ABSTRACT
Keywords: Cell factory Thermococcus onnurineus NA1 Coproduction Hydrogen Thermophilic enzyme
To develop a thermophilic cell factory system that uses CO gas, we attempted to engineer a hyperthermophilic carboxydotrophic hydrogenic archaeon Thermococcus onnurineus NA1 to be capable of producing thermophilic enzymes along with hydrogen (H2). The mutant strains 156T-AM and 156T-POL were constructed to have another copy of a gene encoding α-amylase or DNA polymerase, respectively, and exhibited growth rates and H2 production rates distinct from those of the parental strain, 156T, in gas fermentation using 100% CO or coalgasified syngas. Purified α-amylase displayed starch-hydrolyzing activity, and whole-cell extracts of 156T-AM showed saccharifying activity for potato peel waste. PCR amplification was used to demonstrate that purified DNA polymerase was free from bacterial DNA contamination, in contrast to commercial bacteria-made enzymes. This study demonstrated that this archaeal strain could coproduce enzymes and H2 using CO-containing gas, providing a basis for cell factories to upcycle industrial waste gas.
1. Introduction With limited fossil resources receiving increasing attention, microbial engineering efforts have been made to develop cell factories to produce various substances such as chemicals, proteins and biofuels using renewable resources (Gong et al., 2017; Gustavsson and Lee, 2016). Representatively, Clostridium species, Saccharomyces cerevisiae, Escherichia coli, Corynebacterium glutamicum and Bacillus species are widely being applied to produce useful biocommodities (Li et al., 2018; Liu et al., 2017; Nielsen, 2019; Pérez-García and Wendisch, 2018; Wang et al., 2017). Recently, (hyper)thermophiles have been studied as potentially valuable platforms for metabolic engineering because they can serve as a source of stable enzymes and unique biomaterials in harsh environments (Atalah et al., 2019; Atomi et al., 2011; Demirjian et al., 2001). In particular, (hyper)thermophilic bioprocesses are advantageous in reducing contamination, enhancing substrate solubility and increasing metabolic rates and protein expression compared to those of conventional mesophilic bioprocess (Quehenberger et al., 2017). Despite interest, a lack of genetic manipulation or high-cell-density culture techniques has been a major hurdle for potentiating the thermophilic bioprocess as a cell factory. Over the past several decades, numerous studies have been performed to produce commodity chemicals or fuels such as alcohols,
acids, short-chain fatty acids and hydrogen (H2) using carboxydotrophic microorganisms capable of oxidizing carbon monoxide (CO) (Henstra et al., 2007; Kim et al., 2013; Lee et al., 2008; Park et al., 2017; Wainaina et al., 2018; Younesi et al., 2005). As cost-effective alternative sources for CO, synthetic gas (syngas) or industrial waste gas consisting primarily of CO and H2 have been utilized to produce various microbial metabolites (Phillips et al., 2017). However, no research on the production of value-added proteins has been reported in the field of CO fermentation to date. Thermococcus onnurineus NA1 is a hyperthermophilic archaeon with carboxydotrophic metabolism and oxidizes CO through the water–gas shift reaction (CO + H2O → H2 + CO2, ΔG°′ = −20 kJ/mol) (Amend and Shock, 2001; Bae et al., 2006; Kim et al., 2013; Lee et al., 2008). In our previous report, T. onnurineus NA1 produced H2 using CO or formate in a growth-associated manner (Kim et al., 2013, 2010). Genetic engineering of the species significantly enhanced H2 production (Kim et al., 2015; Lee et al., 2015, 2014; Rittmann et al., 2015). In particular, adaptive engineering was quite effective in improving growth and H2 production (Jung et al., 2017; Lee et al., 2016). In this study, we attempted to demonstrate that the hyperthermophilic strain T. onnurineus NA1 156T could serve as a useful platform for CO fermentation to produce both thermophilic enzymes and H2 at the same time. To evaluate the potential for a cell factory, strain type,
Corresponding author at: Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Busan 49111, Republic of Korea. E-mail address: [email protected] (S.G. Kang). 1 These authors contributed equally to this study. ⁎
https://doi.org/10.1016/j.biortech.2020.123067 Received 11 December 2019; Received in revised form 17 February 2020; Accepted 18 February 2020 Available online 20 February 2020 0960-8524/ © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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target enzyme, and gas composition should be considered. First, the T. onnurineus NA1 156T was employed as a parental strain, which showed high CO conversion activity to produce H2 with an optical cell density of 5.5 in a pH-stat batch culture using 100% CO (Lee et al. 2016). Second, DNA polymerase and α-amylase were selected as target enzymes. Third, as T. onnurineus NA1 can utilize CO-containing gases with various compositions such as steel mill waste gas and coal-gasified syngas (Kim et al., 2013, 2016; Lee et al., 2016), 100% CO and coalgasified syngas were tested for gas fermentation. We also investigated the potential of whole-cell lysates and purified enzymes obtained from the constructed cell factory for biotechnological application.
process. The concentrations of enzymes were determined by the colorimetric assay of Bradford (Bradford, 1976), and sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis was conducted to determine the enzyme purity (Laemmli, 1970). The activity of α-amylase was measured by quantitative analysis of the amount of reducing sugar released during enzymatic hydrolysis from 1% soluble starch in 50 mM sodium acetate buffer (pH 6) at 80 °C for 15 min (Bernfeld, 1955; Lim et al., 2007). The specific activity of DNA polymerase was not determined and is referred to as previously reported (Kim et al., 2007). 2.4. Pretreatment of potato peel waste and measurement of hydrolysis activity of whole-cell extracts
2. Materials and methods
Potato was purchased from a local market, and potato peel was generated by cutting with a knife before cooking; the peel was finely ground after drying at 50 °C for 48 h in the laboratory. Ground peel was stored at −20 °C until use. Then, 5% (w/v) potato peel in 50 mM sodium acetate buffer (pH 6) was prepared, and the hydrolysis activity of cell extract (10 µg) derived from the cultivation in a serum bottle or bioreactor using 100% CO was measured. The mixture of potato peel and the enzyme was incubated at 80 °C, and supernatant (100 µL) after hydrolysis was prepared at the indicated time points (0.5, 1, 1.5, 2 and 3 h) by centrifugation at 13,000 × g for 5 min for the measurement of released reducing sugar, which was determined by a modified dinitrosalicylic acid method (Bernfeld, 1955). Additionally, 156T-AM cell extract was prepared at culture times of 4, 4.75, 5, 6 and 6.5 h in a bioreactor using syngas, and 40 µg of cell extract was used to determine hydrolysis activity using starch or potato peel waste.
2.1. Strain, media and culture conditions The 156T strain developed by adaptive engineering of T. onnurineus NA1 (Lee et al., 2016) was employed as the parental strain in this study. MM1 medium supplemented with CO was prepared and used for cultivation of 156T and mutant strains under anaerobic conditions at 80 °C as previously reported (Kim et al., 2013). Seed cultures were cultured in serum bottles sealed with sterilized bromobutyl rubber stoppers and aluminum caps, and the working volume was 50 mL. For bioreactor cultures, MM1 medium was prepared as described before (Kim et al., 2010) without autoclaving and purged with pure argon gas (99.999%) for 60 min to maintain anaerobic conditions at 80 °C. 100% CO or coalgasified syngas directly obtained from a coal gasification pilot-plant at the Institute for Advanced Engineering in the Republic of Korea was continuously supplied after inoculation of cells. The agitation speed was 900 rpm, and the working volume was 1 L. The pH was adjusted to 6.1 to 6.2 using 0.2 N NaOH in 3.5% NaCl. The gas flow rates of 100% CO or syngas were 50 mL/min to 200 mL/min or 360 mL/min, respectively.
2.5. DNA polymerases used in this study and PCR assays Bacterial genomic DNA was isolated from cell pellets of E. coli (DH5α) using a DNA extraction kit (G-spinTM, iNtRON Biotechnology Inc., Sungnam, Republic of Korea) following the manufacturing protocols. A PCR buffer was prepared to measure the activity of Ex Taq (TaKaRa, Korea Biomedical Inc., Seoul, Republic of Korea), nPfu-Forte (Enzynomics Co. Ltd., Daejeon, Republic of Korea) and the T. onnurineus NA1 DNA polymerase purified in this study. PCR mixtures (25 µL) were prepared as previously described (Kim et al., 2007), and PCR was performed with 1 µL of DNA template (50 ng) or without DNA template. We used 200 µL of RNase- and DNase-free microcentrifuge tubes with flat-top caps (Axygen Inc., Union city, USA). Oligonucleotide primers for bacterial 16S rRNA PCR were synthesized (Bionics Inc., Seoul, Republic of Korea). The primers listed in Table 1 were used as previously described (Niimi et al., 2011).
2.2. Construct for overexpression of a thermophilic enzyme in 156T strain To integrate the gene encoding the mature form of α-amylase (TON_1660) with a C-terminus 6X-histag and DNA polymerase (TON_0001) with a C-terminus 6X-histag between the TON_1126 and TON_1127 genes in the 156T genome, inverse PCR was performed using the vector pQRc, which includes a strong promoter (PTON_0157) with hmgpfu cassette plasmid DNA constructed as previously reported (Kim et al., 2015). The gene encoding the mature form of α-amylase was amplified from the plasmid pET-amylm, which was constructed as previously reported (Lim et al., 2007), using pUC_amylase_slic_F and pUC_amylase_slic_R primers, and the DNA polymerase-encoding gene was amplified from T. onnurineus NA1 genomic DNA using pUC_POL_slic_F and pUC_POL_Chis_slic_R primers listed in Table 1. The final vectors were constructed by a one-step sequence- and ligation-independent cloning (SLIC) method (Jeong et al., 2012), and mutants were generated via homologous recombination by modifying a gene insertion protocol as previously described (Kim et al., 2015; Matsumi et al., 2007).
2.6. Analytical methods Cell growth was determined using the optical density at 600 nm (OD600) with a BioPhotometer plus a UV–Visible spectrophotometer (Eppendorf, Hamburg, Germany). Briefly, the concentration of H2 in the headspace was measured by sampling the headspace gas (100 μL) using gas-tight syringes and a YL6100GC gas chromatograph (GC) (YL Instrument Co., Anyang, Republic of Korea) equipped with a Molsieve 5A column (Supelco, Bellefonte, USA), a Porapak N column (Supelco), a thermal conductivity detector and a flame ionization detector as described previously (Lee et al., 2019). Argon was used as a carrier gas at a flow rate of 30 mL/min. The total volume of outlet gas in a bioreactor was measured using a wet gas meter (Shinagawa, Tokyo, Japan).
2.3. Protein purification and measurement of enzyme activity Mutant strains were cultured in a 3 L bioreactor with a continuous supply of 100% CO at 200 mL/min until the cell densities (OD600) reached 1–1.5. For purification of the enzymes α-amylase and DNA polymerase, cells were harvested by centrifugation at 5000 × g for 30 min at 4 °C under aerobic conditions. Harvested cells were resuspended using buffer as described before (Kim et al., 2007; Lim et al., 2007), and cells were disrupted by sonication. The resulting supernatant was loaded into a column containing TALON metal affinity resin (BD Biosciences Clontech, Palo Alto, USA) to purify the enzyme via a one-step purification protocol without an additional purification
3. Results and discussion 3.1. Construction of mutants for coproduction of thermophilic enzymes and H2 The potential of T. onnurineus NA1 156T as a thermophilic cell 2
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Table 1 Primers used in this study. Primer
Oligonucleotide sequence
Construction of plasmid DNA Blunt_inverse_his_F Complementation_inverse_1016rbs_R pUC_amylase_slic_F pUC_amylase_Chis_slic_R pUC_1126-27-inv_Chis_F pUC_1126-27-inv_Chis_R pUC_HMG-M-inv-AvrII-F pUC_HMG-M-inv-AvrII-R pUC_POL_slic_F pUC_POL_Chis_slic_R Confirmation of gene insertion 1126_LA_in_AM_F1 1126_RA_in_AM_R1 1126_LA_in_POL_F2 1126_RA_in_POL_R2 HMG_in_AM_F1 HMG_in_AM_R1 HMG_in_POL_F2 HMG_in_POL_R2 Bacterial 16S rRNA gene PCR B16S rRNA_287_F B16S rRNA_287_R
5′-caccatcaccactgactgcaggcat-3′ 5′-ccccaacacctatgaaagag-3′ 5′-tcataggtgttggggatggcggaaacac-3′ 5′-tcagtggtgatggtgatgatggccaacaccacagt-3′ 5′-caccatcaccaccatcactga-3′ 5′-cctaggtcatctcccaagcat-3′ 5′-cctagggtggcgtcaacgat-3′ 5′-cctaggtcatctcccaagca-3′ 5′-gggagatgacctaggatgatcctcgacgtcgatta-3′ 5′-cgccaccctaggtcagtgatggtggtgatggtg-3′ 5′-cttctcttcttcctcgcaga-3′ 5′-attttacgaccgtaaaatag-3′ 5′-tcttttgcggacgtactccg-3′ 5′-gattagagttggtggcaatc-3′ 5′-attggccactactcaattga-3′ 5′-ttccgtaatttgagcctcatc-3′ 5′-tatgcaaacgtctaactggg-3′ 5′-ttccgtaatttgagcctcatc-3′ 5′-agcagccgcggtaata-3′ 5′-ggactaccagggtatctaatcct-3′
Fig. 1. Schematic representation of biological process to coproduce H2 and thermophilic enzymes in genetically engineered T. onnurineus NA1 156T by CO fermentation.
one-step purification, a hexa-histidine (6xHis) tag was attached to the C-terminus of the mature form of target proteins (Fig. 2a). The resulting mutants, 156T-AM and 156T-POL, were verified by PCR (Fig. 2b) using the primers listed in Table 1.
Table 2 FPKM values of TON_1126 and TON_1127 genes in wild-type and 156T strains. Locus tag
TON_1126 TON_1127 a
FPKMa Wild type
156T
0.00 3.04
0.00 6.16
3.2. Cell growth and H2 production of gene-overexpressing mutants For each gene-overexpressing mutant, we investigated physiological changes by measuring the cell density and H2 production rate in a bioreactor with a continuous supply of 100% CO. There were some differences in growth and H2 production between these two mutants and the parental strain, 156T (Fig. 3a and 3b). Although the 156T-AM mutant grew to an OD600 of 6.5, which is similar to that achieved by the 156T strain (OD600 = 6.6), 156T-AM exhibited a higher growth rate than the 156T strain (Table 2). This result shows that overexpression of the gene encoding α-amylase had a positive effect on the growth of cells; however, we do not know the mechanism for growth enhancement at present. On the other hand, 156T-POL grew only until the culture reached an OD600 of 1.54 (Fig. 3a), corresponding to only 23.7% of the OD600 reached by the 156T strain, and the 156T-POL maximum specific growth rate was 30% lower than that of the 156T strain (Table 3). This result implies that overexpression of the gene encoding DNA polymerase is likely to be harmful to cells. When we compared the
FPKM values were adapted from a previous study (Lee et al., 2016).
factory system that is capable of using CO can be proved by demonstrating that an enzyme can be stably produced by a genetically modified strain along with hydrogen production (Fig. 1). To construct mutants producing a high level of thermophilic enzyme, a copy of a gene encoding α-amylase (TON_1660) or DNA polymerase (TON_0001) was introduced with a strong constitutive promoter driving expression of the gene encoding T. onnurineus NA1 glutamate dehydrogenase (TON_0157) into an intergenic genomic region between a gene encoding hypothetical phosphonate metabolism protein PhnP (TON_1126) and a gene encoding hypothetical protein (TON_1127). The selected region was poorly transcribed under CO in the T. onnurineus NA1 156T strain and the wild-type strain (Table 2). To facilitate 3
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Fig. 2. Construction of mutants and confirmation of genotype by PCR. (a) The positions of primers used for verification of constructed mutants are marked by arrows. (b) PCR confirmation of the region between TON_1126 and TON_1127 genes (two upper panels) and HMG cassette gene (two lower panels) using the primer pairs listed in Table 1. Pgdh, promoter of the glutamate dehydrogenase gene; HMG: 3-hydroxy-3-methylglutaryl coenzyme A reductase gene of Pyrococcus furiosus; C6xHis, hexa-histidine tag; kb, kilobase; M, molecular marker; V, vector as a positive control.
Fig. 3. Changes in growth and H2 production rate in the mutant strains during CO-containing gas fermentation. Growth (a) and H2 production rate (b) of strains were monitored in a bioreactor with 100% CO supplied at a flow rate of 50 to 200 mL/min. Growth (c) and H2 production rate (d) of strains were monitored in a bioreactor with coal-gasified syngas supplied at a flow rate of 50 to 360 mL/min. The data of the 156T strain cultured using coal-gasified syngas were adapted from a previous work (Lee et al., 2016).
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obtained. The proteins, α-amylase and DNA polymerase, purified from native host strains appeared on SDS-PAGE with molecular sizes corresponding to the mature proteins, 50,531 and 90,000 Da, respectively, as reported for the proteins recombinantly produced and purified from E. coli (Kim et al., 2007; Lim et al., 2007) (Fig. 4). The purified α-amylase showed a specific activity of 3048 ± 85.5 U/mg toward soluble starch, approximately 50% lower than that of the recombinant one (Lim et al., 2007). It is possible that an unknown protein with a molecular size of approximately 25 kDa co-purified with the α-amylase, as shown in SDSPAGE (Fig. 4a), led to miscalculation of the protein content and a decrease in the apparent specific activity. Both values were found to be higher than that of any other α-amylase reported in enzyme database, Brenda (https://www.brenda-enzymes.org/).
Table 3 Comparison of kinetic parameters between the parental 156T strain and mutant strains. 156T-POL
156Ta
156T-AM
100% CO 0.7 0.8
0.5
Syngas 0.8
0.9
410.0 303.6
153.9 276.8
198.4 254.3
178.1 231.3
Kinetic parameter
156T
μmax (h−1) rmax (mmol/L/h) qmax (mmol/g/h)
156T-AM
381.4 272.4
μmax, maximum specific growth rate; rmax, maximum H2 production rate; qmax, maximum specific H2 production rate. a The value was adapted from a previous study (Lee et al., 2016).
H2 production rates of the two mutants and the 156T strain, the maximum H2 production rates of 156T-AM (381.4 mmol/L/h) and 156TPOL (153.9 mmol/L/h) were 93% and 38% of that of the 156T strain (410.0 mmol/L/h), respectively (Fig. 3b). Surprisingly, however, the maximum specific H2 production rates of the mutants and 156T were similar, within 10%, and furthermore, there was little difference between 156T-AM (272.4 mmol/g/h) and 156T-POL (276.8 mmol/g/h) mutants (Table 3). These data indicate that the capacity of each cell to produce H2 was not greatly affected by the overexpression of those genes. Under the same conditions but with a coal-gasified syngas (composition, 39.09% CO, 20.4% H2, 1.4% CH4, 6.95% CO2 and 32.12% N2) at a higher feeding rate than the previous experiment, the 156T-AM mutant was cultured, and growth and H2 production were monitored. The maximum value of the cell density and H2 production rate of the mutant were significantly decreased in comparison with those of the corresponding culture with 100% CO (Fig. 3c and d). Interestingly, the maximum specific H2 production rate was decreased to a similar rate (85%) as that observed for the 156T strain in a previous study (Table 3). Hence, the 156T-AM mutant seems to be appropriate for coproducing H2 and α-amylase using the coal-gasified syngas supplied in this experiment.
3.4. Saccharification using whole-cell extracts In addition to providing a purified enzyme, the α-amylase-overexpressing strain 156T-AM can be applicable as a whole-cell biocatalyst to saccharify starch. We used potato peel wastes as a source of starch because they contain a high amount of starch (33–52%) and nonstarch polysaccharides (Arapoglou et al., 2010; Camire et al., 1997; Khawla et al., 2014; Liang et al., 2014). The saccharifying activity of the whole-cell extract of 156T-AM toward potato peel was measured and compared with that of the parental strain. No activity was detected in the 156T strain. The whole-cell extract of 156T-AM prepared from a culture in a serum bottle with 100% CO generated reducing sugars from potato peel (Fig. 5a). To investigate the time course of enzyme activity during cultivation, wholecell extracts of 156T-AM were tested during gas fermentation using syngas. Starch was hydrolyzed in a growth-dependent manner (Fig. 5b), and the whole-cell extracts of 156T-AM harvested at 4.75 h of culture time, as shown in Fig. 3c, also showed hydrolysis activity toward potato peel waste (Fig. 5c). These results indicate that not only purified enzymes but also whole-cell extracts can be independently utilized as a biocatalyst whether they were prepared using 100% CO or syngas as a CO source.
3.3. Purification and enzymatic characterization of thermophilic enzymes
3.5. PCR system free from bacterial DNA contamination
To purify the enzymes from mutant cells grown by CO fermentation, cells were harvested when the OD600 reached 1–1.5. The use of metal affinity chromatography resulted in 0.8–1.0 mg/L protein being
Sometimes the use of DNA polymerases purified from bacterial hosts such as E. coli is problematic due to DNA contamination, causing falsepositive PCR results in detecting the presence of bacteria. DNA polymerase purified from the 156T-POL mutant was used in a PCR assay to amplify the bacterial 16S rRNA gene without the addition of template DNA. Dose-dependent amplification of the 16S rRNA gene with or without the addition of bacterial genomic DNA was tested using the primer pairs listed in Table 1. When purified DNA polymerase was used, the 16S rRNA gene was amplified in only a sample with externally added bacterial genomic DNA as a template and not in samples without template (Fig. 6a and b). In contrast, with commercially available Ex Taq (Fig. 6c) and nPfu-Forte (Fig. 6d) DNA polymerases, 16S rRNA gene amplification was observed in all the samples without the addition of a bacterial genomic DNA template. This result indicates that DNA polymerase purified in our system can be utilized to provide DNA polymerase activity without bacterial DNA contamination. 3.6. Implications of this study In this study, we demonstrated that T. onnurineus NA1 156T can produce thermophilic enzymes along with H2 during fermentation using 100% CO or coal-gasified syngas. Integration of the production of biofuels and bioproducts of value can be considered a cost-effective approach in the bioeconomy. Hyperthermophilic enzymes have been applied to a broad array of industrial processes (Atomi et al., 2011; Quehenberger et al., 2017). Many enzymes have been successfully
Fig. 4. SDS-PAGE of (a) α-amylase and (b) DNA polymerase purified from 156T-AM and 156T-POL, respectively, cultured by CO-containing gas fermentation. Three micrograms of each protein was loaded onto the SDS-PAGE. 5
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Fig. 5. Saccharification activity of whole-cell extracts of 156T-AM toward potato peel or starch. (a) Quantitative measurement of reducing sugars released from potato peel through incubation with whole-cell extract (10 µg) of 156T-AM cultured in a serum vial using 100% CO. (b) Quantitative measurement of reducing sugars released from 1% starch through incubation at 80 °C for 15 min with whole-cell extract (40 µg) of 156T-AM. The strain was cultured using syngas and harvested at 4, 5, 6, 6.5 h of culture time as shown in Fig. 3c. (c) Quantitative measurement of reducing sugars released from potato peel waste through incubation with 156T-AM whole-cell extract (40 µg) of 156T-AM cultured from syngas fermentation and harvested at 4.75 h of culture time as shown in Fig. 3c. Error bars indicate the standard deviation of three independent experiments.
expressed and purified from heterologous hosts such as E. coli. However, thermophilic hosts have been pursued to harness their advantage in producing thermophilic proteins composed of multiple subunits, involving maturation processes, or requiring cofactors (Atalah et al., 2019). Despite the necessity, low cell density and a lack of a genetic toolbox have been hurdles to developing thermophilic hosts. This study demonstrated that the T. onnurineus NA1 156T strain could be utilized as a good cell factory to produce thermophilic enzymes because of its high cell density and genetic manipulation system. To date, a few hyperthermophilic archaea, such as Sulfolobus spp., Pyrococcus furiosus and Thermococcus kodakarensis, have been extensively studied as thermophilic platforms to produce value-added products. Comparison of cell density and biomass productivity revealed that maximum cell density of 2.4 g/L observed in this study seems to be quite high among obligate anaerobic hyperthermophiles, which is similar to the biomass in the dialysis membrane reactor of P. furiosus (Krahe et al., 1996). Furthermore, biomass productivity of 0.3 g/L/h in this study is comparable to the data obtained by various cultivation studies of aerobic
Sulfolobus species such as dialysis reactor culture of S. shibatae B12 (Krahe et al., 1996), fed-batch culture of S. solfataricus P2 (Park and Lee, 1997), and microfiltration fed-batch culture of S. solfataricus Gθ (Schiraldi et al., 1999). To harness these merits and enhance the production yield of thermophilic enzymes, it would be necessary to further develop extensive genetic tools, such as promoters and induction systems. In this study, we designed the stable expression of thermophilic enzymes by inserting the gene of interest in a chromosome. The growth and H2 production of the α-amylase-overexpressing mutant were even better than those of the parental strain, indicating that production of the enzyme was not inhibitory to the strain. However, the other mutant, overexpressing the DNA polymerase gene, showed significant defects in growth and H2 production. Inhibitory effects of overexpressing DNA polymerase genes on growth have also been reported (Chan et al., 2007; Uchida et al., 2008). Therefore, the effect of gene overexpression on the cell factory should be considered when selecting an enzyme as a coproduction target. Fig. 6. Amplification of the 16S rRNA gene with or without a DNA template. PCR amplification of the 16S rRNA gene was performed using DNA polymerase purified from the 156T-POL mutant (a and b) or commercially available DNA polymerase (c and d) without DNA template (E. coli genomic DNA). Primer pairs for PCR are listed in Table 1.
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To avoid biased data for PCR amplification of bacteria-specific 16S rRNA genes by bacterial DNA contamination, removal of bacterial DNA contamination from commercial DNA polymerases has been studied in previous reports (Carroll et al., 1999; Eshleman and Smith, 2001). However, DNA polymerases purified from eukaryotic organisms, such as plants or yeast, were found to be safe from bacterial DNA contamination (Niimi et al., 2011). Regarding this issue, DNA polymerase obtained by employing archaeal strains is also beneficial.
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A novel CO-responsive transcriptional regulator and enhanced H2 production by an engineered Thermococcus onnurineus NA1 strain. Appl. Environ. Microbiol. 81, 1708–1714. Kim, M.S., Bae, S.S., Kim, Y.J., Kim, T.W., Lim, J.K., Lee, S.H., Choi, A.R., Jeon, J.H., Lee, J.H., Lee, H.S., Kang, S.G., 2013. CO-dependent H2 production by genetically engineered Thermococcus onnurineus NA1. Appl. Environ. Microbiol. 79, 2048–2053. Kim, T.W., Bae, S.S., Lee, J.W., Lee, S.M., Lee, J.H., Lee, H.S., Kang, S.G., 2016. A biological process effective for the conversion of CO-containing industrial waste gas to acetate. Bioresour. Technol. 211, 792–796. Kim, Y.J., Lee, H.S., Bae, S.S., Jeon, J.H., Lim, J.K., Cho, Y., Nam, K.H., Kang, S.G., Kim, S.J., Kwon, S.T., Lee, J.H., 2007. Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1. J. Microbiol. Biotechnol. 17, 1090–1097. 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Lee, J., Lee, J.W., Chae, C.G., Kwon, S.J., Kim, Y.J., Lee, J.H., Lee, H.S., 2019. Domestication of the novel alcohologenic acetogen Clostridium sp. AWRP: from isolation to characterization for syngas fermentation. Biotechnol. Biofuels. 12, 228. Lee, S.H., Kim, M.-S., Jung, H.C., Lee, J., Lee, J.-H., Lee, H.S., Kang, S.G., 2015. Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon. Appl. Microbiol. Biotechnol. 99, 4085–4092. Lee, S.H., Kim, M.S., Bae, S.S., Choi, A.R., Lee, J.W., Kim, T.W., Lee, J.H., Lee, H.S., Kang, S.G., 2014. Comparison of CO-dependent H2 production with strong promoters in Thermococcus onnurineus NA1. Appl. Microbiol. Biotechnol. 98, 979–986. Lee, S.H., Kim, M.S., Lee, Jae Hak, Kim, T.W., Bae, S.S., Lee, S.M., Jung, H.C., Yang, T.J., Choi, A.R., Cho, Y.J., Lee, Jung Hyun, Kwon, K.K., Lee, H.S., Kang, S.G., 2016. Adaptive engineering of a hyperthermophilic archaeon on CO and discovering the underlying mechanism by multi-omics analysis. Sci. Rep. 6, 22896. Li, J., Chi, X., Zhang, Y., Wang, X., 2018. Enhanced coproduction of hydrogen and butanol from rice straw by a novel two-stage fermentation process. Int. Biodeterior. Biodegrad. 127, 62–68. Liang, S., McDonald, A.G., Coats, E.R., 2014. Lactic acid production with undefined mixed culture fermentation of potato peel waste. Waste Manag. 45, 51–56. Lim, J.K., Lee, H.S., Kim, Y.J., Bae, S.S., Jeon, J.H., Kang, S.G., Lee, J.H., 2007. Critical factors to high thermostability of an α-amylase from hyperthermophilic archaeon Thermococcus onnurineus NA1. J. Microbiol. Biotechnol. 17, 1242–1248. Liu, Y., Li, J., Du, G., Chen, J., Liu, L., 2017. Metabolic engineering of Bacillus subtilis fueled by systems biology: recent advances and future directions. Biotechnol. Adv. 35, 20–30. Matsumi, R., Manabe, K., Fukui, T., Atomi, H., Imanaka, T., 2007. Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance. J. Bacteriol. 189, 2683–2691. Nielsen, J., 2019. Yeast systems biology: model organism and cell factory. Biotechnol. J. 14, e1800421. Niimi, H., Mori, M., Tabata, H., Minami, H., Ueno, T., Hayashi, S., Kitajima, I., 2011. A novel eukaryote-made thermostable DNA polymerase which is free from bacterial DNA contamination. J. Clin. Microbiol. 49, 3316–3320. Park, C.B., Lee, S.B., 1997. Constant-volume fed-batch operation for high density
4. Conclusion Here, we demonstrated that the production of thermophilic enzymes could be integrated with hydrogen production by a hyperthermophilic archaeon through gas fermentation. The thermophilic enzymes selected in this study showed considerable potential for application in saccharification or DNA amplification with their use in purified form or as a crude cell extract. This study also showed that syngas was useful as a cost-effective source of CO gas for the coproduction of value-added proteins and hydrogen. Further research is required for the production of exogenous thermophilic enzymes in the thermophilic cell factory developed in this study. CRediT authorship contribution statement Seong Hyuk Lee: Formal analysis, Investigation, Data curation, Writing - original draft. Sung-Mok Lee: Investigation, Formal analysis, Writing - original draft. Jung-Hyun Lee: Conceptualization. Hyun Sook Lee: Conceptualization, Data curation, Writing - review & editing. Sung Gyun Kang: Conceptualization, Data curation, Writing - review & editing, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work is supported by the Korea Institute of Ocean Science and Technology (KIOST) in-house program (PE99822) and the study on operational stability and risk assessment of CO-converting hyperthermophilic archaeon program of the Ministry of Oceans and Fisheries in the Republic of Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2020.123067. References Amend, J.P., Shock, E.L., 2001. Energetics of overall metabolic reactions of thermophilic and hyperthermophilic archaea and bacteria. FEMS Microbiol. Rev. 25, 175–243. Arapoglou, D., Varzakas, T., Vlyssides, A., Israilides, C., 2010. Ethanol production from potato peel waste (PPW). Waste Manag. 30, 1898–1902. Atalah, J., Cáceres-Moreno, P., Espina, G., Blamey, J.M., 2019. Thermophiles and the applications of their enzymes as new biocatalysts. Bioresour. Technol. 280, 478–488. Atomi, H., Sato, T., Kanai, T., 2011. Application of hyperthermophiles and their enzymes. Curr. Opin. Biotechnol. 22, 618–626. Bae, S.S., Kim, Y.J., Yang, S.H., Lim, J.K., Jeon, J.H., Lee, H.S., Kang, S.G., Kim, S.J., Lee, J.H., 2006. Thermococcus onnurineus sp. nov., a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent area at the PACMANUS field. J. Microbiol. Biotechnol. 16, 1826–1831. Bernfeld, P., 1955. Amylases, α and β. Methods Enzymol. 1, 149–158. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Camire, M.E., Violette, D., Dougherty, M.P., McLaughlin, M.A., 1997. Potato peel dietary fiber composition: effects of peeling and extrusion cooking processes. J. Agric. Food.
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S.H. Lee, et al. cultivation of hyperthermophilic aerobes. Biotechnol. Tech. 11, 277–281. Park, S., Yasin, M., Jeong, J., Cha, M., Kang, H., Jang, N., Choi, I.G., Chang, I.S., 2017. Acetate-assisted increase of butyrate production by Eubacterium limosum KIST612 during carbon monoxide fermentation. Bioresour. Technol. 245, 560–566. Pérez-García, F., Wendisch, V.F., 2018. Transport and metabolic engineering of the cell factory Corynebacterium glutamicum. FEMS Microbiol. Lett. 365, fny166. Phillips, J.R., Huhnke, R.L., Atiyeh, H.K., 2017. Syngas fermentation: a microbial conversion process of gaseous substrates to various products. Fermentation 3 (2), 28. Quehenberger, J., Shen, L., Albers, S.V., Siebers, B., Spadiut, O., 2017. Sulfolobus - a potential key organism in future biotechnology. Front. Microbiol. 12, 2474. Rittmann, S.K.M.R., Lee, H.S., Lim, J.K., Kim, T.W., Lee, J.H., Kang, S.G., 2015. Onecarbon substrate-based biohydrogen production: microbes, mechanism, and productivity. Biotechnol. Adv. 33, 165–177. Schiraldi, C., Marulli, F., Lernia, I. Di, Martino, A., De Rosa, M., 1999. A microfiltration
bioreactor to achieve high cell density in Sulfolobus solfataricus fermentation. Extremophiles 3, 199–204. Uchida, K., Furukohri, A., Shinozaki, Y., Mori, T., Ogawara, D., Kanaya, S., Nohmi, T., Maki, H., Akiyama, M., 2008. Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal. Mol. Microbiol. 70, 608–622. Wainaina, S., Horváth, I.S., Taherzadeh, M.J., 2018. Biochemicals from food waste and recalcitrant biomass via syngas fermentation: a review. Bioresour. Technol. 248, 113–121. Wang, C., Pfleger, B.F., Kim, S.W., 2017. Reassessing Escherichia coli as a cell factory for biofuel production. Curr. Opin. Biotechnol. 45, 92–103. Younesi, H., Najafpour, G., Mohamed, A.R., 2005. Ethanol and acetate production from synthesis gas via fermentation processes using anaerobic bacterium, Clostridium ljungdahlii. Biochem. Eng. J. 27, 110–119.
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Case Study
Biological process for coproduction of hydrogen and thermophilic enzymes during CO fermentation Seong Hyuk Leea,1, Sung-Mok Leea,1, Jung-Hyun Leea,b, Hyun Sook Leea,b, Sung Gyun Kanga,b, a b
T
⁎
Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Busan 49111, Republic of Korea Department of Marine Biotechnology, University of Science and Technology, Daejeon 34113, Republic of Korea
ARTICLE INFO
ABSTRACT
Keywords: Cell factory Thermococcus onnurineus NA1 Coproduction Hydrogen Thermophilic enzyme
To develop a thermophilic cell factory system that uses CO gas, we attempted to engineer a hyperthermophilic carboxydotrophic hydrogenic archaeon Thermococcus onnurineus NA1 to be capable of producing thermophilic enzymes along with hydrogen (H2). The mutant strains 156T-AM and 156T-POL were constructed to have another copy of a gene encoding α-amylase or DNA polymerase, respectively, and exhibited growth rates and H2 production rates distinct from those of the parental strain, 156T, in gas fermentation using 100% CO or coalgasified syngas. Purified α-amylase displayed starch-hydrolyzing activity, and whole-cell extracts of 156T-AM showed saccharifying activity for potato peel waste. PCR amplification was used to demonstrate that purified DNA polymerase was free from bacterial DNA contamination, in contrast to commercial bacteria-made enzymes. This study demonstrated that this archaeal strain could coproduce enzymes and H2 using CO-containing gas, providing a basis for cell factories to upcycle industrial waste gas.
1. Introduction With limited fossil resources receiving increasing attention, microbial engineering efforts have been made to develop cell factories to produce various substances such as chemicals, proteins and biofuels using renewable resources (Gong et al., 2017; Gustavsson and Lee, 2016). Representatively, Clostridium species, Saccharomyces cerevisiae, Escherichia coli, Corynebacterium glutamicum and Bacillus species are widely being applied to produce useful biocommodities (Li et al., 2018; Liu et al., 2017; Nielsen, 2019; Pérez-García and Wendisch, 2018; Wang et al., 2017). Recently, (hyper)thermophiles have been studied as potentially valuable platforms for metabolic engineering because they can serve as a source of stable enzymes and unique biomaterials in harsh environments (Atalah et al., 2019; Atomi et al., 2011; Demirjian et al., 2001). In particular, (hyper)thermophilic bioprocesses are advantageous in reducing contamination, enhancing substrate solubility and increasing metabolic rates and protein expression compared to those of conventional mesophilic bioprocess (Quehenberger et al., 2017). Despite interest, a lack of genetic manipulation or high-cell-density culture techniques has been a major hurdle for potentiating the thermophilic bioprocess as a cell factory. Over the past several decades, numerous studies have been performed to produce commodity chemicals or fuels such as alcohols,
acids, short-chain fatty acids and hydrogen (H2) using carboxydotrophic microorganisms capable of oxidizing carbon monoxide (CO) (Henstra et al., 2007; Kim et al., 2013; Lee et al., 2008; Park et al., 2017; Wainaina et al., 2018; Younesi et al., 2005). As cost-effective alternative sources for CO, synthetic gas (syngas) or industrial waste gas consisting primarily of CO and H2 have been utilized to produce various microbial metabolites (Phillips et al., 2017). However, no research on the production of value-added proteins has been reported in the field of CO fermentation to date. Thermococcus onnurineus NA1 is a hyperthermophilic archaeon with carboxydotrophic metabolism and oxidizes CO through the water–gas shift reaction (CO + H2O → H2 + CO2, ΔG°′ = −20 kJ/mol) (Amend and Shock, 2001; Bae et al., 2006; Kim et al., 2013; Lee et al., 2008). In our previous report, T. onnurineus NA1 produced H2 using CO or formate in a growth-associated manner (Kim et al., 2013, 2010). Genetic engineering of the species significantly enhanced H2 production (Kim et al., 2015; Lee et al., 2015, 2014; Rittmann et al., 2015). In particular, adaptive engineering was quite effective in improving growth and H2 production (Jung et al., 2017; Lee et al., 2016). In this study, we attempted to demonstrate that the hyperthermophilic strain T. onnurineus NA1 156T could serve as a useful platform for CO fermentation to produce both thermophilic enzymes and H2 at the same time. To evaluate the potential for a cell factory, strain type,
Corresponding author at: Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Busan 49111, Republic of Korea. E-mail address: [email protected] (S.G. Kang). 1 These authors contributed equally to this study. ⁎
https://doi.org/10.1016/j.biortech.2020.123067 Received 11 December 2019; Received in revised form 17 February 2020; Accepted 18 February 2020 Available online 20 February 2020 0960-8524/ © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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target enzyme, and gas composition should be considered. First, the T. onnurineus NA1 156T was employed as a parental strain, which showed high CO conversion activity to produce H2 with an optical cell density of 5.5 in a pH-stat batch culture using 100% CO (Lee et al. 2016). Second, DNA polymerase and α-amylase were selected as target enzymes. Third, as T. onnurineus NA1 can utilize CO-containing gases with various compositions such as steel mill waste gas and coal-gasified syngas (Kim et al., 2013, 2016; Lee et al., 2016), 100% CO and coalgasified syngas were tested for gas fermentation. We also investigated the potential of whole-cell lysates and purified enzymes obtained from the constructed cell factory for biotechnological application.
process. The concentrations of enzymes were determined by the colorimetric assay of Bradford (Bradford, 1976), and sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis was conducted to determine the enzyme purity (Laemmli, 1970). The activity of α-amylase was measured by quantitative analysis of the amount of reducing sugar released during enzymatic hydrolysis from 1% soluble starch in 50 mM sodium acetate buffer (pH 6) at 80 °C for 15 min (Bernfeld, 1955; Lim et al., 2007). The specific activity of DNA polymerase was not determined and is referred to as previously reported (Kim et al., 2007). 2.4. Pretreatment of potato peel waste and measurement of hydrolysis activity of whole-cell extracts
2. Materials and methods
Potato was purchased from a local market, and potato peel was generated by cutting with a knife before cooking; the peel was finely ground after drying at 50 °C for 48 h in the laboratory. Ground peel was stored at −20 °C until use. Then, 5% (w/v) potato peel in 50 mM sodium acetate buffer (pH 6) was prepared, and the hydrolysis activity of cell extract (10 µg) derived from the cultivation in a serum bottle or bioreactor using 100% CO was measured. The mixture of potato peel and the enzyme was incubated at 80 °C, and supernatant (100 µL) after hydrolysis was prepared at the indicated time points (0.5, 1, 1.5, 2 and 3 h) by centrifugation at 13,000 × g for 5 min for the measurement of released reducing sugar, which was determined by a modified dinitrosalicylic acid method (Bernfeld, 1955). Additionally, 156T-AM cell extract was prepared at culture times of 4, 4.75, 5, 6 and 6.5 h in a bioreactor using syngas, and 40 µg of cell extract was used to determine hydrolysis activity using starch or potato peel waste.
2.1. Strain, media and culture conditions The 156T strain developed by adaptive engineering of T. onnurineus NA1 (Lee et al., 2016) was employed as the parental strain in this study. MM1 medium supplemented with CO was prepared and used for cultivation of 156T and mutant strains under anaerobic conditions at 80 °C as previously reported (Kim et al., 2013). Seed cultures were cultured in serum bottles sealed with sterilized bromobutyl rubber stoppers and aluminum caps, and the working volume was 50 mL. For bioreactor cultures, MM1 medium was prepared as described before (Kim et al., 2010) without autoclaving and purged with pure argon gas (99.999%) for 60 min to maintain anaerobic conditions at 80 °C. 100% CO or coalgasified syngas directly obtained from a coal gasification pilot-plant at the Institute for Advanced Engineering in the Republic of Korea was continuously supplied after inoculation of cells. The agitation speed was 900 rpm, and the working volume was 1 L. The pH was adjusted to 6.1 to 6.2 using 0.2 N NaOH in 3.5% NaCl. The gas flow rates of 100% CO or syngas were 50 mL/min to 200 mL/min or 360 mL/min, respectively.
2.5. DNA polymerases used in this study and PCR assays Bacterial genomic DNA was isolated from cell pellets of E. coli (DH5α) using a DNA extraction kit (G-spinTM, iNtRON Biotechnology Inc., Sungnam, Republic of Korea) following the manufacturing protocols. A PCR buffer was prepared to measure the activity of Ex Taq (TaKaRa, Korea Biomedical Inc., Seoul, Republic of Korea), nPfu-Forte (Enzynomics Co. Ltd., Daejeon, Republic of Korea) and the T. onnurineus NA1 DNA polymerase purified in this study. PCR mixtures (25 µL) were prepared as previously described (Kim et al., 2007), and PCR was performed with 1 µL of DNA template (50 ng) or without DNA template. We used 200 µL of RNase- and DNase-free microcentrifuge tubes with flat-top caps (Axygen Inc., Union city, USA). Oligonucleotide primers for bacterial 16S rRNA PCR were synthesized (Bionics Inc., Seoul, Republic of Korea). The primers listed in Table 1 were used as previously described (Niimi et al., 2011).
2.2. Construct for overexpression of a thermophilic enzyme in 156T strain To integrate the gene encoding the mature form of α-amylase (TON_1660) with a C-terminus 6X-histag and DNA polymerase (TON_0001) with a C-terminus 6X-histag between the TON_1126 and TON_1127 genes in the 156T genome, inverse PCR was performed using the vector pQRc, which includes a strong promoter (PTON_0157) with hmgpfu cassette plasmid DNA constructed as previously reported (Kim et al., 2015). The gene encoding the mature form of α-amylase was amplified from the plasmid pET-amylm, which was constructed as previously reported (Lim et al., 2007), using pUC_amylase_slic_F and pUC_amylase_slic_R primers, and the DNA polymerase-encoding gene was amplified from T. onnurineus NA1 genomic DNA using pUC_POL_slic_F and pUC_POL_Chis_slic_R primers listed in Table 1. The final vectors were constructed by a one-step sequence- and ligation-independent cloning (SLIC) method (Jeong et al., 2012), and mutants were generated via homologous recombination by modifying a gene insertion protocol as previously described (Kim et al., 2015; Matsumi et al., 2007).
2.6. Analytical methods Cell growth was determined using the optical density at 600 nm (OD600) with a BioPhotometer plus a UV–Visible spectrophotometer (Eppendorf, Hamburg, Germany). Briefly, the concentration of H2 in the headspace was measured by sampling the headspace gas (100 μL) using gas-tight syringes and a YL6100GC gas chromatograph (GC) (YL Instrument Co., Anyang, Republic of Korea) equipped with a Molsieve 5A column (Supelco, Bellefonte, USA), a Porapak N column (Supelco), a thermal conductivity detector and a flame ionization detector as described previously (Lee et al., 2019). Argon was used as a carrier gas at a flow rate of 30 mL/min. The total volume of outlet gas in a bioreactor was measured using a wet gas meter (Shinagawa, Tokyo, Japan).
2.3. Protein purification and measurement of enzyme activity Mutant strains were cultured in a 3 L bioreactor with a continuous supply of 100% CO at 200 mL/min until the cell densities (OD600) reached 1–1.5. For purification of the enzymes α-amylase and DNA polymerase, cells were harvested by centrifugation at 5000 × g for 30 min at 4 °C under aerobic conditions. Harvested cells were resuspended using buffer as described before (Kim et al., 2007; Lim et al., 2007), and cells were disrupted by sonication. The resulting supernatant was loaded into a column containing TALON metal affinity resin (BD Biosciences Clontech, Palo Alto, USA) to purify the enzyme via a one-step purification protocol without an additional purification
3. Results and discussion 3.1. Construction of mutants for coproduction of thermophilic enzymes and H2 The potential of T. onnurineus NA1 156T as a thermophilic cell 2
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Table 1 Primers used in this study. Primer
Oligonucleotide sequence
Construction of plasmid DNA Blunt_inverse_his_F Complementation_inverse_1016rbs_R pUC_amylase_slic_F pUC_amylase_Chis_slic_R pUC_1126-27-inv_Chis_F pUC_1126-27-inv_Chis_R pUC_HMG-M-inv-AvrII-F pUC_HMG-M-inv-AvrII-R pUC_POL_slic_F pUC_POL_Chis_slic_R Confirmation of gene insertion 1126_LA_in_AM_F1 1126_RA_in_AM_R1 1126_LA_in_POL_F2 1126_RA_in_POL_R2 HMG_in_AM_F1 HMG_in_AM_R1 HMG_in_POL_F2 HMG_in_POL_R2 Bacterial 16S rRNA gene PCR B16S rRNA_287_F B16S rRNA_287_R
5′-caccatcaccactgactgcaggcat-3′ 5′-ccccaacacctatgaaagag-3′ 5′-tcataggtgttggggatggcggaaacac-3′ 5′-tcagtggtgatggtgatgatggccaacaccacagt-3′ 5′-caccatcaccaccatcactga-3′ 5′-cctaggtcatctcccaagcat-3′ 5′-cctagggtggcgtcaacgat-3′ 5′-cctaggtcatctcccaagca-3′ 5′-gggagatgacctaggatgatcctcgacgtcgatta-3′ 5′-cgccaccctaggtcagtgatggtggtgatggtg-3′ 5′-cttctcttcttcctcgcaga-3′ 5′-attttacgaccgtaaaatag-3′ 5′-tcttttgcggacgtactccg-3′ 5′-gattagagttggtggcaatc-3′ 5′-attggccactactcaattga-3′ 5′-ttccgtaatttgagcctcatc-3′ 5′-tatgcaaacgtctaactggg-3′ 5′-ttccgtaatttgagcctcatc-3′ 5′-agcagccgcggtaata-3′ 5′-ggactaccagggtatctaatcct-3′
Fig. 1. Schematic representation of biological process to coproduce H2 and thermophilic enzymes in genetically engineered T. onnurineus NA1 156T by CO fermentation.
one-step purification, a hexa-histidine (6xHis) tag was attached to the C-terminus of the mature form of target proteins (Fig. 2a). The resulting mutants, 156T-AM and 156T-POL, were verified by PCR (Fig. 2b) using the primers listed in Table 1.
Table 2 FPKM values of TON_1126 and TON_1127 genes in wild-type and 156T strains. Locus tag
TON_1126 TON_1127 a
FPKMa Wild type
156T
0.00 3.04
0.00 6.16
3.2. Cell growth and H2 production of gene-overexpressing mutants For each gene-overexpressing mutant, we investigated physiological changes by measuring the cell density and H2 production rate in a bioreactor with a continuous supply of 100% CO. There were some differences in growth and H2 production between these two mutants and the parental strain, 156T (Fig. 3a and 3b). Although the 156T-AM mutant grew to an OD600 of 6.5, which is similar to that achieved by the 156T strain (OD600 = 6.6), 156T-AM exhibited a higher growth rate than the 156T strain (Table 2). This result shows that overexpression of the gene encoding α-amylase had a positive effect on the growth of cells; however, we do not know the mechanism for growth enhancement at present. On the other hand, 156T-POL grew only until the culture reached an OD600 of 1.54 (Fig. 3a), corresponding to only 23.7% of the OD600 reached by the 156T strain, and the 156T-POL maximum specific growth rate was 30% lower than that of the 156T strain (Table 3). This result implies that overexpression of the gene encoding DNA polymerase is likely to be harmful to cells. When we compared the
FPKM values were adapted from a previous study (Lee et al., 2016).
factory system that is capable of using CO can be proved by demonstrating that an enzyme can be stably produced by a genetically modified strain along with hydrogen production (Fig. 1). To construct mutants producing a high level of thermophilic enzyme, a copy of a gene encoding α-amylase (TON_1660) or DNA polymerase (TON_0001) was introduced with a strong constitutive promoter driving expression of the gene encoding T. onnurineus NA1 glutamate dehydrogenase (TON_0157) into an intergenic genomic region between a gene encoding hypothetical phosphonate metabolism protein PhnP (TON_1126) and a gene encoding hypothetical protein (TON_1127). The selected region was poorly transcribed under CO in the T. onnurineus NA1 156T strain and the wild-type strain (Table 2). To facilitate 3
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Fig. 2. Construction of mutants and confirmation of genotype by PCR. (a) The positions of primers used for verification of constructed mutants are marked by arrows. (b) PCR confirmation of the region between TON_1126 and TON_1127 genes (two upper panels) and HMG cassette gene (two lower panels) using the primer pairs listed in Table 1. Pgdh, promoter of the glutamate dehydrogenase gene; HMG: 3-hydroxy-3-methylglutaryl coenzyme A reductase gene of Pyrococcus furiosus; C6xHis, hexa-histidine tag; kb, kilobase; M, molecular marker; V, vector as a positive control.
Fig. 3. Changes in growth and H2 production rate in the mutant strains during CO-containing gas fermentation. Growth (a) and H2 production rate (b) of strains were monitored in a bioreactor with 100% CO supplied at a flow rate of 50 to 200 mL/min. Growth (c) and H2 production rate (d) of strains were monitored in a bioreactor with coal-gasified syngas supplied at a flow rate of 50 to 360 mL/min. The data of the 156T strain cultured using coal-gasified syngas were adapted from a previous work (Lee et al., 2016).
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obtained. The proteins, α-amylase and DNA polymerase, purified from native host strains appeared on SDS-PAGE with molecular sizes corresponding to the mature proteins, 50,531 and 90,000 Da, respectively, as reported for the proteins recombinantly produced and purified from E. coli (Kim et al., 2007; Lim et al., 2007) (Fig. 4). The purified α-amylase showed a specific activity of 3048 ± 85.5 U/mg toward soluble starch, approximately 50% lower than that of the recombinant one (Lim et al., 2007). It is possible that an unknown protein with a molecular size of approximately 25 kDa co-purified with the α-amylase, as shown in SDSPAGE (Fig. 4a), led to miscalculation of the protein content and a decrease in the apparent specific activity. Both values were found to be higher than that of any other α-amylase reported in enzyme database, Brenda (https://www.brenda-enzymes.org/).
Table 3 Comparison of kinetic parameters between the parental 156T strain and mutant strains. 156T-POL
156Ta
156T-AM
100% CO 0.7 0.8
0.5
Syngas 0.8
0.9
410.0 303.6
153.9 276.8
198.4 254.3
178.1 231.3
Kinetic parameter
156T
μmax (h−1) rmax (mmol/L/h) qmax (mmol/g/h)
156T-AM
381.4 272.4
μmax, maximum specific growth rate; rmax, maximum H2 production rate; qmax, maximum specific H2 production rate. a The value was adapted from a previous study (Lee et al., 2016).
H2 production rates of the two mutants and the 156T strain, the maximum H2 production rates of 156T-AM (381.4 mmol/L/h) and 156TPOL (153.9 mmol/L/h) were 93% and 38% of that of the 156T strain (410.0 mmol/L/h), respectively (Fig. 3b). Surprisingly, however, the maximum specific H2 production rates of the mutants and 156T were similar, within 10%, and furthermore, there was little difference between 156T-AM (272.4 mmol/g/h) and 156T-POL (276.8 mmol/g/h) mutants (Table 3). These data indicate that the capacity of each cell to produce H2 was not greatly affected by the overexpression of those genes. Under the same conditions but with a coal-gasified syngas (composition, 39.09% CO, 20.4% H2, 1.4% CH4, 6.95% CO2 and 32.12% N2) at a higher feeding rate than the previous experiment, the 156T-AM mutant was cultured, and growth and H2 production were monitored. The maximum value of the cell density and H2 production rate of the mutant were significantly decreased in comparison with those of the corresponding culture with 100% CO (Fig. 3c and d). Interestingly, the maximum specific H2 production rate was decreased to a similar rate (85%) as that observed for the 156T strain in a previous study (Table 3). Hence, the 156T-AM mutant seems to be appropriate for coproducing H2 and α-amylase using the coal-gasified syngas supplied in this experiment.
3.4. Saccharification using whole-cell extracts In addition to providing a purified enzyme, the α-amylase-overexpressing strain 156T-AM can be applicable as a whole-cell biocatalyst to saccharify starch. We used potato peel wastes as a source of starch because they contain a high amount of starch (33–52%) and nonstarch polysaccharides (Arapoglou et al., 2010; Camire et al., 1997; Khawla et al., 2014; Liang et al., 2014). The saccharifying activity of the whole-cell extract of 156T-AM toward potato peel was measured and compared with that of the parental strain. No activity was detected in the 156T strain. The whole-cell extract of 156T-AM prepared from a culture in a serum bottle with 100% CO generated reducing sugars from potato peel (Fig. 5a). To investigate the time course of enzyme activity during cultivation, wholecell extracts of 156T-AM were tested during gas fermentation using syngas. Starch was hydrolyzed in a growth-dependent manner (Fig. 5b), and the whole-cell extracts of 156T-AM harvested at 4.75 h of culture time, as shown in Fig. 3c, also showed hydrolysis activity toward potato peel waste (Fig. 5c). These results indicate that not only purified enzymes but also whole-cell extracts can be independently utilized as a biocatalyst whether they were prepared using 100% CO or syngas as a CO source.
3.3. Purification and enzymatic characterization of thermophilic enzymes
3.5. PCR system free from bacterial DNA contamination
To purify the enzymes from mutant cells grown by CO fermentation, cells were harvested when the OD600 reached 1–1.5. The use of metal affinity chromatography resulted in 0.8–1.0 mg/L protein being
Sometimes the use of DNA polymerases purified from bacterial hosts such as E. coli is problematic due to DNA contamination, causing falsepositive PCR results in detecting the presence of bacteria. DNA polymerase purified from the 156T-POL mutant was used in a PCR assay to amplify the bacterial 16S rRNA gene without the addition of template DNA. Dose-dependent amplification of the 16S rRNA gene with or without the addition of bacterial genomic DNA was tested using the primer pairs listed in Table 1. When purified DNA polymerase was used, the 16S rRNA gene was amplified in only a sample with externally added bacterial genomic DNA as a template and not in samples without template (Fig. 6a and b). In contrast, with commercially available Ex Taq (Fig. 6c) and nPfu-Forte (Fig. 6d) DNA polymerases, 16S rRNA gene amplification was observed in all the samples without the addition of a bacterial genomic DNA template. This result indicates that DNA polymerase purified in our system can be utilized to provide DNA polymerase activity without bacterial DNA contamination. 3.6. Implications of this study In this study, we demonstrated that T. onnurineus NA1 156T can produce thermophilic enzymes along with H2 during fermentation using 100% CO or coal-gasified syngas. Integration of the production of biofuels and bioproducts of value can be considered a cost-effective approach in the bioeconomy. Hyperthermophilic enzymes have been applied to a broad array of industrial processes (Atomi et al., 2011; Quehenberger et al., 2017). Many enzymes have been successfully
Fig. 4. SDS-PAGE of (a) α-amylase and (b) DNA polymerase purified from 156T-AM and 156T-POL, respectively, cultured by CO-containing gas fermentation. Three micrograms of each protein was loaded onto the SDS-PAGE. 5
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Fig. 5. Saccharification activity of whole-cell extracts of 156T-AM toward potato peel or starch. (a) Quantitative measurement of reducing sugars released from potato peel through incubation with whole-cell extract (10 µg) of 156T-AM cultured in a serum vial using 100% CO. (b) Quantitative measurement of reducing sugars released from 1% starch through incubation at 80 °C for 15 min with whole-cell extract (40 µg) of 156T-AM. The strain was cultured using syngas and harvested at 4, 5, 6, 6.5 h of culture time as shown in Fig. 3c. (c) Quantitative measurement of reducing sugars released from potato peel waste through incubation with 156T-AM whole-cell extract (40 µg) of 156T-AM cultured from syngas fermentation and harvested at 4.75 h of culture time as shown in Fig. 3c. Error bars indicate the standard deviation of three independent experiments.
expressed and purified from heterologous hosts such as E. coli. However, thermophilic hosts have been pursued to harness their advantage in producing thermophilic proteins composed of multiple subunits, involving maturation processes, or requiring cofactors (Atalah et al., 2019). Despite the necessity, low cell density and a lack of a genetic toolbox have been hurdles to developing thermophilic hosts. This study demonstrated that the T. onnurineus NA1 156T strain could be utilized as a good cell factory to produce thermophilic enzymes because of its high cell density and genetic manipulation system. To date, a few hyperthermophilic archaea, such as Sulfolobus spp., Pyrococcus furiosus and Thermococcus kodakarensis, have been extensively studied as thermophilic platforms to produce value-added products. Comparison of cell density and biomass productivity revealed that maximum cell density of 2.4 g/L observed in this study seems to be quite high among obligate anaerobic hyperthermophiles, which is similar to the biomass in the dialysis membrane reactor of P. furiosus (Krahe et al., 1996). Furthermore, biomass productivity of 0.3 g/L/h in this study is comparable to the data obtained by various cultivation studies of aerobic
Sulfolobus species such as dialysis reactor culture of S. shibatae B12 (Krahe et al., 1996), fed-batch culture of S. solfataricus P2 (Park and Lee, 1997), and microfiltration fed-batch culture of S. solfataricus Gθ (Schiraldi et al., 1999). To harness these merits and enhance the production yield of thermophilic enzymes, it would be necessary to further develop extensive genetic tools, such as promoters and induction systems. In this study, we designed the stable expression of thermophilic enzymes by inserting the gene of interest in a chromosome. The growth and H2 production of the α-amylase-overexpressing mutant were even better than those of the parental strain, indicating that production of the enzyme was not inhibitory to the strain. However, the other mutant, overexpressing the DNA polymerase gene, showed significant defects in growth and H2 production. Inhibitory effects of overexpressing DNA polymerase genes on growth have also been reported (Chan et al., 2007; Uchida et al., 2008). Therefore, the effect of gene overexpression on the cell factory should be considered when selecting an enzyme as a coproduction target. Fig. 6. Amplification of the 16S rRNA gene with or without a DNA template. PCR amplification of the 16S rRNA gene was performed using DNA polymerase purified from the 156T-POL mutant (a and b) or commercially available DNA polymerase (c and d) without DNA template (E. coli genomic DNA). Primer pairs for PCR are listed in Table 1.
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To avoid biased data for PCR amplification of bacteria-specific 16S rRNA genes by bacterial DNA contamination, removal of bacterial DNA contamination from commercial DNA polymerases has been studied in previous reports (Carroll et al., 1999; Eshleman and Smith, 2001). However, DNA polymerases purified from eukaryotic organisms, such as plants or yeast, were found to be safe from bacterial DNA contamination (Niimi et al., 2011). Regarding this issue, DNA polymerase obtained by employing archaeal strains is also beneficial.
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Microbiology of synthesis gas fermentation for biofuel production. Curr. Opin. Biotechnol. 18, 200–206. Jeong, J.Y., Yim, H.S., Ryu, J.Y., Lee, H.S., Lee, J.H., Seen, D.S., Kang, S.G., 2012. Onestep sequence-and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl. Environ. Microbiol. 78, 5440–5443. Jung, H.C., Lee, S.H., Lee, S.M., An, Y.J., Lee, J.H., Lee, H.S., Kang, S.G., 2017. Adaptive evolution of a hyperthermophilic archaeon pinpoints a formate transporter as a critical factor for the growth enhancement on formate. Sci. Rep. 7, 6124. Khawla, B.J., Sameh, M., Imen, G., Donyes, F., Dhouha, G., Raoudha, E.G., Oumèma, N.E., 2014. Potato peel as feedstock for bioethanol production: a comparison of acidic and enzymatic hydrolysis. Ind. Crops Prod. 52, 144–149. Kim, M.-S., Choi, A.R., Lee, S.H., Jung, H.-C., Bae, S.S., Yang, T.-J., Jeon, J.H., Lim, J.K., Youn, H., Kim, T.W., Lee, H.S., Kang, S.G., 2015. 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4. Conclusion Here, we demonstrated that the production of thermophilic enzymes could be integrated with hydrogen production by a hyperthermophilic archaeon through gas fermentation. The thermophilic enzymes selected in this study showed considerable potential for application in saccharification or DNA amplification with their use in purified form or as a crude cell extract. This study also showed that syngas was useful as a cost-effective source of CO gas for the coproduction of value-added proteins and hydrogen. Further research is required for the production of exogenous thermophilic enzymes in the thermophilic cell factory developed in this study. CRediT authorship contribution statement Seong Hyuk Lee: Formal analysis, Investigation, Data curation, Writing - original draft. Sung-Mok Lee: Investigation, Formal analysis, Writing - original draft. Jung-Hyun Lee: Conceptualization. Hyun Sook Lee: Conceptualization, Data curation, Writing - review & editing. Sung Gyun Kang: Conceptualization, Data curation, Writing - review & editing, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work is supported by the Korea Institute of Ocean Science and Technology (KIOST) in-house program (PE99822) and the study on operational stability and risk assessment of CO-converting hyperthermophilic archaeon program of the Ministry of Oceans and Fisheries in the Republic of Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2020.123067. References Amend, J.P., Shock, E.L., 2001. Energetics of overall metabolic reactions of thermophilic and hyperthermophilic archaea and bacteria. FEMS Microbiol. Rev. 25, 175–243. Arapoglou, D., Varzakas, T., Vlyssides, A., Israilides, C., 2010. Ethanol production from potato peel waste (PPW). Waste Manag. 30, 1898–1902. Atalah, J., Cáceres-Moreno, P., Espina, G., Blamey, J.M., 2019. Thermophiles and the applications of their enzymes as new biocatalysts. Bioresour. Technol. 280, 478–488. Atomi, H., Sato, T., Kanai, T., 2011. Application of hyperthermophiles and their enzymes. Curr. Opin. Biotechnol. 22, 618–626. Bae, S.S., Kim, Y.J., Yang, S.H., Lim, J.K., Jeon, J.H., Lee, H.S., Kang, S.G., Kim, S.J., Lee, J.H., 2006. Thermococcus onnurineus sp. nov., a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent area at the PACMANUS field. J. Microbiol. Biotechnol. 16, 1826–1831. Bernfeld, P., 1955. Amylases, α and β. Methods Enzymol. 1, 149–158. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Camire, M.E., Violette, D., Dougherty, M.P., McLaughlin, M.A., 1997. Potato peel dietary fiber composition: effects of peeling and extrusion cooking processes. J. Agric. Food.
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