WO2019045416A2 - 에탄올 비생산성 아세토젠 균주를 에탄올 생성균주로 전환하는 방법 및 상기 에탄올 생성균주로부터 일산화탄소를 이용한 에탄올의 제조방법 - Google Patents
에탄올 비생산성 아세토젠 균주를 에탄올 생성균주로 전환하는 방법 및 상기 에탄올 생성균주로부터 일산화탄소를 이용한 에탄올의 제조방법 Download PDFInfo
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Definitions
- the present invention relates to a transformant strain having an ethanol production ability prepared by introducing a gene for producing ethanol into Eubacterium limosum , which is an ethanol-free acetogen , and a method for producing ethanol from the strain using carbon monoxide .
- Waste gas is a mixture gas composed of carbon monoxide (CO), carbon dioxide (CO 2 ), and hydrogen (H 2 ) obtained through gasification processes of various carbon-based raw materials such as waste, coal, coke, low hydrocarbon gas, naphtha, Is referred to as syngas or waste gas.
- the group of microorganisms that produce acetic acid by anaerobic metabolism using synthetic gas or sugar as a carbon and energy source is called 'Acetogen'.
- Acetogen uses the waste gas as carbon and energy source, (HL Drake et al., Annals of the New York Academy of Sciences, 1125: 100, 2008).
- organic acids such as butyric acid and bioalcohols such as ethanol and butanol.
- the present inventors have made intensive efforts to develop an acetogenic strain capable of producing ethanol from a syngas containing carbon monoxide. As a result, they have found that a high expression of an acetogen, Eubacterium limosum KCTC13263BP, which does not produce ethanol, The present inventors have completed the present invention by confirming that the transformant strain produces ethanol through a pathway specific to a carbon monoxide substrate by preparing a transformant strain into which an aldehyde alcohol dehydrogenase gene is externally introduced so that the expression can be regulated by the promoter.
- It is another object of the present invention is to provide an oil cake Te Solarium remote island (Eubacterium limosum) oil cake Te Solarium remote island (Eubacterium limosum) vector for expression strain containing the constitutive high expression promoter derived.
- the present invention provides a method for producing an Eubacterium limosum having an ethanol- producing ability and a gene encoding a bifunctional aldehyde alcohol dehydrogenase is introduced into an Eubacterium limosum having no ethanol- Lt; / RTI >
- the present invention also provides a method for producing ethanol, comprising: (a) culturing the transformed Eubacterium limosum strain in the presence of a carbon monoxide-containing gas to produce ethanol; And (b) obtaining the resultant ethanol.
- the present invention also provides a promoter derived from Eubacterium limosum having the nucleotide sequence shown in SEQ ID NO: 2 and a vector for expressing Eubacterium limosum strain shown in SEQ ID NO: 1.
- Figure 1 shows the process of preparing a shuttle vector embryo for the smaller size Eubacterium limosum KCTC13263BP strain by recombining the major region sequence of the pJIR418 vector.
- FIG. 2 is a histogram of a total of 3,611 total transcripts of the genes in the genome of the strain, based on the transcript analysis results obtained during the logarithmic growth period under the three culturing conditions of the strain Eubacterium limosum KCTC13263BP , And genes with high / intermediate / low transcript expression values, respectively.
- FIG. 3 shows the expression levels of ⁇ -glucuronidase (GUS) expression after H1, H2, M1 and L1, respectively, at the upper end of the gene having high (H) / intermediate (M) / low
- GUS ⁇ -glucuronidase
- FIG. 4 is a graph showing the results obtained by introducing a vector in which four kinds of autonomous promoters (H1, H2, M1, L1) are located in front of the promoter of GUS expression gene into Eubacterium limosum KCTC13263BP strain, The GUS activity was measured with the wild-type strain.
- A is a graph showing the absorbance change of each sample at 405 nm
- B is a graph showing the activity of GUS of the four kinds of transformant and wild-type strain-derived samples.
- FIG. 5 shows an example of a recombinant vector (pECPH2) inserted with a gene fragment in which H2 promoter is arranged at the upper end of the expression gene of two kinds of bifunctional aldehyde alcohol dihydrogenase (AdhE1, AdhE2), using the succinic vector pELM for Eubacterium limosum KCTC13263BP as a backbone :: AdhE1 or pECPH2 :: AdhE2).
- AdhE1, AdhE2 bifunctional aldehyde alcohol dihydrogenase
- FIG. 6 shows that the transformants transformed with the recombinant vectors (pECPH2 :: AdhE1 and pECPH2 :: AdhE2) were transformed into the transformants of the present invention, and A was transformed with the AdhE1 and AdhE2 insertion genes B was obtained by introducing the vector extracted from each transformant into the E. coli strain and then treating the reextracted vector with the restriction enzyme , Showing the result of checking the size of the vector fragment, indicating that the transformant of Eubacterium limosum KCTC13263BP strain into which each recombinant vector was introduced was successfully obtained.
- FIG. 7 shows the results of analysis of growth and metabolite production under glucose substrate culture conditions with the wild type Eubacterium limosum KCTC13263BP strain and the above transformant expressing AdhE1 and AdhE2, respectively, and A shows the growth and metabolism of the wild type strain B and C show the results of growth and metabolite production analysis for the transformants expressing AdhE1 and AdhE2, respectively.
- FIG. 8 shows the results of analysis of growth and metabolite production under the condition of carbon monoxide substrate culture with the transformant strain expressing the wild type Eubacterium limosum KCTC13263BP strain and AdhE1 and AdhE2, respectively, wherein A is the growth and metabolism product of the wild type strain And B and C are the results of growth and metabolite production analysis for the transformant strain expressing AdhE1 and AdhE2, respectively.
- A is AdhE1 enzyme Or an acetyl-CoA under the catalysis of an AdhE2 enzyme directly through acetaldehyde
- B represents a pathway for producing ethanol by the aldehyde ferredoxin oxidoreductase (AOR; ELI_0332, ELI_1752, ELI_3389), followed by acetic acid reuse ethanol production pathway where acetaldehyde is converted to ethanol by the AdhE1 or AdhE2 enzyme.
- metabolic engineering for producing high-value-added ethanol from carbon monoxide using ethanol-non- producing acetobenzene ( Eubacterium limosum ) KCTC13263BP was carried out.
- Acetic acid was converted into acetaldehyde Production of Ethanol Applied to Metabolic Engineering Based on the Existence of Aldehyde Ferredoxin Oxidoreductase (AOR) Expression Gene Expressing Conversion Catalyzed Acid Aldehyde Ferredoxin Oxidoreductase (AOR) It was confirmed that ethanol was produced in an energetically favorable direction by reusing acetic acid produced by AOR while maintaining the production route.
- the ethanol production pathway through the reuse of acetic acid produces ethanol by reusing the reduced form of ferredoxin (Fd2-), which is produced in the state of preserving ATP obtained through substrate-level phosphorylation in the initial acetic acid production, Compared to the metabolic pathways that make ethanol through acetaldehyde directly from Acetyl-CoA, ATP can be conserved to minimize energy loss.
- Fd2- ferredoxin
- ATP can be conserved to minimize energy loss.
- the E. limosum strain used as an acetogen is a strain producing a useful organic acid such as acetic acid and butyric acid while being highly resistant to carbon monoxide and actively growing using carbon monoxide as a unique carbon source (Chang IS et al , J. Microbiol . Biotechnol., 8: 134, 1998, J Inoue Sup Kor J. Appl. Microbiol. Bitoechnol., 25: 1, 1997).
- ELI_0332 ELI_1752 (SEQ ID NO: 1)
- AOR aldehyde ferredoxin oxidoreductase
- ELI_1752 shows very high homology with the major conserved bases and motifs on the amino acid sequence of AOR of the Pyrococcus furiosus strain that was first discovered as a catalyst of AOR. Therefore, The AOR of the strain will also play a similar role to the previously known AOR (Kletzin A et al., J Bacteriol., 177 (16): 4817-9, 1995).
- an external gene introduction and expression system suitable for the E. coli strain E. lososum is constructed, and a gene encoding a bifunctional aldehyde alcohol dehydrogenase is introduced into the wild-type strain, Was prepared.
- the present invention relates to a transformed E. limosum strain having an ethanol producing ability in one aspect, the aldehyde-functional gene coding for alcohol dehydrogenase transferred to E. limosum strain that does not have an ethanol producing ability is introduced.
- the bifunctional aldehyde alcohol dehydrogenase was selected from AdhE1 and AdhE2 derived from Clostridium autoethanogenum. These were selected from the group consisting of C. ljungdahlii and C. carboxidivorans, and AdhE1 and AdhE2, It has homology.
- GUS GUS expression gene
- the gene encoding the bifunctional aldehyde alcohol dehydrogenase may be transcribed by a promoter represented by the nucleotide sequence of SEQ ID NO: 2.
- Eubacterium limosum may be characterized by being a strain of Eubacterium limosum KCTC13263BP.
- the introduction of the foreign gene into the E. limosum strain can be characterized in that it is introduced using the shuttle vector pELM represented by the nucleotide sequence of SEQ ID NO: 1.
- the present invention provides a method for producing ethanol, comprising: (a) culturing the transformed E. limosum strain in the presence of a carbon monoxide-containing gas to produce ethanol; And (b) obtaining the resulting ethanol.
- the transformed E. limosum strain of the present invention can be used not only for the direct conversion of acetic acid, the main metabolite of the wild type strain, to ethanol, under autotrophic CO substrate conditions, but also for the production of butanol without producing another metabolite, It was confirmed that the product can be produced as a single product.
- Transformed E. limosum strain of the present invention consumed 11.5 mmols (milli moles) of carbon monoxide under the autotrophic substrate condition, not the heterotrophic substrate condition, without the manipulation of the separate genomic DNA, so that a significant concentration of ethanol Which is the only one produced.
- the transformed strains containing AdhE1 obtained through the above-described method of the present invention are applied to the synthesis gas process in the future, it is possible to omit or simplify the product separation process in the down-stream of the synthesis gas process, As well as the role of
- the present invention proposes an optimum ethanol production pathway in terms of energy acquisition efficiency under a carbon monoxide substrate condition, and in fact, it is industrially very effective that the transformant strain produces only ethanol as a single product without any other competitive metabolites, And confirmed the valuable characteristics.
- a shuttle vector suitable to introduce foreign genes of E. limosum strain when the existing introduced in E. limosum strain, identified as being stably replicated pJIR418 vector (J Sloan et al., Elsevier, 27: 3, 1992) using the antibiotic resistance cassette, only the major gene parts in pJIR412 vector recombination as Gram-negative origin of replication, a Gram-positive origin of replication and the size is represented by the base sequence of SEQ ID NO: 1
- a shuttle vector pELM for the reduced E. limosum strain was constructed.
- the external gene expression vector may include a high expression promoter for the strain represented by the nucleotide sequence of SEQ ID NO: 2 and a vector backbone represented by the nucleotide sequence of SEQ ID NO: 1.
- Electroporation was used as a transformation method for introducing a foreign gene into E. limosum strain KCTC13263BP (hereinafter referred to as "Elm strain"), and the method used by Ching Leang et al was partially modified ( Appl Environ Microbiol., 79: 1102, 2013).
- Electro-competent cells of the Elm strain were prepared by the following procedure for transformation by electroporation.
- the cells were inoculated into 500 ml HBBM-Glc medium of the same composition containing 20 mM DL-threonine,
- the cultured Elm strain was washed twice with SMP buffer (270 mM sucrose, 1 mM MgCl 2, 7 mM sodium phosphate, 3.17 mM L-cysteine hydrochloride, pH 7.4) and resuspended in 5 ml of final SMP buffer. It was prepared by concentrating more than 100 times.
- the pJIR418 vector (Sloan J et al., Elsevier, 27: 3, 1992 ) was used to recombine only the major gene part of the pJIR412 vector, such as the antibiotic resistance cassette, the gram-negative cloning start point, and the gram positive cloning start point, to generate the reduced-size shuttle vector pELM (SEQ ID NO: 1) for the Elm strain (Fig. 1).
- Example 2 Eubacterium limosum KCTC13263BP strain self-promoter screening for high expression of ethanol producing enzyme
- a constant promoter of a gene showing a high expression rate constantly in the Elm strain was screened to select a constant high promoter.
- transcript analysis of each substrate was performed to determine the expression level of 4,579 genes in the genome of Elm strain.
- Elm strains were cultured under four different substrate conditions (glucose, CO, CO / CO 2 and H 2 / CO 2 ) and were identified by mid-log phase and early- As a result, transcript analysis was performed by sampling the culture with 3 replicates in each of 8 environmental conditions. Finally, in each of these 8 conditions, the RPM of each of the 4,579 genes annotated in the Elm strain Million mapped reads values were obtained. The RPKM mean value of each gene was obtained under eight environmental conditions, and histograms were obtained to obtain a normal distribution graph (FIG. 2).
- the standard deviation of the RPKM value for each substrate and growth condition among the genes within the upper 3% with high average RPKM value is low, and the upstream 100 bp sequence at the start codon of the gene does not overlap with the ORF of the preceding gene (ELI_4394, ELI_3815) was selected as a candidate gene having a strong endogenous promoter, and the promoter predicted region of the upstream of the ORF of the two genes was determined as a promoter region of the promoter H1 (SEQ ID NO: 3), and promoter H2 (SEQ ID NO: 2).
- ELI_4394 and ELI_3815 As a control group for the above two genes (ELI_4394 and ELI_3815) having a high average expression value, a gene having an intermediate expression value (ELI_0016) and a gene having a low expression value (ELI_1842) were also selected as above, Promoter predicted intervals were designated as promoter M1 (SEQ ID NO: 4) and promoter L1 (SEQ ID NO: 5), respectively.
- Promoter M1 (SEQ ID NO: 4)
- Promoter L1 (SEQ ID NO: 5)
- Example 3 Identification of activity of Eubacterium limosum KCTC13263BP strain-derived promoter
- Example 2 In order to confirm the intensity of the four promoters selected in Example 2, a reporter gene assay was performed.
- As a reporter gene ⁇ -glucuronidase (GUS) expression gene of E. coli strain BL21 was selected.
- the shuttle vector for the Elm strain prepared in Example 1 was used as a pELM backbone and the four types of promoter candidate groups (H1, H2, M1, L1) selected in Example 2 were inserted into the GUS expression gene Gene fragments were constructed and inserted into pELM vectors, respectively (Fig. 3).
- GUS activity was assessed by fluorometric method using 4-NPG (4-Nitrophenyl ⁇ -D-glucuronide) as a substrate using the four confirmed Elm strain transformants and a wild-type Elm strain as a control. Cells were harvested from the transformants and wild-type Elm strains of each strain to obtain a crude enzyme. The GUS assay was performed using the final 2 mM 4-NPG as a substrate (Fig. 4A) The GUS sample expressed by the promoter exhibited the highest activity (20.35 mU / mg) (Fig. 4B).
- the GUS sample expressed by the H1 promoter showed 5.64 mU / mg activity and the wild type strain extract
- the GUS samples expressed by the remaining M1 or L1 promoters all exhibited very low activity of less than 1 mU / mg.
- the H2 promoter among the promoter candidate groups H1 and H2, which had a high expression value on the transcript analysis result of Example 2 was finally selected as a promoter of a high expression gene for Elm strain.
- Example 4 Bifunctional alcohols with non-alcoholic productivity Eubacterium limosum KCTC13263BP Production of ethanol through introduction of aldehyde alcohol dihydrogenase (AdhE)
- a gene expressing a bifunctional aldehyde alcohol dehydrogenase (AdhE1, AdhE2) gene from the acetone strain C. autoethanogenum DSM10061 CAETHG_3747, CAETHG_3748) was selected as a target gene for introduction into the strain.
- AdhE1 and AdhE2 genes were synthesized as an insertion gene for cloning by linking with Elm gene high expression promoter H2.
- the insert gene fragment to which the H2 promoter and the AdhE1 gene (SEQ ID NO: 6) and the AdhE2 gene (SEQ ID NO: 7) are linked is shown in Table 2 as a backbone for the Elm strain shuttle vector (pELM), and two kinds of recombinant vector pECPH2 :: AdhE1 8) and pECPH2 :: AdhE2 (SEQ ID NO: 9) (FIG. 5).
- pECPH2 :: AdhE1 and pECPH2 :: AdhE2 were introduced into the Elm strain through the electro-invasion method, and then Elm strain transformants containing the respective recombinant vectors were selected using the same sorting method as in Example 3, and the AdhE1 gene And the presence of the AdhE2 gene were also genetically confirmed (Fig. 6).
- FIG. 6A each of the AdhE1 and AdhE2-containing transformants was subjected to colony PCR using the other transformants containing the GUS expression gene as a control, PCR products of the desired size could be obtained only from the transformant template.
- plasmid DNA was extracted from each of the AdhE1 and AdhE2-containing transformant cultures, and then each of the extracted vectors was back-transformed into E. coli strains, and plasmid DNA was re-extracted and extracted from each E. coli transformant culture (Fig. 6B), with the results of Fig. 6A, by applying confirmation methods such as specific restriction enzyme treatment to the vector.
- the identified transformants were cultivated for analysis of growth characteristics and production of metabolites including ethanol in each HBBM-Glc medium supplemented with erythromycin and HBBM-CO (CO medium) medium for analysis.
- the wild-type Elm strain as a control group was cultured in HBBM-Glc medium and HBBM-CO medium not containing erythromycin.
- each transformant (Fig. 7B and Fig. 7C) containing AdhEl and AdhE2 produced ethanol in contrast to the wild-type strain (Fig. 7A), and the transformants containing AdhEl Producing a high concentration of ethanol.
- Transformants containing AdhE1 consumed glucose at a concentration of 21.2 mM and produced ethanol at a concentration of up to 10.5 mM.
- the strain growth rate and maximum growth (ODmax) showed no significant difference between the wild type strain and the transformant strain, but the production of metabolites such as ethanol and butyric acid except for acetic acid showed a great difference.
- Acetic acid production patterns and maximum yields showed no significant differences between wild type strains and transformants.
- Butyric acid a 4-carbon metabolite of the strain, was scarcely produced in AdhE1-containing transformants.
- AdhE2-containing transformants a small amount of butyric acid was produced at a concentration of 1.1 mM as compared with 4.5 mM in the wild-type strain.
- FIG. 8A The autotrophic growth of transformants and wild type strains containing AdhE1 and AdhE2, respectively, under CO substrate conditions is shown in FIG.
- the production of ethanol was not detected in the wild type strain as in the case of the glucose substrate condition (Fig. 8A), and ethanol was produced only in the transformant strain (Fig. 8B, Fig. 8C).
- both of the transformants containing AdhE1 and AdhE2 consumed a total of 11.5 mmols of CO to produce ethanol at a concentration of about 28 mM.
- the transformant strain containing AdhE1 produced the highest ethanol production was faster.
- the transforming strains clearly demonstrated the ability to produce and consume acetic acid, the major metabolite of the strain, in the CO substrate conditions.
- acetic acid was produced at a maximum concentration of 1.8 mM only at the early stage of growth, and the acetic acid produced was gradually consumed again and production was not detected at the background level after the stationary phase.
- AdhE2-containing transformants also showed acetic acid production and re-consumption, similar to those found in the previous AdhE1-containing transformants. However, acetic acid was slower to be consumed again than the AdhE1-containing transformants, It was not completely consumed. Both transformants did not produce any butyric acid during the growth. In addition, there was no significant difference in growth rate or maximum growth (ODmax) between the wild type strain and the transformant strain as in the case of the preceding glucose substrate condition.
- the Elm strain produces both L-lactate and D-lactate, -form produced total lactate at a maximum of 13 mM under a 20 mM glucose substrate condition at a production rate of about 1: 1.5. Furthermore, lactate was not produced under autotrophic CO substrate conditions and was only produced under heterotrophic glucose substrate conditions. According to the genetic information in the strain, the lactate production pathway is produced from pyruvate under the catalyst of lactate dehydrogenase (LDH; ELI_3346, ELI_4443), where NADH serves as an electron donor .
- LDH lactate dehydrogenase
- lactate is produced for effective reductive power consumption because of NAD + / NADH balance and the like.
- the autotrophic CO gas metabolic pathway is a consumptive reaction of NADH and ATP, it can be interpreted that lactate is not produced unlike heterotrophic substrate conditions.
- Example 5 Ethanol production using CO substrate Ethanol production pathway and bioenergetics model in Eubacterium limosum KCTC13263BP transformant strain
- the CO substrate-based ethanol production using the Elm transformant strain prepared in Example 4 was carried out in the same manner as in Example 4 except that the pathway for producing ethanol via acetaldehyde immediately from acetyl-CoA (FIG. 9A) and the acetate (FIG. 9B), which reuses acetaldehyde and produces ethanol.
- the path from CO to the synthesis of acetyl-CoA and the route to produce ethanol from acetaldehyde are the same, but the routes for the synthesis of acetaldehyde from acetyl-CoA are different.
- NADH of the same molecule is required for each molecule of acetyl-CoA in the pathway for synthesizing acetaldehyde from acetyl-CoA, and acetaldehyde is reused after acetyl-CoA is preferentially produced from acetal-
- the pathway for the synthesis requires a reduced form of ferredoxin (Fd2-) of the same molecule per molecule of acetyl-CoA and at the same time ATP of the same molecule is produced.
- the Elm transformant strain containing the AdhE1 expression gene under autotrophic CO substrate conditions, not only converts acetic acid, which is the main metabolite of the wild-type strain, directly to ethanol, It was confirmed that ethanol alone can be produced without production of the product butyric acid.
- the transfected strain containing AdhE1 produced ethanol at a significant concentration of about 28 mM under the condition of heterotrophic substrate and no manipulation of genomic DNA under autotrophic substrate conditions.
- the transformed strains containing AdhE1 obtained through the above-described method of the present invention are applied to the synthesis gas process in the future, it is possible to omit or simplify the product separation process in the down-stream of the synthesis gas process, As well as the role of
- high value-added ethanol can be produced from carbon monoxide contained in the waste gas using Eubacterium limosum , which is an acetogen that has no ethanol-producing ability.
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Abstract
Description
Claims (8)
- 에탄올 생성능을 가지고 있지 않은 유박테리움 리모섬(Eubacterium limosum)에 이관능성 알데히드 알코올 디하이드로게나아제를 코딩하는 유전자가 도입되어 있는 에탄올 생성능을 가지는 형질전환 유박테리움 리모섬(Eubacterium limosum) 균주.
- 제1항에 있어서, 이관능성 알데히드 알코올 디하이드로게나아제를 코딩하는 유전자는 클로스트리듐 오토아세타노게눔 유래의 AdhE1 또는 AdhE2인 것을 특징으로 하는 형질전환 유박테리움 리모섬(Eubacterium limosum) 균주.
- 제1항에 있어서, 상기 이관능성 알데히드 알코올 디하이드로게나아제를 코딩하는 유전자는 서열번호 2의 염기서열로 표시되는 프로모터에 의해서 전사조절되는 것을 특징으로 하는 형질전환 유박테리움 리모섬(Eubacterium limosum) 균주.
- 제1항에 있어서, 상기 Eubacterium limosum는 Eubacterium limosum KCTC13263BP 균주인 것을 특징으로 하는 형질전환 유박테리움 리모섬(Eubacterium limosum) 균주.
- 제1항에 있어서, 상기 유전자의 도입은 서열번호 1의 염기서열로 표시되는 셔틀벡터 pELM을 이용하여 도입되는 것을 특징으로 하는 형질전환 유박테리움 리모섬(Eubacterium limosum) 균주.
- 다음 단계를 포함하는 에탄올의 제조방법:(a) 일산화탄소 함유 가스 존재 하에서 제1항 내지 제5항 중 어느 한 항의 형질전환 유박테리움 리모섬(Eubacterium limosum) 균주를 배양하여 에탄올을 생성시키는 단계; 및(b) 상기 생성된 에탄올을 수득하는 단계.
- 서열번호 2 또는 서열번호 3으로 표시되는 염기서열을 가지는 유박테리움 리모섬(Eubacterium limosum) 유래 항시 고발현 프로모터.
- 제7항에 있어서, 서열번호 2 또는 서열번호 3의 염기서열로 표시되는 프로모터와 서열번호 1의 염기서열로 표시되는 셔틀벡터 백본을 함유하는 유박테리움 리모섬(Eubacterium limosum) 균주 발현용 벡터.
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