KR101734957B1 - Highly efficient Recombinant Escherichia coli strains for co-production of hydrogen and ethanol and highly efficient method for co-production of hydrogen and ethanol from glucose - Google Patents

Abstract

The present invention relates to a method for simultaneous production of hydrogen and ethanol using high-efficiency recombinant E. coli strain and glucose for the simultaneous production of hydrogen and ethanol. In the present invention, These mutants enable the cell growth in the anaerobic condition and the simultaneous production of hydrogen and ethanol by the overexpression of the enzymes in the adaptive evolution or rate step of these mutants. The recombinant E. coli according to the present invention simultaneously produced hydrogen and ethanol with high efficiency from fermentation using glucose. The maximum yields of hydrogen and ethanol from glucose were about 1.7 and 1.4 mol / mol, respectively. The yield of ethanol production was at least 70% higher than that of wild strains. In addition, the activating strain of the PP pathway obtained by the present invention can be used for research on the production of biofuel or useful chemicals such as NAD (P) H produced through the PP pathway, which requires a reducing power.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a high-efficiency recombinant Escherichia coli strains for co-production of hydrogen and ethanol, and ethanol from glucose}

The present invention relates to a method for simultaneous production of hydrogen and ethanol using high efficiency recombinant E. coli strain and glucose for the simultaneous production of hydrogen and ethanol.

Biological hydrogen production methods include photolysis, photo-fermentation, anaerobic fermentation (dark fermentation), and water-gas shift reaction. Among them, hydrogen production through anaerobic fermentation is recognized as the most realistic biological process among various biological production methods. However, because of the low yield of hydrogen production compared to glucose, there are many difficulties in industrial application. Researches on metabolic engineering and enzyme engineering have been actively carried out to solve this problem. In the case of anaerobic microorganisms such as Escherichia coli, various fermentations such as lactate, acetate, succinate, formate, carbon dioxide and hydrogen are generally used under anaerobic fermentation conditions in which no external electron acceptor such as oxygen is supplied. Mixed-acid fermentation to produce the product. At this time, ethanol, which is produced with hydrogen, is also one of the spotlight biofuels. Glucose is converted to the intermediate metabolite pyruvate via corresponding processes such as Embden-Meyerhof-Parnas (EMP), and pyruvate is again converted to pyruvate-formate lyase PFL) to acetyl-CoA and formic acid. Under typical fermentation conditions, wild-type E. coli strains produce equimolar ratios of acetic acid and ethanol, and formic acid is degraded into hydrogen and carbon dioxide by a formate-hydrogen lyase (FHL) complex. There have been considerable studies to eliminate the reaction pathways that compete with the formic acid production pathway so far to improve the yield of hydrogen production. For example, lactate dehydrogenase (LDH), which catalyzes the production of lactic acid in pyruvic acid, may be deleted, or fumaric acid reductase (FFA), which catalyzes the conversion of pyruvic acid, phosphoenolpyruvate (PEP) fumarate reductase). In addition, the yield of hydrogen production has been increased by eliminating the uptake hydrogenase that consumes the produced hydrogen again. However, despite these efforts, the theoretical yield of hydrogen production is limited to not exceed 2 mol hydrogen per mole of glucose, that is, 2 mol H2 / mol glucose.

In order to overcome the limit of theoretical yield, synthetic biology techniques have been studied to improve hydrogen production by using mesophilic microorganisms such as E. coli as a host. A typical method is to introduce a specific hydrogenation enzyme capable of receiving electrons from NAD (P) H to improve hydrogen production. However, the NAD (P) H-dependent hydrogenase has thermodynamic and kinetic problems in which the hydrogen partial pressure is extremely low or its activity appears at high temperatures. Hydrogen production reported through these studies only confirmed the concept that NAD (P) H could be used as an electron donor, and the yield of hydrogen production was very low. Characteristically, all of the strains produced through the present study show very low energy recovery compared to the energy content of the raw materials such as glucose.

Two-stage fermentation processes have also been actively studied as an alternative to overcome low hydrogen yield and energy recovery. Typical of these is the one-stage hydrogen production and this stage methane production, the Hythane process. In this process, hydrogen and organic acid are produced at a high rate in the first stage, and methane is produced in the second stage by using the organic acid produced in the first stage. This process has been conducted at a considerable level and some commercialization has proceeded. However, it takes a long time to produce methane, the methane conversion of organic acid requires a lot of reducing power, and the methane value is significantly lower than that of hydrogen. In particular, it is pointed out that the two-step methane fermentation tank needs to be 3-4 times larger than the first-stage fermentation tank because of the difference in the rate of the first stage hydrogen fermentation and the second stage methane fermentation. In another two-stage process, there are processes for hydrogen production in both stages 1 and 2. In this case, the first stage is anaerobic fermentation, and the second stage is photo fermentation. In this case, both stages 1 and 2 have the advantage of producing hydrogen. In addition, there is an advantage that the reducing power for the second stage hydrogen production can be obtained from light. However, light fermentation is slower than methane fermentation and has not been studied on a commercial scale because it is expensive to produce a photobioreactor. In addition, the first stage anaerobic fermentation and the second stage electrochemical reactor have been studied as a two stage hydrogen production reactor. In this case, in order to replace the slow photobioreactor, it is necessary to reduce the proton to hydrogen by electrically supplying energy in the second step reaction (activating low potential electrons when oxidizing the organic acid etc. at the anode). This reactor also succeeded in showing its concept on a small scale, but due to the nature of the electrochemical reactor, it failed to economically design and operate a large scale reactor. Overall, all of these efforts have in common use of a two-stage reactor to address the problem of low hydrogen production and energy recovery in anaerobic fermentation.

Korean Patent Laid-open No. 10-2011-0000775 (published Jan. 6, 2011)

An object of the present invention is Escherichia to produce a high efficiency at the same time hydrogen and ethanol under anaerobic culture conditions SH5p_Z coli recombinant strain (KCTC 12762BP) or Escherichia coli recombinant strains SH8AE to provide a (KCTC 12764BP).

Another object of the present invention is to provide a method for producing the Escherichia SH5p_Z coli recombinant strain (KCTC 12762BP) to the 6-phosphate gluconate dehydrogenase; were introduced by the over-expression transformants (6-phosphogluconate dehydrogenase Gnd), Escherichia , which at the same time with high efficiency production of hydrogen and ethanol under anaerobic culture conditions coli SH5p_ZG recombinant strain (KCTC 12763BP).

Another object of the present invention is to provide a method for producing the Escherichia An overexpression of an E. coli SH8AE recombinant strain (KCTC 12764BP) by transfection with glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd) Escherichia , which produces hydrogen and ethanol simultaneously under high culture conditions coli SH8AE_ZG recombinant strain (KCTC 12765BP).

It is another object of the present invention to provide a method for simultaneously producing hydrogen and ethanol at a high efficiency by culturing the recombinant strain.

In order to accomplish the above object, the present invention provides a method for producing hydrogen and ethanol simultaneously in an anaerobic culture condition using Escherichia coli SH5p_Z provides a recombinant strain (KCTC 12762BP).

The present invention also relates to a method of producing Escherichia coli (Escherichia coli) which produces both hydrogen and ethanol at high efficiency under anaerobic culture conditions by overexpressing 6-phosphogluconate dehydrogenase (Gnd) The E. coli SH5p_ZG recombinant strain (KCTC 12763BP) is provided.

In addition, the present invention relates to a method for producing hydrogen and ethanol simultaneously in an anaerobic culture condition with Escherichia E. coli SH8AE recombinant strain (KCTC 12764BP).

The present invention also relates to a method for producing a recombinant microorganism by overexpressing the recombinant strain by transfecting glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd) Escherichia , which produces hydrogen and ethanol simultaneously under high culture conditions The E. coli SH8AE_ZG recombinant strain (KCTC 12765BP) is provided.

Also, the present invention provides a method for producing a recombinant microorganism, comprising: culturing the recombinant strain in a medium containing a carbon source; And simultaneously obtaining hydrogen and ethanol from the cultured culture liquid. The present invention also provides a method for simultaneously producing hydrogen and ethanol at a high efficiency.

The present invention relates to a method for simultaneous production of hydrogen and ethanol using a high-efficiency recombinant E. coli strain and glucose for simultaneous production of hydrogen and ethanol, and an efficient simultaneous production of hydrogen and ethanol using the recombinant E. coli strain according to the present invention It is possible. This means the development of a high-efficiency, one-stage fermentation process with improved energy recovery for raw materials in useful alternative biofuel production. In addition, PP pathway - activated strains in anaerobic conditions can be used for the production of various chemicals that require NAD (P) H produced by the PP pathway in the future as reductive power.

Figure 1 shows a simplified representation of three corresponding pathways for glucose metabolism in E. coli and a reaction pathway for the simultaneous production of hydrogen and ethanol. In the present invention, the PP path to be activated is indicated in a box surrounded by a dotted line. Abbreviation: GLU, glucose; G6P, glucose-6-phosaphate; F6P, fructose-6-phosphate; 6PG, 6-phosphogluconate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; PG, phosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; AcCoA, acetyl-CoA; FOR, formate; ACE, acetate; ETH, ethanol; 6PG, 6-phospho gluconate; RL5P, Ribulose-5-phosphate; X5P, xylulose-5-phosphate; R5P, ribose-5-phosphate; S7P, sedoheptuloas-7-phosphate; E4P, erythrose-4-phosphate; KDPG, 2-keto-3-deoxy-6-phosphogluconate.
Figure 2 shows the change in cell growth rate through adaptive evolution of the SH8 strain.
FIG. 3 shows the results of SDS-PAGE of the expression of Zwf and Gnd proteins simultaneously overexpressed in the SH5ped strain. Soluble proteins in cell lysate were isolated and analyzed. 1: protein marker, 2: comparative strain (0 mM IPTG), 3: induction strain (0.1 mM IPTG)
Figure 4 shows the yields of ethanol and acetic acid production yields obtained through glucose fermentation of SH5pZ and SH5pZG.
Figure 5 shows the results of Metabolic Flux Analysis (MFA) through in silico modeling through analysis of fermentation metabolites of SH5pZ and SH5pZG.

Therefore, the present inventors have recognized that it is impossible to produce hydrogen with high efficiency and high energy yield by a biological method using a one-stage reactor, and as a new concept replacing a two-stage reactor, And ethanol at the same time. In other words, considering that the coarse anaerobic microorganism produces hydrogen and ethanol, acetic acid and the like simultaneously, researching and developing a method of converting the production of acetic acid into ethanol without changing the hydrogen production. In this case, since hydrogen is a gas and ethanol is a liquid, there is no particular difficulty in separation and purification, and it is advantageous to produce ethanol instead of low-energy energy methane, and to use only one anaerobic reactor without using expensive photobioreactors or electrochemical reactors There are advantages. In the present invention, various recombinant E. coli strains with modified glucose metabolism pathway were developed and its performance was confirmed through actual fermentation. Specifically, we have tried various strategies to maximize ethanol production instead of acetic acid production by using strain SH5, a gene-deficient E. coli strain developed in the previous research, as a parent strain. The key to this strategy is to convert the carbon stream from acetyl-CoA to acetic acid to ethanol (Figure 1). The first thing to do for this was to lose the genes of the acetic acid production pathway. But this strategy did not help at all. First, the acetic acid production pathway is an important means of supplying ATP under anaerobic conditions. Second, even if the acetic acid pathway is blocked, it requires additional reducing power such as NADH for ethanol production. Particularly, in order to produce 1 mole of ethanol from acetyl-CoA, 2 moles of NADH is required and thus supply thereof is indispensable. On the other hand, the strategy for maximizing the supply of NAD (P) H is (i) using a pentose phosphate (PP) circuit rather than an EMP pathway, or (ii) using a TCA circuit (TriCarboxylic Acid Cycle). However, it is impossible to use the TCA circuit for the simultaneous production of hydrogen and ethanol, which only works under aerobic conditions and requires enzymes to function in anaerobic conditions. Therefore, it is a practical strategy to activate the PP path through (partial) deactivation of the EMP path and thereby improve the supply of NAD (P) H.

In the present invention, by using the PP pathway as the corresponding pathway of glucose, the present inventor produced sufficient reducing power necessary for ethanol production. It is not easy to activate the PP pathway instead of the EMP pathway, which is the most preferred pathway for most microorganisms. Moreover, research to activate PP pathway in anaerobic conditions has not been attempted worldwide yet. In the present invention, the inactivation strategy of phosphoglucose isomerase (Pgi) enzyme or phosphofructokinase (Pfk) enzyme, which is a key enzyme that drives EMP, is used to prevent EMP pathway. Pgi is the only enzyme that catalyzes the bidirectional reaction from glucose-6-phosphate to fructose-6-phosphate, and the deletion of this enzyme completely blocks the EMP pathway. On the other hand, Pfk is an enzyme that catalyzes the normal reaction of fructose (fructose) -1,6-phosphate to fructose (fructose) -6-phosphate, and PfkA and PfkB are isozyme. Of these, PfkA is known to be the major child element. The overproduction of glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), also known as the rate-limiting reaction of PP pathway, .

The present invention provides Escherichia coli SH5p_Z recombinant strain (KCTC 12762BP) which simultaneously produces hydrogen and ethanol at high efficiency under anaerobic culture conditions.

Specifically, the recombinant strains include hycA , hyaAB , hybBC , ldhA , frdAB, and pgi Escherichia with genetic defects E. coli BW25113 strain ( Escherichia coli ? hycA ? hyaAB ? hybBC ? ldhA ? frdAB ? pgi ; Escherichia E. coli SH5p) can be overexpressed by transfection with glucose-6-phosphate dehydrogenase (Zwf).

In particular, the strain SH5, which is the parent strain used in the present invention, is a gene-deleted E. coli strain developed by the present inventors in a previous study (international journal of hydrogen energy 34 (2009) 7417-7427).

On the other hand, the strains used in the present invention are described in detail in Table 1. Especially, Escherichia coli SH5p strains contain hycA , hyaAB , hybBC , ldhA , frdAB and pgi Escherichia with genetic defects E. coli BW25113 strain ( Escherichia coli ? hycA ? hyaAB ? hybBC ? ldhA? frdAB ? pgi ).

hycA is a regulatory gene of formate-hydrogen lyase (FhlA), hyaAB and hybBC are coding for hydrogenase, and ldhA is coding for lactate dehydrogenase. In addition, frdAB is encoding a fumarate reductase and pgi is encoding a phosphoglucose isomerase.

In addition, the present invention relates to the above Escherichia SH5p_Z coli recombinant strain (KCTC 12762BP) to the 6-phosphate gluconate dehydrogenase; were introduced by the over-expression transformants (6-phosphogluconate dehydrogenase Gnd), Escherichia , which at the same time with high efficiency production of hydrogen and ethanol under anaerobic culture conditions The E. coli SH5p_ZG recombinant strain (KCTC 12763BP) is provided.

In addition, the present invention relates to a method for producing hydrogen and ethanol simultaneously in an anaerobic culture condition with Escherichia coli SH8AE provides a recombinant strain (KCTC 12764BP).

Specifically, the recombinant strains include hycA , hyaAB , hybBC , ldhA , frdAB , pta -ackA and pfkA Escherichia with genetic defects E. coli BW25113 strain ( Escherichia coli ? hycA ? hyaAB ? hybBC ? ldhA ? frdAB ? pta - ackA ? pfkA ; Escherichia The strains may be produced by the adaptive evolution (adaptive evolution) of coli SH8).

Meanwhile, Escherichia The E. coli SH8 strains include hycA , hyaAB , hybBC , ldhA , frdAB , pta -ackA and pfkA Escherichia with genetic defects E. coli BW25113 strain ( Escherichia an ackA △ pfkA) - △ coli h y cA hyaAB △ △ △ hybBC ldhA frdAB △ △ pta. pta - ackA encodes a phosphotransacetylase - acetate kinase, and pfkA encodes a phosphofructokinase.

In detail, the recombinant strain may have improved cell growth rate by using glucose as a carbon source under anaerobic culture conditions through adaptive evolution.

In addition, the present invention relates to the above Escherichia An overexpression of an E. coli SH8AE recombinant strain (KCTC 12764BP) by transfection with glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd) Escherichia coli SH8AE_ZG recombinant strain (KCTC 12765BP), which produces hydrogen and ethanol simultaneously under high culture conditions, is provided.

Also, the present invention provides a method for producing a recombinant microorganism, comprising: culturing the recombinant strain in a medium containing a carbon source; And simultaneously obtaining hydrogen and ethanol from the cultured medium. Preferably, the step of culturing can be carried out in an anaerobic condition, and the carbon source is added to the grape But is not limited thereto.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

One. Experimental strain

(1) Gene cloning

Polymerase chain reaction (PCR) was used to amplify the target gene in large quantities. First, primers required for PCR were designed to contain restriction enzymes at both ends of the target gene. Using this primer Escherichia The target gene was amplified from the E. coli BW25113 chromosome. The amplified PCR product was electrophoresed on agarose gel to separate and purify the PCR fragment. The isolated and purified PCR fragment was digested with each restriction enzyme and ligated to the vector using T4 DNA ligase. The ligation products were transformed using a heat shock method.

(2) gene deletion

In the present invention, the λ red recombinase method and the pKOV method were used to remove the gene from the chromosome.

i) λ red recombinase method

An antibiotic cassette (FRT-kan-FRT) containing a part of the gene to be removed was amplified by PCR and cloned into pKD4 vector. The recombinant pKD4 vector was transformed by electroporation. Colonies resistant to kanamycin were confirmed to have been removed by PCR.

ii) pKOV method

PCR was used to amplify a portion of the gene to be removed and the PCR product was cloned into a pKOV vector. The recombinant pKOV vector was transformed by electroporation and plated on an agar plate containing chloramphenicol for about 16 hours at 42 ° C. to induce integration. PCR was performed to screen for strains that had been integrated, and then plated on an agar plate containing sucrose and incubated at 30 ° C for about 16 hours to allow curing to occur. Curing was detected by PCR.

2. Culture conditions

Luria Bertani (LB) medium was used for the development and maintenance of the recombinant strains. Cell culture for simultaneous production proceeded in three steps. First, a single colony is taken from the plate and cultured in LB medium under aerobic conditions for about 10 hours. The culture was then inoculated into an anaerobic flask containing a modified M9 culture medium containing 5 g / L glucose and 1 g / L yeast extract. The overnight culture was transferred to an anaerobic flask containing M9 culture medium and cell growth and co - production were investigated. Secondary and tertiary anaerobic cultures were initiated by sealing the serum bottle containing the M9 culture medium with rubber caps and aluminum caps, replacing with argon gas, and then inoculating the cultures in which the anaerobic conditions were established and cultured in advance. Inoculation was performed at a cell concentration of 0.05-0.1 OD, and all cultures were carried out at 37 ° C and 200 rpm. The expression of overexpressed genes was initiated by incubation with 0.1-0.2 mM IPTG. Kanamycin (50 / / mL), chloramphenicol (25 / / mL) was used as needed.

3. Analysis method

Cell growth was measured by measuring the absorbance of the cells at 600 nm using a spectrophotometer.

Gas production such as hydrogen and carbon dioxide was analyzed using gas chromatography equipped with a TCD detector. Molecular sieve 5A and Hayesep Q (Alltech Deerfield, IL, USA) were used for gas analysis and oven, injector and detector temperatures were 80, 90 and 120 ° C, respectively. The carbon source and metabolites contained in the culture were analyzed by liquid chromatography (Agilent Technologies, HP, 1160 series) equipped with a refractive index (RI) detector and a photodiode array (PDA) detector. The column used here was Aminex 87H (Bio-Rad).

The SDS-PAGE analysis was performed by centrifugation of the cultured cells, washing with potassium phosphate buffer (50 mM, pH 7.0), resuspension in the same buffer and concentration, and disruption using a French press. The disrupted cell lysate was centrifuged and the supernatant was taken and electrophoresis analysis was performed. Protein bands were stained with Coomassie Brillaint Blue and analyzed using an image analyzer (BIO_RAD Gel Doc 2000).

4. Quantification PCR

For quantitative PCR analysis, cells in the exponential growth phase were harvested, treated with RNA protect reagent (Qiagen, Inc, USA), RNA was extracted, and cDNA synthesis was carried out using this. For cDNA synthesis, SuperScript III first-strand synthesis system (Invitrogen, USA) was used. Real-time quantitative PCR was performed using the Step One Real Time PCR system (Applied Biosystems, USA) using the SYBR green method. The total amount of each reaction sample was 20 uL, and 300 ng of cDNA, 10 uL of 2x Power SYBR Green PCR Master Mix (Applied Biosystems, UK), 5 pmol forward primer, reverse primer, And DEPC-treated water. Quantitative PCR conditions are as follows. Denaturation: 1 cycle, 95 캜, 30 sec; Amplification: 40 cycles, 95 ° C, 15 sec / 62 ° C, 30 sec / 72 ° C, 30 sec. All quantitative PCR analyzes were duplicate.

< Example  1> Development of recombinant Escherichia coli strains for high efficiency simultaneous production of hydrogen and ethanol

Hydrogen and ethanol are produced by fermentation of mixed acid of E. coli using sugar. However, production yields of these biofuels are considerably low when wild-type strains are used as biocatalysts, and a large metabolic pathway is required to improve the yield. As is well known, hydrogen is produced through the oxidation of formic acid in anaerobic fermentation conditions after conversion of pyruvate produced through the pathway to acetyl-CoA and formate. In addition, ethanol is produced from acetyl-CoA through the action of alcohol dehydrogenase (AdhE). However, the conversion of acetyl-CoA to ethanol requires 2 moles of NADH, a reduced form of cofactor, per mole of ethanol produced. On the other hand, NAD ⁺ regenerated by oxidation of NADH is used to convert glucose into pyruvate, which is the driving force for the process. Under normal glucose fermentation conditions, the NAD ⁺ / NADH balance is well maintained when 1 mol of ethanol and 1 mol of acetic acid are produced in 1 mol of glucose. Acetic acid is also produced from acetyl-CoA and does not require reducing power. Conversion to acetic acid is used for the growth of anaerobic cells with ATP production. Therefore, in order to produce ethanol instead of acetic acid in acetyl-CoA, NAD (P) H, which is a reducing power, should be additionally supplied.

In general, Escherichia coli mainly uses the EMP pathway for glucose metabolism. Through the EMP pathway, 1 mol of glucose is converted to 2 mol of pyruvic acid, yielding 2 mol of NADH. Therefore, the maximum amount of ethanol that can be produced from 1 mole of glucose is not more than 1 mole.

Thus, the present invention attempted to activate the PP pathway through partial or complete blockage of the EMP pathway (Fig. 1) in order to provide a sufficient supply of cofactor (NAD (P) H) for ethanol production. The EMP pathway is completely blocked by deletion of the first gene of the EMP pathway pGI and the carbon pathway is induced to the PP pathway and the metabolite of the PP pathway is converted to fructose-6-phosphate or glyceraldehyde- -3-phosphate form, it is possible to maximize the production of reducing power such as pyruvate and NAD (P) H for simultaneous production. Alternatively, deletion of the pfkA gene may be expected to reduce the carbon flow through the EMP pathway and instead increase the carbon flow to the PP pathway, or use some carbon flow to operate the complete PP circuitry, thereby increasing the production of reducing power .

Table 1 shows the strains and plasmids developed and investigated in the present invention based on this strategy. In particular, the strain SH5, which is the parent strain used in the present invention, is a gene-deleted E. coli strain developed by the present inventors in a previous study (international journal of hydrogen energy 34 (2009) 7417-7427).

The strains and plasmids used in the present invention The strains used and
Plasmid Genetic characteristic Strain SH5 BW25113 Δ hyca Δ hya AB Δ hyb BC Δ ldh A Δ frd AB SH6 SH5 Δ pta - acka SH5p SH5 Δ pgi Km resistant SH5p_Z SH5p_pZwf SH5p_ZG SH5p_pZwGn SH5ped SH5p Δ edd Δ eda SH5ped_Z SH5ped_pZwf SH5ped_ZG SH5ped_pZwGn SH8 SH6 Δ PFKA Km resistant SH8AE SH8 with adaptive evolution SH8AE_ZG SH8AE_pZwGn SH8AEed SH8AE Δ edd Δ eda SH8AEed_ZG SH8AEed_pZwGn Plasmid pDK7 Cm resistant pZwf pDK7 carries zwf of Escherichia coli pZwGn pDK7 carries zwf , gnd of Escherichia coli

< Example  2> Improvement of ethanol production through elimination of acetic acid production pathway

Acetyl-CoA produced by PFL action from pyruvic acid as described in Example 1 is converted to acetic acid or ethanol. First, to improve the ethanol yield, the pTA-ackA gene, an acetic acid producing enzyme, was deleted (SH6). The cell growth rate of the SH6 strain was significantly reduced compared to that of the SH5 strain. This seems to be due to a decrease in the yield of ATP produced through the acetic acid production reaction and the accumulation of acetyl-CoA. In this case, acetic acid production was observed to be negligible. Table 2 compares the metabolite production results of SH5 and SH6. Despite the loss of the acetic acid production pathway, the yield of ethanol production remained at about 10%. On the other hand, the yield of hydrogen production decreased more than 50%. The production of pyruvic acid, an intermediate metabolite of the process, was observed to be very high. The secretion of pyruvic acid occurs when acetyl-CoA is not continuously metabolized to ethanol and acetic acid. When carbon metabolism is clogged in pyruvic acid, the production yield of hydrogen and ethanol can not be high. From these results, it can be seen that the simple deletion of the acetic acid production pathway inhibits the production of acetic acid, but does not contribute to the conversion of acetyl-CoA to ethanol. The main reason that acetyl-CoA is not converted to ethanol is the lack of NADH supply, and activation of the PP pathway is essential to overcome this.

Comparison of metabolite yields of SH5, SH6 and SH8AE strains SH5
( Glucose ) SH6
( Glucose ) SH8AE
( Glucose ) SH8AE
( Sorbitol ) Yield  ( mol / mol glucose ) H ₂ 1.42 0.69 1.03 1.25 Ethanol 0.84 0.95 1.04 1.49 Acetate 0.73 0.02 0.04 0.05 Pyruvate 0.02 0.73 0.74 0.24 Formate 0.12 0.19 0.11 0.25 Carbon distribution  (%) Pyruvate 1.07 37.67 39.17 11.31 Ethanol 28.58 31.25 34.64 51.60 Acetate 24.86 2.45 1.47 1.68 Formate 2.09 2.47 1.80 4.22 Biomass 18.39 8.74 8.15 5.96 CO ₂ 24.23 11.50 17.25 19.79 Recovery  (%) 99.22 94.10 101.44 94.56

< Example  3> EMP  Pathway gene ( pgi , pfkA ) Cell growth of deletion strain

Activation of the PP pathway was attempted through partial or complete blockage of the EMP pathway to provide the reductive power needed for ethanol production. This resulted in partial blocking of the EMP pathway through deletion of the pfkA gene and complete blocking of the EMP pathway through deletion of the pgi gene in the SH5 strain, respectively. First, cell growth of these pfkA and pgi deletion strains was investigated under aerobic and anaerobic conditions. The deletion of pfkA is difficult to block the complete EMP pathway due to the presence of pfkB isozymes, but the pfkA gene deletion reduces the carbon flow to the EMP pathway and reduces the carbon flow to the PP or ED pathway, (SH8 strain). When the SH8 strain was cultured under aerobic conditions, the cell growth rate was lowered but growth was possible, and it was hardly grown under anaerobic conditions. This is the result of confirming that pfkB acts as major isozyme even though pfkB isozyme exists.

On the other hand, in order to completely block EMP, deletion of pgi gene was attempted in SH5 in which pta-ackA gene was live (SH5p strain). The deletion strain was able to grow cells using all carbon sources (glucose, gluconate, fructose) used in aerobic conditions. However, in anaerobic conditions, no growth of glucose or glucose consumption was observed at all. Fermentation was attempted using gluconate and fructose as carbon sources (Table 3). Unlike glucose, cell growth using fructose and gluconate was found to be well performed in anaerobic conditions. These results confirm that deletion of the pgi gene does not affect the remainder of the EMP pathway. In particular, the difference in the growth of pgi- and pgi + under the gluconate carbon source is due to the conversion of glucose-6-phosphate into 6-phosphogluconate, which is essential for PP and ED pathways in E. coli It means not smooth.

Comparison of cell growth of SH5p strain Carbon source Condition Relative Cell growth rate Biomass yield
(g cell / g carbon) Glucose + O ₂ +++ 0.37 - O ₂ - - Gluconate + O ₂ +++ 0.15 - O ₂ +++ 0.09 Fructose + O ₂ +++ 0.29 - O ₂ ++ 0.16

< Example  4> pfkA  Through adaptive evolution of defective strains anaerobe  Acquisition of microorganisms and simultaneous production using them

SH8, a pfkA-deficient strain of Example 3, could hardly grow glucose as a carbon source under anaerobic conditions. We have attempted adaptive evolution method to convert this strain into a strain that can grow in anaerobic fermentation condition. For adaptive evolution, M9 culture medium containing 5 g / L glucose and 3 g / L yeast extract was used. The adaptive evolution of SH8 is a method to improve the rate of cell growth by repeated passage of cells in the exponential growth phase in anaerobic conditions (Fig. 2). Repeatedly subculturing is took place in the dilution 1/10 ⁴ ~ 1/10 ^{of 5} range according to changes in the growth rate, repeated passaging experiment for adaptive evolution was carried out for about 40 days. Through adaptive evolution, the growth rate of SH8 strain continuously increased, and the final SH8 strain showed higher cell growth rate than the parent strain SH6 strain in anaerobic condition. The finally obtained adaptive evolution SH8 strain was named SH8AE (KCTC 12764BP).

On the other hand, production of products from glucose was investigated using SH8AE strain obtained through adaptive evolution. Hydrogen production of SH8AE was increased by more than 40% compared to that of SH6, but a considerable amount of pyruvic acid was secreted and there was little change in ethanol yield (Table 2). This means that the supply of NAD (P) H required for ethanol production is still insufficient. It was surprising that the production of ethanol was not increased under the condition where EMP was almost shut off, and pyruvic acid still accumulated in a large amount. Among the various possibilities, it was estimated that the shortage of NADH supply was the highest because the ED path could be activated instead of activating PP pathway with EMP blocked. The ED pathway does not improve NADH production compared to EMP. To confirm the lack of NADH supply, SH8AE fermentation was investigated with sorbitol as a carbon source. Sorbitol is a carbon source that is reduced form of glucose and produces 1 mol of NADH during the pyruvic acid production process. The cell growth rate and the carbon source consumption rate were lower than that of glucose in the case of using sorbitol, but the yields of ethanol and hydrogen production were greatly increased to 1.49 and 1.25 mol / mol, respectively. In addition, the amount of pyruvic acid was significantly reduced from 0.74 mol / mol to 0.24 mol / mol. These results suggest that glucose is metabolized via the ED pathway rather than the PP pathway and that the supply of NADH is not increased despite the blockage of the EMP pathway. The in silico MFA (Metabolic Flux Analysis) result of the SH8AE metabolite analysis also confirmed this fact. The SH8AE strain was reduced by more than 70% of the carbon flow to the EMP pathway by pfkA deletion, but these carbons were found to flow to the ED pathway rather than the PP pathway.

< Example  5> Zwf  Over-expression pgi  Cell growth of the deletion strain

In Example 3, it was confirmed that the carbon flow from glucose-6-phosphate to 6-phosphogluconate was not smooth in the SH5p strain in which the pgi gene was deleted. The enzymes involved in this reaction step are Zwf and Pgl, and Zwf is known as a general rate-limiting step in the PP pathway. Therefore, in order to enable the metabolism of pgi-deficient strains in anaerobic conditions, the conversion of glucose-6-phosphate, the first enzyme in the PP pathway, to 6-phosphogluconolactone Overexpressing the zwf gene catalyzing (SH5p_Z; KCTC 12762BP). Zwf overexpression was achieved at the same time as cell growth from the beginning of fermentation.

Zwf overgrowth strains were able to grow using glucose even under anaerobic conditions. This suggests that the lower enzyme activity of Zwf than Pgl is a factor preventing the anaerobic growth of pgi-deficient strains.

However, the simultaneous productivity of hydrogen and ethanol was not as high as that of the SH5 strain, despite the modification of the process due to the deletion of the EMP pathway (Table 4). It was assumed that the pgi deletion mutant strain, like the pfkA deletion mutant described in Example 4, metabolized most of the carbon to the ED pathway rather than the PP pathway. In silico MFA results based on glucose metabolism analysis of SH5p_Z strain also support this fact (Fig. 5). This means that if the EMP pathway, which is the central metabolic pathway of the process, is blocked, E. coli prefer the ED pathway rather than the PP pathway in anaerobic conditions. It also means that it is very difficult to block the EMP and activate the PP path.

On the other hand, the yield of ethanol production was lower than that of SH5p_Z and the yield of acetic acid production was high in the case of SH5p strain gluconate fermentation. This means that NADPH produced by Zwf reaction has a direct effect on ethanol production. Gluconate is metabolized via PP pathway or ED pathway without going through Zwf and produces less NAD (P) H than glucose because Zwf process is omitted.

Simultaneous productivity comparison of SH5p_Z, SH5, and SH5p strains Strains Carbon source Yield (mol / mol) H ₂ Ethanol Acetate SH5 Glucose 1.67 0.86 0.85 SH5p Gluconate 1.64 0.57 1.29 SH5p_Z Glucose 1.71 0.81 0.83

< Example  6> ED  Path edd , eda  Gene deletion

According to the metabolite production yields and MFA analyzes observed in Examples 4 and 5, the pfkA, pgi deletion strain was dependent on the ED pathway rather than the PP pathway to grow in anaerobic conditions. Therefore, to convert the carbon metabolic flow from the ED pathway to the PP pathway, the two genes edd and eda in the ED pathway were deleted (SH5ped, SH8AEed) in the SH5p and SH8AE strains, respectively.

In the SH5p strain, when the edd and eda genes were additionally deleted (SH5ped_Z), no cell growth was observed in the glucose medium despite overexpression of Zwf. Even when gluconate was used as a carbon source, there was no cell growth. These results confirm once again the fact that the pgi deletion strain metabolizes glucose through the ED pathway in anaerobic conditions. In addition, it did not grow in gluconate, suggesting that another task is needed to activate the PP pathway. This suggests that some enzymes in the non-oxidative PP pathway after the 6-phosphogluconate node where ED and PP pathways are separated may be rate-limiting steps. To confirm this, SH 5ped strain was used to overexpress Gnd (6-phosphogluconate dehydrogense) or TktA (transketolase) in the non-oxidative PP pathway. Growth of each over-expressing strain was examined using gluconate as a carbon source. Gnd overexpressed strain (SH5ped_ZG) was able to grow in anaerobic condition using gluconate. However, the final biomass obtained was considerably lower. On the other hand, TktA overexpression had no effect on cell growth of SH5ped_ZT strain. This suggests that overexpression of Gnd is important for PP pathway activation as Zwf.

However, the mutant overexpression of Zwf and Gnd in the SH5ped strain failed to grow using glucose. First, the overexpression of Zwf and Gnd enzymes in the overexpressed strain was confirmed. After culturing in complex medium or aerobic condition, activity was measured using SDS-PAGE analysis and cell lysate. SDS-PAGE analysis and enzyme activity measurement confirmed that these proteins had no problem in intracellular expression. FIG. 3 shows the result of simultaneous overexpression of these enzymes by SDS-PAGE analysis.

In the SH8AE strain, when the eda and edd genes were deleted (SH8AEed), cell growth was greatly inhibited. In addition, cell growth rate was not improved by Zwf, Gnd overexpression (SH8AEed_ZG).

These results suggest that E. coli does not prefer to use the PP pathway as the only process in anaerobic conditions and that the cells do not grow well using the PP pathway alone. For that reason, the first thing you can think of is the imbalance between NADPH production and consumption. In other words, when the PP pathway is operated with the corresponding pathway, it is likely that an increase in the concentration of NADHP produced by an enzyme such as Zwf or Gnd causes an imbalance between the overall NADPH production and consumption, . To investigate this possibility, Udh, an enzyme that converts NADPH into NADH, was overexpressed in SH5ped_ZG and SH8AEed_ZG strains. However, this case also did not improve cell growth. Theoretical calculations show that if all of the carbon in the glucose is degraded through the PP pathway, the excess NAD (P) H is produced even if only ethanol is produced without acetic acid production. If surplus NAD (P) H is present, it is assumed that the oxidation-reduction imbalance can completely stop carbon metabolism.

Another possibility is that the produced NADPH accumulates intracellularly and degrades the activity of key enzymes in the PP pathway at the enzymatic level, thereby stopping the PP pathway. In order to confirm NADPH inhibition at the enzyme level, the effect of overexpression, separation and purification of Zwf and Gnd enzymes on NADPH concentration was investigated. The activity of NADPH decreased with increasing NADPH concentration and decreased by 50% at 150 uM NADPH concentration in the mixed solution containing Mg ²⁺ ion (1.0 mM) same as that used in the medium.

Based on these results, it was found that using only the PP pathway in the anaerobic process causes serious problems both in the redox imbalance and the inhibition at the enzyme level. Finally, to produce hydrogen and ethanol at the same time, we confirmed that it is essential to bring the process into the PP pathway, if possible, and open the ED or EMP appropriately to prevent redox imbalance and prevent enzyme-level inhibition.

< Example  7> SH8AE  Strain PP  Pathway enzymes ( Zwf , Gnd Overexpression

Activation of the PP pathway was attempted using the SH8AE strain in which the acetic acid producing enzyme was inactivated and the pfkA gene deletion reduced the carbon source flow through the EMP pathway. The activation of the PP pathway was the simultaneous overexpression of Zwf and Gnd irradiated with the rate-limiting enzyme (SH8AE_ZG, KCTC 12765BP). In this case, edd and eda were not eliminated, and it was predicted that all three routes would be used due to overexpression of Zwf and Gnd. Table 5 shows the effect of overexpression of Zwf and Gnd on co-production by metabolite production. In the case of SH8AE, pyruvate (0.74 mol / mol) was produced due to the deletion of acetic acid production pathway. However, production yields of hydrogen and ethanol increased to 1.38 and 1.17 mol / mol respectively due to the overexpression of Zwf and Gnd. This is presumed to be the result of proper activation of the EMP and ED pathways at the same time as PP pathway activation. Not only the yield of simultaneous production was improved, but the secretion of by-product pyruvic acid was reduced by about 50%. These results are not as efficient as those using sorbitol. However, it is possible to activate the PP pathway in anaerobic conditions, which shows that it can be used to produce reduced forms of fuel or chemical such as ethanol.

Effect of overexpression of Zwf and Gnd on the simultaneous production of SH8AE Yield (mol / mol glucose) H ₂ Ethanol Pyruvate SH8AE 1.03 1.04 0.74 SH8AE_ZG 1.38 1.17 0.39

Table 6 shows the results of quantitative PCR comparing SH8AE_ZG gene expression with SH8AE strain. For the mRNA quantification, a house-keeping gene rpoD encoding sigma factor 70 in the RNA polymerase was used as a reference. The transcription level of the deleted pfkA gene was not observed, but the expression of pfkB, minor isozyme, was observed. Overexpression of the Zwf and Gnd genes reduced the expression level of the pgi gene encoding the first enzyme of the EMP pathway by 80% or more. However, there was no significant difference in the expression of gapA gene. Also, edd, the first gene in the ED pathway, and pflB, the enzyme gene that degrades pyruvic acid in anaerobic conditions, also increased. However, the expression level of udhA, a soluble transhydrogenase gene known to convert NADPH and NAD ⁺ into NADP ⁺ and NADH, respectively, did not increase. Interestingly, the expression of the adhE gene involved in ethanol production increased 1.8-fold.

Comparison of gene expression according to Zwf, Gnd overexpression of SH8AE strain Gene SH8AE SH8AEZG pgi 13.7 2.5 pfkA 0.0 0.0 pfkB 125.9 55.1 gapA 68.9 61.2 edd 0.3 0.7 zwf 1.0 5316.6 gnd 8.1 4689.6 tktA 8.7 6.1 pflB 73.6 116.1 fhlA 0.3 0.2 udhA 0.4 0.1 adhE 9.1 16.7

< Example  8> SH5p  Strain PP  Pathway enzymes ( Zwf , Gnd Overexpression

Similar to SH8AE, the activation of the PP pathway was attempted using a SH5p strain with an ED pathway remaining (but with EMP completely blocked). Activation of the PP pathway consisted of simultaneous overexpression of Zwf and Gnd irradiated with rate-limiting enzymes (SH5p_ZG; KCTC 12763BP) in the same manner as SH8AE. Glucose was used as a carbon source and these Zwf and Gnd enzymes were overexpressed with fermentation initiation under anaerobic conditions. Table 7 compares anaerobic fermentation results of SH5p_Z strain and SH5p_ZG strain. The production of ethanol and acetic acid was almost similar for SH5p_Z, which is expected to depend on the ED pathway for most carbon sources. However, the SH5p_ZG strain with additional expression of Gnd increased ethanol production and decreased acetic acid production. Also, the yield of hydrogen production was remarkably increased, and the production of pyruvic acid was remarkably lowered. As seen in SH8AE_ZG, activation of the PP pathway confirms the increased supply of cofactors such as NAD (P) H. It also shows that instead of completely closing the acetic acid production pathway, the carbon stream can be converted from acetic acid to ethanol by increasing the supply of cofactors for ethanol production.

Theoretical yield calculations show that the amount of hydrogen and ethanol that can be obtained using only the PP pathway is 1.67 molecules per glucose molecule, which is considerably larger than the theoretical yield 80-90% is predicted as realistic achievable value. On the other hand, the carbon yield for ethanol and acetic acid or hydrogen production can be increased by reducing the amount of carbon lost (in the Gnd reaction) to CO ₂ in the PP pathway by metabolizing some carbon sources via the ED pathway, (1 g / L) of yeast extract in the case of SH5p_ZG, we concluded that we could achieve the simultaneous production of hydrogen and ethanol which we intended. The results in Table 7 show that these goals are almost achieved.

Simultaneous production of hydrogen and ethanol using Zwf or Zwf and Gnd over-expressed SH5p Yield (mol / mol glucose) H ₂ Ethanol Acetate Pyruvate SH5p_Z 1.71 0.81 0.83 - SH5p_ZG 1.74 1.39 0.29 -

Figure 4 shows the amount of acetate and ethanol production in SH5p_Z and SH5p_ZG strains compared. Gnd expression increased ethanol production and increased IPTG concentration and increased the expression level.

The amount of gene expression by quantitative PCR was also compared (Table 8). The amount of pgkA gene expression was not observed at all and the amount of pfkA expression was increased when PP pathway due to Zwf and Gnd overexpression was activated. This seems to be due to an increase in carbon flow to the EMP intermediate path as the carbon flow increases from the ED path to the PP path (Fig. 1). The expression of gapA was also increased, which is considered to be similar to the increased expression of pfkA. On the other hand, the expression level of edd gene in ED pathway tended to decrease slightly. As with SH8AE, there was no significant change in udhA expression and expression of the adhE gene, an ethanol-producing enzyme, increased more than three-fold.

To support these results, intracellular Metabolic Flux analysis (MFA) was performed through fermentation product analysis (Fig. 5). When Zwf alone is over expressed, most of the carbon is metabolized through the ED pathway. However, when Gnd was additionally expressed, it was predicted to take approximately half of the carbon flow from the 6-phosphogluconate branch into the PP pathway. In addition, by opening the acetate pathway properly, it was possible to inhibit pyruvic acid production while increasing ethanol production. That is, the carbon flow in the pyruvate node can be controlled appropriately.

Comparison of gene expression of SH5p_Z and SH5p_ZG strains Gene SH5p _Z SH5p _ ZG
(0.1 mM IPTG ) SH5p _ ZG
(0.2 mM IPTG ) pgi 0.0 0.0 0.0 pfkA 2.2 3.6 5.5 pfkB 2.6 1.3 1.2 gapA 34.1 43.7 80.9 edd 1.4 1.1 0.9 zwf 2090.9 2656.5 8001.8 gnd 6.0 2090.7 6041.7 tktA 12.2 5.7 7.3 pflB 43.0 21.7 37.0 fhlA 0.4 0.5 0.4 udhA 0.4 0.5 0.3 adhE 4.5 7.2 13.8

Korea Biotechnology Research Institute KCTC12762BP 20150216 Korea Biotechnology Research Institute KCTC12763BP 20150216 Korea Biotechnology Research Institute KCTC12764BP 20150216 Korea Biotechnology Research Institute KCTC12765BP 20150216

Claims (10)

Escherichia which simultaneously produces hydrogen and ethanol under anaerobic culture conditions E. coli SH5p_Z recombinant strain (KCTC 12762BP). According to claim 1, wherein said recombinant strain is hycA, hyaAB, hybBC, ldhA, frdAB pgi gene and a deletion Escherichia E. coli BW25113 strain ( Escherichia coli ? hycA ? hyaAB? hybBC ? ldhA ? frdAB ? pgi ; Escherichia coli SH5p) to glucose-6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase; Escherichia , characterized in that over-expression was introduced to transform the Zwf) E. coli SH5p_Z recombinant strain (KCTC 12762BP). A method of producing Escherichia coli, which produces hydrogen and ethanol simultaneously under anaerobic culture conditions by transfecting the recombinant strain of claim 1 with 6-phosphogluconate dehydrogenase (Gnd) E. coli SH5p_ZG recombinant strain (KCTC 12763BP). delete delete delete delete Culturing the recombinant strain of any one of claims 1 to 3 in a medium containing a carbon source; And
And simultaneously obtaining hydrogen and ethanol from the cultured medium. 9. The method according to claim 8, wherein said culturing is carried out under anaerobic conditions. 9. The method according to claim 8, wherein the carbon source is glucose.

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