WO2018105790A1 - Thermococcus onnurineus wtf-156t having mutation in formate transporter and methods of hydrogen production using thereof - Google Patents

Abstract

The present invention provides Thermococcus onnurineus WTF-156T strain (accession no. KCTC13132BP) having increased hydrogen production ability, wherein there is the mutation at the position 52 of formate transporter from alanine to threonine. In addition, the present invention provides a method for producing hydrogen by using the strain. Thermococcus onnurineus WTF-156T strain according to the present invention shows greater hydrogen production ability form formate than wild-type strain. The method for hydrogen production according to the present invention can produce hydrogen efficiently with lower cost.

Description

THERMOCOCCUS ONNURINEUS WTF-156T HAVING MUTATION IN FORMATE TRANSPORTER AND METHODS OF HYDROGEN PRODUCTION USING THEREOF

The present invention relates to Thermococcus onnurineus WTF-156T and method for producing hydrogen using the same.

Hydrogen energy has drawn attention as an alternative energy source^{1 ,2}. Currently, the annual production of H₂　is approximately 0.1 Gtons, of which 98% comes from the reforming of fossil fuels³: 40% of hydrogen　is produced from natural gas, 30% is produced from heavy oil and naphtha, 18% is produced from coal, 4% is produced from electrolysis and approximately 1% is produced from biomass⁴. Due to the advantage of environmentally friendliness and cost-effectiveness compared with conventional chemical methods, biological hydrogen production has been extensively studied over several decades^{5 ,6}.

Formate can be produced efficiently from various inexpensive resources or as an end product of microbial activity, and a number of studies on formate-dependent hydrogen production have been carried^{7 -9}. A variety of microbes with formate hydrogen lyase (FHL), such as the FHL complex from Escherichia coli ^{10 -12} have been identified in phylogenetically diverse groups of archaea and bacteria⁹.

Oxidation of formate to CO₂ and H₂ under anoxic conditions is an endergonic process under standard conditions at 25 °C (HCOO^- + H₂O HCO₃ ^- + H₂, ΔG_{25 °C} = +1.3 kJ/mol). In anaerobic syntrophic formate oxidation, the reaction is made thermodynamically possible by removal of the end product H₂ using a methanogenic or sulfate-reducing partner^{13 -16}. No pure culture has ever been shown to grow on formate with hydrogen production. However, we demonstrated that T. onnurineus NA1 isolated from a deep-sea hydrothermal vent can grow on formate to produce hydrogen^17-19.

T. onnurineus NA1 encodes three copies of formate dehydrogenase gene clusters, including the fdh1 - mfh1 - mnh1 cluster (TON_0282-0266), fdh2 - mfh2 -mnh2 cluster (TON_1563-1580) and fdh3 - sulfI cluster (TON_0534-0540). Among those gene clusters, the fdh2 - mfh2 - mnh2 gene cluster was shown to be solely essential for formate-driven growth^{17 ,19}: the Fdh2 module oxidizes formate and the Mfh2 module transfers electrons to protons, thereby generating a proton gradient across the membrane. This gradient is used by the Mnh2 module to produce a secondary sodium ion gradient that drives ATP synthesis, catalyzed by a Na⁺-ATP synthase. A formate transporter (TON_1573) gene is encoded in the downstream region of the fdh2 - mfh2 - mnh2 gene cluster, which presumably plays a role in importing formate into the cytoplasm¹⁷. Although the formate transporter in the members of Thermococcales is largely uncharacterized, it is predicted to belong to the formate/nitrite transporter (FNT) family, a family of evolutionarily related transmembrane bacterial and archaebacterial proteins²⁰.

The present inventors employed adaptive laboratory evolution to investigate molecular changes to enhance the cell growth of T. onnurineus NA1 on formate. Adaptive laboratory evolution allows the selection of desirable phenotypes in a laboratory environment against an applied stress and can be a powerful way to develop beneficial phenotypic characteristics in strains²¹. During adaptation, genetic variations occur all over the chromosome, and beneficial mutations can improve the ability to handle the stress²². Previously, the serial transfer of T. onnurineus NA1 in a CO condition greatly improved tolerance and growth of the strain on CO²³, accompanied by mutations that included a mutation at TON_1525, a putative DNA-binding protein.

To obtain an integrative picture of physiological and molecular changes during adaptation, physiological changes during the transfer were monitored, and the whole genome sequence of the adapted mutant was determined in comparison with the parent strain. The effect of the identified mutations on the adapted strain was determined. Hydrogen production of the mutant was assessed in a bioreactor in comparison with the parent strain.

The inventors of present invention have discovered that a single mutation in formate transporter increases hydrogen production from formate in T. onnurineus NA1, and achieved the present invention.

Korean Patent Application No.10-2011-0021390 discloses a method for hydrogen gas production using Thermococcus strain in anaerobic condition.

(Patent Literature2) Korean Patent Application No.10-2011-7014737 discloses a novel hydrogenase isolated from a novel thermophilc Thermococcus strain, gene encoding the same and method for producing hydrogen gas using the same.

US patent No. 8,993,291 discloses Thermococcus mutant having improved hydrogen production from formate and methods of hydrogen production by using thereof.

Bae S.S. et al. discloses a method for producing hydrogen from carbon monoxide, formic acid or starch using a thermophilic strain, Thermococcus onnurineus (Biotechnol Lett. 2012 Jan;34(1):75-9).

Moon Y.J. et al. discloses a proteome analysis data using Thermococcus onnurineus strain (Mol Cell Proteomics. 2012 Jun;11(6):M111.015420).

One purpose of present invention to provide new Thermococcus onnurineous strain to produce hydrogen.

The other purpose of present invention to provide new method of production of hydrogen by using the new Thermococcus onnurineus strain.

In one aspect, the present invention provides Thermococcus onnurineus strain having amino acid mutation in formate transporter (TON_1573). Preferably, the Thermococcus onnurineus strain is Thermococcus onnurineus WTF-156T strain (deposit accession number of KCTC13132BP).

As used herein, the term "formate transporter" is intended to mean a protein translocating formate across cytoplasmic membrane. Preferably, the formate transporter is originated from Themococcus sp. More preferably, the formate transporter is the protein having Sequence ID. No. 1.

As used here, the term "amino acid mutation" is intended to mean to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and　modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics e.g. promoting formate translocation in from Themococcus sp. Preferred amino acid mutations are amino acid substitutions. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like.

Preferably, the said mutation is that the alanine at the position of 52 in the formate transporter (TON-1573) is mutated to threonine or glutamine. More preferably the mutation is from alanine to threonine.

In another aspect, the present invention provides a method for producing hydrogen by using the strain having mutation in formate transporter (TON-1573). Preferably, the method is that the said mutation is from alanine to threonine or glutamate at 52 position in the formate transporter (TON-1573). More preferably, the method is that the said mutation is from alanine to threonine at 52 position in the formate transporter (TON-1573).

In another embodiment, the method of producing hydrogen from formate by using the Thermococcus onnurineus WTF-156T strain (deposit accession number of KCTC13132BP). Preferably, the culture condition of the strain for hydrogen production comprises temperatures between 60 to 90 ℃ and/or at pressures of 1 - 3 atm.

Thermococcus onnurineus WTF-156T strain according to the present invention shows greater hydrogen production ability form formate than wild-type strain. And the method for hydrogen production according to the present invention can produce hydrogen efficiently with lower cost.

FIG. 1 illustrates physiological changes of T. onnurineus NA1 following serial transfers into fresh MM1 medium containing 147 mM sodium formate. After 2 (closed circle), 32 (open square), 62 (closed inverted triangle), 92 (open triangle), 122 (closed square) and 156 (open circle) transfers, the cell density (expressed as optical density at 600 nm) (a) was determined at the indicated time points. Formate consumption rates (b) and H₂ production rates (c) were determined during the exponential phase. All experiments were conducted independently in duplicate.

FIG. 2 illustrates time profiles of cell growth (a) and H₂ production rate (b) in the wild-type (closed symbol) and WTF-156T (open symbol). (c), Changes of residual formate (square) and hydrogen production (inverted triangle) in the wild-type (closed symbol) and WTF-156T (open symbol) during the batch culture on 400 mM sodium formate. The pH was adjusted to 6.1-6.2 using 2 N HCl containing 3.5% NaCl as a pH-adjusting agent.

FIG 3 illustrates mutations found in the genome of WTF-156T. The numbers inside and outside the circle represent genome position (Mb) and locus tag, respectively. Mutations are summarized in Table 3.

FIG. 4 illustrates the effect of each mutation was determined by restoring each mutation in WTF-156T. Cell growth (a), formate consumption (b) and H₂ production (c) in the revertants were analyzed in comparison with those of wild-type and WTF-156T at the late exponential phase (after 6 h incubation). Error bars indicate the standard deviation from three independent experiments.

FIG. 4 illustrates the effect of each mutation was determined by restoring each mutation in WTF-156T. Cell growth (a), formate consumption (b) and H₂ production (c) in the revertants were analyzed in comparison with those of wild-type and WTF-156T at the late exponential phase (after 6 h incubation). Error bars indicate the standard deviation from three independent experiments.

FIG. 5 illustrates the effect of the A52T mutation and deletion of TON_1573. Changes of cell density (optical density at 600 nm) (a), formate consumption (b) and H₂ production (c) were determined in the wild-type (closed circle), TON_1573 (A52T) (open circle) and deletion mutant of TON_1573 (closed inverted triangle) during the batch culture. Error bars indicate the standard deviations of independent duplicate experiments.

FIG. 6 shows 3D model structure of the wild-type formate transporter (TON_1573) (a), A52T mutant (b) and A52E mutant (c). The predicted two constriction sites in the closed central pore are highlighted in red and the amino acid residues that contribute to the two constriction sites, Phe 81/Phe 212 and Leu 85/Leu 94, are predicted by the multiple alignment shown in Table 5.

FIG. 7 illustrates the effect of alteration at formate transporter (TON_1573) on formate conversion using resting cell suspensions. Formate consumption (a) and H₂ production (b) of A52T mutant and deletion mutant of TON_1573 were determined in comparison with those of the wild-type and WTF-156T strains using resting cell assay. Error bars indicate the standard deviations of independent duplicate experiments.

Hereinafter, the present invention will be described in further detail with reference to examples. However, these examples are for illustrative purposes only and it is to be understood by those with common knowledge in the technical field the present invention is part of, that these examples are not to be construed to limit the scope of the present invention.

EXAMPLE 1. Physiological changes of T. onnurineus NA1 during serial transfers on formate

Strain, medium, and culture condition

The T. onnurineus strain NA1 (KCTC 10859) was isolated from a deep-sea hydrothermal vent area in a Papua New Guinea-Australia-Canada-Manus (PACMANUS) field31. This strain was routinely cultured in modified medium 1 (MM1)^17,32. The pH of the medium was adjusted to 6.5 with 2 N HCl. The medium was kept in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI, USA) filled with an anoxic gas mixture (N₂/H₂/CO₂, 90:5:5) to equilibrate after autoclaving. For the adaptive laboratory evolution study, the parent strain was cultured on MM1 medium with 1% yeast extract and 147 mM sodium formate at 80 °C for 15 h and transferred to fresh medium. The pH-stat fed-batch culture of T. onnurineus NA1 was anaerobically conducted in a 3-L fermentor (Fermentec, Cheongwon, Korea) with a working volume of 1.5 L using MM1 medium with 4 g·L^-1 of yeast extract and 400 mM sodium formate. The culture temperature and agitation speed were 80 °C and 300 rpm, respectively, and the pH was controlled at 6.1-6.2 by automatic titration with 2N HCl in 3.5% NaCl as a pH-adjusting agent. The medium of the fermentor was flushed with argon gas for at least 30 min to maintain anaerobic conditions before inoculation.

Analytical methods

Cell growth was monitored by measuring the optical density at 600 nm (OD600) with a UV/Vis spectrophotometer (Biophotometer Plus, Eppendorf, Hamburg, Germany). Biomass concentration was determined by the correlation of dry cell weight (DCW) with OD600 as in a previous report18. H₂ was measured using a YL6100GC gas chromatograph (YL Instrument Co., Anyang, South Korea) equipped with a Molsieve 5A column (Supelco, Bellefonte, PA, USA), a Porapak N column (Supelco), a thermal conductivity detector, and a flame ionization detector. Argon was used as the carrier gas at a flow rate of 30 ml/min. The total volume of outlet gas was measured using a wet gas meter (Shinagawa, Tokyo, Japan) at 1 atm, at each time interval. The volumetric H₂ production rate (HER) (mmol L^-1 h^-1) was calculated by the amount of H₂ produced as a function of time. The specific H₂ production rate was calculated by dividing HER by biomass concentration. The concentration of formate was determined using high-performance liquid chromatography equipped with a UV detector and an RSpak KC-811 column (Shodex, Tokyo, Japan) with a mobile phase of 0.1% (vol/vol) H₃PO₄ at a flow rate of 1.0 ml min^-1.

Genome sequencing

For genome re-sequencing, we extracted genomic DNA from cultures of the WTF-156T strain without single-colony isolation. Genome sequencing was performed using PacBio Single Molecule Real-Time (SMRT) sequencing (Pacific Biosciences, Menlo Park, CA, USA)³³. Variants were detected using SAMtools v0.1.18. PacBio SMRT sequencing of a 10-kb insert library providing approximately 100X coverage. Assembly and consensus polishing were performed using SMRTpipe HGAP and SMRTpipe Quiver, respectively. All mutations were verified by PCR and Sanger sequencing, and all primers are listed in Table 1.

Primers	Oligonucleotide Sequences	Sequence ID.
Construction of mutants
pUC118_0282del_HMG_fo_inverse_F	5'-gacctgcaggcatgcaagct-3'	Seq. ID. No. 2
pUC118_0282del_HMG_fo_inverse_R	5'-gactctagaggatccccggg-3'	Seq. ID. No. 3
TON_0820_SLIC_F	5'-ggatcctctagagtccaatactcgggaacctcaag-3'	Seq. ID. No. 4
TON_0820_SLIC_R	5'-gcatgcctgcaggtctctgggccgcgtacctctca-3'	Seq. ID. No. 5
TON_1084_SLIC_F	5'-ggatcctctagagtctcctgtcgcgtgaaggggct-3'	Seq. ID. No. 6
TON_1084_SLIC_R	5'-gcatgcctgcaggtcgctatccttcttccggtctt-3'	Seq. ID. No. 7
TON_1561_SLIC_F	5'-ggatcctctagagtcgatacaacgctggcactcat-3'	Seq. ID. No. 8
TON_1561_SLIC_R	5'-gcatgcctgcaggtccagcgaaataaagccctcag-3'	Seq. ID. No. 9
TON_1573-SLIC-F	5'-tttggtttcctcctgacggtggttgc-3'	Seq. ID. No. 10
TON_1573-SLIC-R	5'-ccgctgcaaccaccgtcaggaggaaa-3'	Seq. ID. No. 11
1573-point-mutation-F	5'-tttggtttcctcctgacggtggttgc-3'	Seq. ID. No. 12
1573-point-mutation-R	5'-ccgctgcaaccaccgtcaggaggaaa-3'	Seq. ID. No. 13
TON_1561_insertion(G)-F	5'-ggacatagtccttaaggggggacttc-3'	Seq. ID. No. 14
TON_1561_insertion(G)-R	5'-tcgaggaagtccccccttaaggacta-3'	Seq. ID. No. 15
Confirmation of constructs
TON_1573_point- confirm-R	5'-tgcaaccaccgt-3'	Seq. ID. No. 16
TON_0820_ point-confirm-R	5'-agaagacgctgc-3'	Seq. ID. No. 17
TON_1084_point-confirm-F	5'-cagaaccccccc-3'	Seq. ID. No. 18
TON_1561_point-confirm-F	5'-cttaagggggg-3'	Seq. ID. No. 19
Confirmation of mutations in coding region
TON_0618-F	5'-cctcatttattccaaaacta-3'	Seq. ID. No. 20
TON_0618-R	5'-ctaaaataaaactttcagga-3'	Seq. ID. No. 21
TON_0820-F	5'-acagaggtgagagagatgcccgttac-3'	Seq. ID. No. 22
TON_0820-R	5'-gaaaaaagcaaaggattacttcctga-3'	Seq. ID. No. 23
TON_1084-F	5'-ataccctacgagcgctggta-3'	Seq. ID. No. 24
TON_1084-R	5'-tgcgttgaagttggccctaa-3'	Seq. ID. No. 25
TON_1138-F	5'-cctctacgggagggtgaaga-3'	Seq. ID. No. 26
TON_1138-R	5'-ccgaacctcgatcccggggg-3'	Seq. ID. No. 27
TON_1555-F	5'-gagatacccctccacagtca-3'	Seq. ID. No. 28
TON_1555-R	5'-tggtgatgttatcctataca-3'	Seq. ID. No. 29
TON_1561-F	5'-caagggaggagctccttgaa-3'	Seq. ID. No. 30
TON_1561-R	5'-tctgcgctctcgcaagcttt-3'	Seq. ID. No. 31
TON_1573-F	5'-atccttcgaacggtcatact-3'	Seq. ID. No. 32
TON_1573-R	5'-gtctccaacgtggccgaaga-3'	Seq. ID. No. 33
TON_1641-F	5'-acagcggtactcctcgcgct-3'	Seq. ID. No. 34
TON_1641-R	5'-ttcctagcgttaatcatata-3'	Seq. ID. No. 35
TON_RS08635-F	5'-tccttaaaattccagttccc-3'	Seq. ID. No. 36
TON_RS08635-R	5'-tagttttttgaacctcaagc-3'	Seq. ID. No. 37
Confirmation of mutations in non-coding region
TON_0901-0902-intergenic region-F	5'-cgccaacccttccgagccgc-3'	Seq. ID. No. 38
TON_0901-0902-intergenic region-R	5'-ttctctgtcagaagtcttcc-3'	Seq. ID. No. 39
TON_1668-1669-intergenic region-F	5'-cccagcgcatagacatggtg-3'	Seq. ID. No. 40
TON_0901-0902-intergenic region-R	5'-cggctattgcagagccgccg-3'	Seq. ID. No. 41

Construction of mutants

Mutants of each revertant (TON_0820, TON_1084, TON_1561, TON_1573), and TON_1561 (insertion G) and TON_1573 (A52T) were made by applying a gene recombination system. Briefly, we designed primer sets for base-pair substitutions and mutated genes by site-directed mutagenesis. Each mutated gene and its flanking regions were ligated by one-step sequence- and ligation-independent cloning (SLIC)³⁴, and subsequent mutants were generated through homologous recombination using an unmarked in-frame deletion³⁵ method and a modified gene disruption system that was previously used for Thermococcus kodakarensis KOD1³⁶. T. onnurineus NA1 cells were transformed and incubated in the presence of 10 μM simvastatin as a selection marker. The sequences of the primers used for gene disruption and construct verification are given in Table 1.

Cell suspension experiment

To prepare cell suspensions, T. onnurineus NA1 was anaerobically cultured in a 2-L Scott-Duran bottle containing 1 L of MM1 medium with 1% yeast extract and 147 mM sodium formate at 80 °C for 12 h. At the end of the culture, cells were harvested by centrifugation at 8,000 × g for 20 min at 25 °C. Cells were resuspended in an anaerobic buffer A containing 20 mM imidazole-HCl (pH 7.5), 600 mM NaCl, 30 mM MgCl₂, and 10 mM KCl. Cells were recollected by centrifugation at 6,000 × g for 20 min at 25 °C and resuspended in buffer A.

For formate consumption and H₂ production, cell suspensions in the MM1 medium without yeast extract and sodium formate at a final cell density of OD₆₀₀ = 0.5 were used. Cell suspensions were preincubated at 80 °C for 30 min. To determine H₂ production, a rubber-sealed glass vial was used. The reaction was initiated by the addition of 50 mM sodium formate. At various time intervals, gas samples were taken and analyzed in a YL6100 GC gas chromatograph (YL Instrument) for H₂ and the concentration of formate was determined using high-performance liquid chromatography.

3D model structure

The TON_1573 protein sequence of Thermococcus onnurineus NA1 was retrieved from the National Center of Biotechnology information (NCBI) Protein sequence database in FASTA format. Swiss model automatic modeling mode was selected, the protein sequence was entered in FASTA format in the space provided and the modeling request was submitted. The most fitting template for the three-dimensional prediction of the constructed model was saved and subjected to assessment. The model thus obtained was edited and visualized using PyMOL.

Results

Previously, it was reported that the hyperthermophilic archaeon T. onnurineus NA1 can grow on formate to produce H₂. The respiratory complex encoded in the fdh2 - mfh2 - mnh2 gene cluster mediated the conversion of formate to hydrogen and generated a successive proton/sodium gradient coupled to ATP generation by Na⁺-specific ATP synthase^{17 ,19}. The present inventors attempted to adapt T. onnurineus NA1 on formate to identify beneficial changes to enhance its formate-driven growth. T. onnurineus NA1 was inoculated into a medium containing formate as a whole energy source and cultured to stationary phase. Then, 2% of the culture was inoculated into the same, fresh medium and the serial transfer was repeated more than 150 times. Through these serial transfers, changes in cell growth, H₂ production and formate consumption were monitored (Fig. 1). It was shown that cell density, H₂ production and formate consumption of T. onnurineus NA1 gradually increased as the serial transfer continued. After 156 transfers, the adapted strain, designated WTF-156T, showed 1.71-, 1.93- and 1.91-fold higher cell density, H₂ production and formate consumption, respectively, than the parent strain.

T. onnurineus NA1 WTF-156T was deposited in Korea Research Institute of Bioscience and Biotechnology (KRIBB) with accession no.: KCTC13132BP on the date of Oct. 24, 2016.

EXAMPLE 2. Kinetic analysis of formate consumption and H₂ production

Even though the strain exhibited enhanced cell growth and hydrogen production on formate-containing medium, it was difficult to characterize the changes quantitatively in a serum vial. The pH in the culture medium rapidly increased, with a final pH of approximately 8 at stationary phase. Therefore, the kinetic properties of WTF-156T were investigated in a pH-controlled bioreactor (pH 6.2) in comparison with those of the parental strain. As shown in Figure 2, the WTF-156T strain reached 0.7-0.8 optical density (OD₆₀₀) after 5 hours. It also showed 1.9- and 3.8-fold higher maximum biomass yield and H₂ production rate, respectively, than the parent strain (Table 2). Notably, WTF-156T exhibited a shorter lag time in the bioreactor culture (Fig. 2a). Formate consumption was well balanced with hydrogen production. The parent strain consumed 176.8 mM formate after 16 hours, while WTF-156T consumed 348.1 mM formate after 7 hours, indicating that the consumed formate was converted to hydrogen in both the parent strain and WTF-156T (Fig. 2c).

Kinetic parameters of the wild-type and WTF -156T

Kinetic parameters	wild-type strain	WTF -156T strain
μmax (h-1)	0.3	1.1 (3.7)c
r max (mmol liter-1 h-1)	31.7	109.0 (3.4)c
Biomass productivity (g liter-1 h-1)a	0.026	0.101 (3.9)c
q max (mmol g-1 h-1)	198.2	345.7 (1.7)c
H2 productivity (mmol liter-1 h- 1)b	9.5	52.3 (5.5)c

Kinetic parameters were calculated with data from the graphs in Fig. 2.μ_max, specific growth rate; r _max, maximum H₂ production rate; q _max, maximum specific H₂ production rate.

^a Biomass productivity was determined by dividing total yield by time difference from 11 to 13 h for the wild-type strain and from 2 to 4 h for WTF-156T strain

^b H₂ Productivity was determined by dividing the total yield by time

^c The number in parenthesis refers to the fold difference in comparison with that of the wild-type strain

EXAMPLE 3. Genome-wide mutation analysis

To understand the cause of the physiological changes, genetic variations in the genomic DNA of WTF-156T were analyzed in comparison with the sequence of the parent strain by genome sequencing using PacBio Single Molecule Real-Time (SMRT) sequencing technology. There were 11 single-base substitutions either at the coding (9 sites) or intergenic regions (2 sites). The mutation list also includes insertions (2 sites), deletions (2 sites) and multiple substitutions (7 sites) (Fig. 3). The base substitution occurred at genes encoding a hypothetical protein (TON_0618), aromatic amino acid permease (TON_0820), another hypothetical protein (TON_1084), 3-phosphoshikimate-1-carboxyvinyltransferate (TON_1138), signal peptidase (TON_1555), F₄₂₀-reducing hydrogenase β subunit (TON_1561), formate transporter (TON_1573), a third hypothetical protein (TON_1641), a short-sequence hypothetical protein (TON_RS08535) and noncoding regions between the amino-acid transporter and biotin-protein ligase (TON_0901 -TON_0902) and between a hypothetical protein and the peptide transporter (TON_1668 -TON_1669) (Table 3). To determine the time of mutation for each mutation during the adaption period, we attempted to determine the distribution of each mutation in the 2^nd, 62^nd, 156^th transferred strains. Out of 11 mutations found in the genome of WTF-156T, 6 mutations were found in the 62^nd transferred strain, while the other 5 mutations were detected only in the 156^th transferred strain .

Mutations in the genome of WTF 156T

Locus_tag	Genome position	Mutation typea	Codon changes	Description
TON_1555	1427744	substitution	Pro to Leu	Peptidase
TON_1573	1446340	substitution	Ala to Thr	Formate transporter
TON_0820	760913	substitution	Gly to Asp	Aromatic amino acid permease
TON_RS08535	1537688	substitution	Gly to Glu	Hypothetical protein
TON_1138	1046432	substitution	-	3-Phosphoshikimate 1-carboxyvinyltransferase
TON_1641	1500400	substitution	-	Hypothetical protein
TON_0618	576102	T deletion	Frame shift	Hypothetical protein
TON_1084	1005110	C insertion	Frame shift	Hypothetical protein
TON_1561	1433065	G insertion	Frame shift	Coenzyme F420 hydrogenase
TON_0901 - 0902	832564	A deletion	-	Between amino acid transporter and Biotin-protein ligase
TON_1668 - 1669	1532991	substitution	-	Between hypothetical protein and peptide transporter

^a All mutations were confirmed by PCR verification and Sanger sequencing

To evaluate the contribution of each mutation to the phenotypic changes, genes such as aromatic amino acid permease (TON_0820), hypothetical protein (TON_1084), F₄₂₀-reducing hydrogenase β subunit (TON_1561) and formate transporter (TON_1573) were selected before embarking on time-consuming empirical analysis. As each mutation of WTF-156T was restored to the sequence of the parental strain, the growth rate of the four revertants decreased on formate media (Fig. 4). In particular, two revertants of the mutation at TON_1561 or TON_1573 caused significant decreases in cell growth and hydrogen production in WTF-156T.

EXAMPLE 3. Mutation of TON_1573 (A52T) increased H₂ production in T. onnurineus NA1

To test whether the TON_1561 (insertion G) and TON_1573 (A52T) mutations were indeed responsible for enhanced growth on formate, we introduced each mutation into the wild-type. The resulting mutant with the alteration at TON_1573 (A52T) displayed enhanced growth, H₂ production and formate consumption (Fig. 5). However, the TON_1561 (insertion G) mutant did not show much change from the wild-type. Previously, we reported that a gene cluster encoding an FHL (formate hydrogenlyase), a cation/proton antiporter and a formate transporter in T. onnurineus NA1 were responsible for growth on exogenous formate¹⁷ and that the expression level of genes in the fdh2 - mfh2 -mnh2 gene cluster significantly increased in the presence of formate. No mutation of genes in the gene cluster, except TON_1573, was found during the adaptation. However, the mutation at the formate transporter could confer the increase in hydrogen production in WTF-156T. On the other hand, the knockout mutant deficient in the TON_1573 gene exhibited a significant decrease in cell growth on formate (Fig. 5).

TON_1573 in the mfh2 gene cluster is predicted to be a formate transporter and shows similarity to FocA in bacterial strains. Therefore, it presumably played a role in transporting exogenous formate to the cytoplasm. Based on the structure of the bacterial FocA (PDB ID: 3KLY), Swiss model software was used to predict the structure of TON_1573 (Fig. 6). The mutated 52^nd residue was predicted to be part of a hydrophobic patch in the axial channel, facing internally towards the central pore. The change from alanine to threonine in the residue could slightly affect hydrophobicity in the patch (Fig. 6 b).

To verify the effect of the change (A52T) in TON_1573, we compared the rate of formate consumption with that of the parent strain during the batch culture (Fig. 5). The mutant displayed enhanced formate consumption and H₂ production, associated with increased cell growth. To measure the rate of formate consumption, resting cell suspensions of the parental strain and the mutant were incubated with formate. After incubating at 80 °C for 5 min, the WTF-156T strain showed 17.4% higher formate uptake (302.4 mM/g/h) than the wild-type (257.6 mM/g/h). The mutant at TON_1573 (A52T) in the parent background showed 9.3% higher formate consumption (281.6 mM/g/h) than the parent strain, while the deletion of TON_1573 significantly decreased formate consumption (187.2 mM/g/h) and hydrogen production (Fig. 7). Taken together, TON_1573 (A52T) is determined to be a beneficial mutation that occurred during the adaptive laboratory evolution.

Previously, we tested cell recycling of T. onnurineus NA1 at a bioreactor scale²⁴, and achieved much higher cell density and H₂ production rate than the wild-type. The kinetic analysis clearly showed the increase of the growth and hydrogen production by the approach (Table 4). We realized that T. onnurineus NA1 in the cell recycling experiment was actually transferred many times in the formate medium. Therefore, we speculated that the cell could be exposed to a genomic change. To address the issue, the genome sequence of the strain in the recycling experiment was determined using PacBio Single Molecule Real-Time (SMRT) sequencing technology. The mutations were listed in Table 5. Interestingly, a mutation at the same residue (52^nd residue) of TON_1573 was identified, but alanine was changed to glutamate in this case (Fig. 6 c). In conclusion, the strain adapted through serial transfer, in the serum vial or repeated batch culture in a bioreactor retained a single mutation at the same residue (52^nd residue) of TON_1573, which was determined to be a critical factor to enhance formate uptake and hydrogen production of T. onnurineus NA1.

The kinetic analysis of the recycling experiment

Kinetic parameters	wild-type strain	strain in the recycling experiment*
μmax (h -1)	0.3	0.43
r max (mmol liter-1h -1)	31.7	85.8
Biomass productivity (g liter-1 h- 1)a	0.026	0.085
q max (mmol g-1 h-1)	198.2	351.6
H2 productivity (mmol liter-1 h- 1)b	9.5	70.9

Kinetic parameters were calculated using the data from graphs in Fig. 2. μ_max , specific growth rate; r _max, maximum H₂ production rate; q _max, maximum specific H₂ production rate.

^aBiomass productivity was determined by dividing the total yield by time difference from 11 to 13 h for the wild-type strain and from 2 to 4 h for repeated batch strains

^bH₂ productivity was determined by dividing the total yield by time

*The data for kinetic analysis were adapted from Bae et al. (2015)

Mutations found in the strain at the recycling experiment

Locus_tag	Genome position	Mutation type	Codon changes	Description
TON_0865	800143	substitution	Leu to Pro	pyridine nucleotide-disulfide oxidoreductase
TON_0916	846887	substitution	Gly to Asp	ATPase C-terminus
TON_1513	1388815	substitution	Ala to Val	orotate phosphoribosyltransferase
TON_1573	1446341	substitution	Ala to Glu	formate transporter
TON_1779	1645564	substitution	Ser to Tyr	ATPase
TON_0902	832911	substitution	-	biotin--protein ligase
TON_1532	1406368	substitution	-	lipoate--protein ligase
TON_1872	1752952	substitution	-	putative vitamin B12 transport protein
TON_1513	1388814	substitution	-	orotate phosphoribosyltransferase
TON_0536	490681	T deletion	Frame shift	cytochrome-c3 hydrogenase subunit gamma

ACKNOWLEDGMENTS

This work was supported by a grant from the KIOST in-house program (PE99413), the C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning in the Republic of Korea (2015M3D3A1A01064884), and the Development of Technology for Biohydrogen Production Using Hyperthermophilic Archaea program of the Ministry of Oceans and Fisheries in the Republic of Korea.

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Claims (6)

1. Thermococcus onnurineus strain having mutation in a formate transporter (TON_1573).
2. The Thermococcus onnurineus according to claim 1, the said mutation is that the alanine at the position of 52 in the formate transporter is mutated to threonine or glutamine.
3. The Thermococcus onnurineus according to claim 2, the said strain is WTF-156T strain (accession no.: KCTC13132BP).
4. A method for hydrogen production from formate by using the Thermococcus onnurineus according to any one of claims 1 to 3.
5. The method of claim 4, wherein culture condition of the strain for hydrogen production comprises temperatures between 60 to 90 ℃.
6. The method of claim 4, wherein culture condition of the strain for hydrogen production comprises providing the strain at pressures of 1 - 3 atm.

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