Escherichia coli engineering strain for synthesizing L-isoleucine by using ethylene glycol and application of escherichia coli engineering strain

Abstract

The invention provides an engineering strain for synthesizing isoleucine by using ethylene glycol and application of the engineering strain. The engineering strain comprises an expression plasmid vector or an expression plasmid vector combination, the vector or combination comprises a first group of polynucleotides and a second group of polynucleotides, and the first group of polynucleotides comprises at least one polynucleotide encoding an enzyme promoting ethylene glycol utilization; the second set of polynucleotides comprising at least one polynucleotide encoding an L-isoleucine synthesis pathway enzyme; and a backbone plasmid capable of autonomously replicating in a host cell. By introducing the first group of polynucleotides, not only is the glucose utilization rate of the engineering strain improved, but also the yield of the L-isoleucine is improved; meanwhile, by overexpressing or strengthening a plurality of L-isoleucine biosynthetic pathway related genes from different sources and knocking out or weakening L-isoleucine degradation pathway related genes, efficient fermentation production of the L-isoleucine is realized.

Description

[0001] The present application claims priority to Chinese patent application CN202410463997.8, filed on April 17, 2024, the contents of the specification and claims of which are incorporated herein by reference in their entirety and are hereby made part of the present application. Applicant further states that Applicant has the right to amend the specification and claims of the present application based on the priority document. TECHNICAL FIELD

[0002] The present application relates to the technical field of biochemical engineering, and particularly relates to an Escherichia coli engineering bacterium for synthesizing L-isoleucine from ethylene glycol and application thereof. BACKGROUND

[0003] Ethylene glycol has wide industrial application value and can be used to manufacture polyester plastics, such as polyethylene terephthalate. As a common industrial fermentation substrate, glucose has a high overlap between the processes of biomass synthesis and the synthesis of high-value chemicals in the metabolic conversion of microbial cell factories, which limits the efficiency of using glucose to synthesize target products. Ethylene glycol can minimize the interaction between biomass and chemical synthesis pathways, thereby improving substrate utilization. In addition, ethylene glycol is relatively price economical and can be obtained through the electrochemical conversion of CO2, the hydrolysis of plastic waste or the hydrogenolysis of cellulose waste, so it is a potential abundant renewable substrate and can be used as an advantageous substrate for microbial production of high-value chemicals.

[0004] L-isoleucine, as one of the eight essential amino acids and one of the three branched-chain amino acids in the human body, plays an important role in human life metabolism due to its special structure and function. L-isoleucine is widely used in the fields of tissue engineering, biological medicine, cosmetics and food industry due to its special structure and function. In traditional industrial production, L-isoleucine is mainly derived from chemical synthesis and natural extraction, and microbial fermentation method has the advantages of mild reaction conditions, low raw material cost and easy large-scale production. L-isoleucine fermentation method mainly includes direct fermentation method and microbial conversion method. Escherichia coli has become the preferred strain for L-isoleucine production due to its clear genetic background and fast growth rate.

[0005] There are the following problems in the production of L-isoleucine by using Escherichia coli:

[0006] 1) High requirement for product separation and purification technology: Due to the presence of proteins and other metabolites in the cells of Escherichia coli, high-efficiency separation and purification technology is needed to obtain high-purity L-isoleucine. This usually involves a complex process flow and high cost.

[0007] 2) Competition between metabolic pathways: There may be competition between metabolic pathways in E. coli, which limits the synthesis of L-isoleucine. For example, key metabolic intermediates such as pyruvate and alpha-ketobutyrate may be involved in multiple metabolic pathways simultaneously, and need to be optimized through metabolic engineering to increase the yield of L-isoleucine.

[0008] 3) Feedback inhibition phenomenon: In the biosynthetic pathway of L-isoleucine, when the product accumulates to a certain extent, it may inhibit the activity of key enzymes in the pathway, thereby limiting the further synthesis of L-isoleucine. This feedback inhibition phenomenon needs to be removed through metabolic engineering to increase the yield.

[0009] 4) Challenge of strain modification: Although E. coli is easy to genetically manipulate, to obtain an engineered strain with high yield of L-isoleucine, precise editing and regulation of multiple genes are required, which is technically challenging.

[0010] Currently, there is no report on the synthesis of L-isoleucine from ethylene glycol. SUMMARY

[0011] In view of the deficiencies in the prior art, the present application provides an E. coli engineering strain for synthesizing L-isoleucine from ethylene glycol and its application, which improves the yield of L-isoleucine, substrate utilization rate, and reduces the cost.

[0012] In E. coli, ethylene glycol can be converted into glycolate, which is then utilized in the metabolic network of E. coli. It is found that three enzymes are involved in the catalytic reaction of ethylene glycol to glycolate, lactal reductase, lactal dehydrogenase and glycolate oxidase, and the coding genes are fucO, aldA and glcDEF, respectively. Ethylene glycol is converted into glycolic aldehyde, glycolic acid, and finally glycolate. Fe 2+ dependent propylene glycol oxidoreductase FucO is prone to oxidative inactivation, and is sensitive to oxygen, and its mutant FucO I6L ,L7V can improve the oxygen stability of FucO. In addition, the NAD-dependent glycerol dehydrogenase encoded by the gldA gene in E. coli can catalyze the reaction of glycerol to produce dihydroxyacetone, and the GldA protein subunit needs Zn 2+ as a cofactor for catalytic reaction. At the same time, GldA also has broad substrate specificity and can react with ethylene glycol as a substrate to produce glycolic aldehyde. In addition, inactivation of ethylene glycol reductase YqhD and glycolate / hydroxyketopropionate reductase YcdW can improve the conversion rate of ethylene glycol.

[0013] The first aspect of the present application provides an expression plasmid vector or an expression plasmid vector combination, comprising: a first group of polynucleotides comprising at least one polynucleotide encoding an enzyme facilitating ethylene glycol utilization; and a second group of polynucleotides comprising at least one polynucleotide encoding an enzyme of L-isoleucine synthesis pathway.

[0014] and a backbone plasmid which can autonomously replicate in a host cell.

[0015] In the detailed description of the present application, the plasmid vector or vector combination is used to facilitate ethylene glycol utilization and / or enhance L-isoleucine expression amount of a host cell.

[0016] In the detailed description of the present application, the first group of polynucleotides comprises a) a gene encoding glycerol dehydrogenase gldA or a mutant thereof, or lactaldehyde reductase fucO or a mutant thereof; and / or b) a gene encoding lactaldehyde dehydrogenase (aldA) or a mutant thereof.

[0017] In the detailed description of the present application, the glycerol dehydrogenase is a glycerol dehydrogenase derived from Escherichia genus or a propanediol / glycerol bifunctional dehydrogenase, such as AAC43051 derived from Escherichia coli, or a glycerol dehydrogenase derived from Escherichia albertii such as OSL28309.1, or a glycerol dehydrogenase derived from Klebsiella pneumoniae such as ABR79620, or a glycerol dehydrogenase derived from Shigella genus such as Shigella boydii or Shigella dysenteriae (such as ABB68416.1 or ABB63725.1), or a glycerol dehydrogenase derived from Streptococcal pneumoniae such as VTQ29411.1, or a glycerol dehydrogenase derived from Bacillus stearothermophilus such as AAA22477, or a glycerol dehydrogenase (alcohol / choline dehydrogenase) derived from Bacillus subtilis such as CAB15083.2, or a glycerol dehydrogenase derived from Pseudomonas putida such as AAC44426, or a glycerol dehydrogenase derived from Clostridium saccharobutylicum such as AAA83520.1, or a glycerol dehydrogenase derived from Clostridium acetobutylicum such as AAK79593.1, or a glycerol dehydrogenase derived from Salmonella genus such as CNU06578.1, or an alcohol dehydrogenase (glycerol dehydrogenase) derived from Corynebacterium glutamicum such as SJM54452.1, or an alcohol dehydrogenase derived from Gluconobacter oxydans AAW60096.1, or a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 derived from Parasutterella excrementihominis genus, and a natural or artificial mutant derived from the same species enzyme. The glycerol dehydrogenase gldA is derived from Escherichia coli; or derived from Klebsiella pneumoniae.

[0018] Preferably, the glycerol dehydrogenase gldA from Escherichia coli is the protein sequence accession number AAC43051 (ec.gldA); preferably, the glycerol dehydrogenase gldA from Klebsiella pneumoniae is the protein sequence accession number ABR79620 (kp.gldA); or an enzyme that maintains the above enzyme activity and has an amino acid sequence that is at least 96% or 97% or 98%, or at least 99% identical to the amino acid sequence represented by the protein accession number AAC43051 or ABR79620.

[0019] In a specific embodiment of the present application, the lactal reductase fucO is derived from Escherichia coli.

[0020] Preferably, the lactal reductase fucO is the protein sequence accession number AAA23825, or an enzyme that maintains the above enzyme activity and has an amino acid sequence that is at least 96% or 97% or 98%, or at least 99% identical to the amino acid sequence represented by the protein accession number AAA23825.

[0021] In a specific embodiment of the present application, the second group of polynucleotides encodes at least one enzyme of at least one L-isoleucine synthesis pathway and / or transport pathway.

[0022] In a specific embodiment of the present application, the L-isoleucine synthesis pathway comprises the pathway from L-threonine to L-isoleucine in KEGG entry M00570.

[0023] In a specific embodiment of the present application, the L-isoleucine synthesis pathway comprises the pathway from 2-oxobutanoate to L-isoleucine in KEGG entry M00019.

[0024] In an embodiment of the present application, the second group of polynucleotides encodes at least one enzyme, including:

[0025] 1) L-isoleucine synthesis pathway, preferably at least one of threonine deaminase ilvA, acetohydroxy acid synthase ilvI, acetohydroxy acid synthase ilvH, dihydroxy acid dehydratase ilvD, acetohydroxy acid reductoisomerase ilvC, branched-chain amino acid transaminase; and / or

[0026] 2) L-isoleucine transport pathway, preferably branched-chain amino acid efflux protein (Z subunit) ygaZ, branched-chain amino acid efflux protein (H subunit) ygaH.

[0027] In the detailed description of the present application, the threonine deaminase ilvA is a mutant of the enzyme having the protein sequence accession number CAA28577 derived from Escherichia coli, and the mutation sites are L447F and L451A.

[0028] In the detailed description of the present application, the acetohydroxy acid synthase ilvH is a mutant of the enzyme having the protein sequence accession number CAA25756 derived from Escherichia coli, and the mutation sites are G14D and S17F.

[0029] In the detailed description of the present application, the acetohydroxy acid reductoisomerase ilvC is a mutant of the enzyme having the protein sequence accession number AAC76779 derived from Escherichia coli, and the mutation sites are L67E, R68F and K75E.

[0030] In the detailed description of the present application, the polynucleotides in the first and second groups of polynucleotides are homologous or heterologous to the host cell.

[0031] In the detailed description of the present application, the polynucleotides in the first and second groups of polynucleotides comprise codon optimization for the host cell.

[0032] The nucleotide sequences, polynucleotides and DNA molecules used in the present application are not limited to functional regions, and can include at least one of expression suppression regions, coding regions, leader sequences, exons, introns and expression cassettes. In addition, the nucleotide sequences or polynucleotides can include double-stranded DNA or single-stranded DNA (i.e. sense and antisense strands constituting double-stranded DNA) or RNA. The polynucleotides containing a specific polynucleotide sequence can include fragments and / or mutants of the specific polynucleotide sequence. The fragments of the polynucleotides refer to a portion of the polynucleotides, which encode a polypeptide providing substantially the same function as the polypeptide encoded by the complete polynucleotide. Examples of the mutants of the specific polynucleotide sequence include naturally occurring allelic mutants, artificial mutants and polynucleotide sequences obtained by deletion, substitution, addition and / or insertion of one or more nucleotides in the specific polynucleotide sequence. It should be understood that such fragments and / or mutants of the specific polynucleotide sequence encode a polypeptide having substantially the same function as the polypeptide encoded by the original specific polynucleotide sequence.

[0033] In the detailed description of the present application, the polynucleotides in the first to second groups of polynucleotides can be operably linked to a promoter, which is homologous or heterologous to the host cell.

[0034] The second aspect of the present application provides transformants comprising one or more of the above-mentioned expression plasmid vectors or combinations thereof in a host cell.

[0035] In the present application, the transformant is a host cell that has been altered by introducing one or more expression plasmid vectors into the host cell, wherein the one or more expression plasmid vectors are the same or different. In certain embodiments, the transformant is obtained by introducing the expression plasmid vector by transformation in a host cell that exhibits competence for the expression plasmid vector.

[0036] In the detailed description of the present application, the transformant is a mutant host cell, and the expression plasmid vector or the combination of expression plasmid vectors is integrated into the chromosome of the host cell.

[0037] In the detailed description of the present application, the mutant host cell comprises a first set of polynucleotides and a second set of polynucleotides integrated into the chromosome of the host cell.

[0038] In the present application, the first set and the second set of polynucleotides can be integrated into the chromosome of the host cell according to methods such as plasmid transformation, phage-mediated transformation, and genome editing.

[0039] In the detailed description of the present application, the expression of one or both of the following genes in the transformant is inhibited, reduced, or eliminated: glyoxylate / hydroxy pyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD.

[0040] In the further preferred embodiments of the present application, the metabolic byproduct production pathways of succinic acid, lactic acid, ethanol, acetic acid, and formic acid, and the L-isoleucine catabolic pathway are inhibited or blocked in the transformant.

[0041] In the further preferred embodiments of the present application, the expression of one, two, or more of the following genes is inhibited, reduced, or eliminated in the genetically engineered strain: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA.

[0042] The present application employs gene knockout to inhibit, reduce, or eliminate the expression of the corresponding enzymes, and those skilled in the art know that other ways to inhibit enzyme expression or reduce / eliminate enzyme functional activity can also be applicable to the construction of genetically engineered strains, such as promoter knockout or replacement, introduction of enzyme inactivation / deactivation mutations, deletion (or partial deletion) of gene transcription / translation essential functional elements or regions, introduction of mutations or nucleic acid sequences that accelerate the degradation of mRNA that directs enzyme synthesis, introduction of mutations or protein tags that accelerate enzyme degradation / decomposition / inactivation, inhibition of signal activation, RNA interference and gene silencing, CRISPRi, etc.; in the detailed description of the present application, the expression amount of the enzyme is reduced by more than 30% or the activity of the enzyme is reduced by more than 30%.

[0043] In the detailed description of the application, the host strain of the transformant is selected from bacteria or fungi; alternatively, the host strain is selected from wild or genetically engineered Escherichia coli, Bacillus, Corynebacterium, Saccharomyces or Streptomyces; alternatively, the host strain is selected from wild or genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; alternatively, the host strain is selected from wild or genetically engineered Escherichia coli. The E. coli cell can be any E. coli strain derived from E. coli K12 (e.g. MG1655, W3110, DH10b, DH1, BW2952 and strains derived therefrom) or E. coli B or strains derived therefrom.

[0044] The backbone plasmid that can autonomously replicate in the host cell can be any plasmid that can replicate in the host cell. In one embodiment, the expression plasmid vector comprises a backbone plasmid that can replicate in E. coli. Examples of backbone plasmids include, but are not limited to, backbone plasmids that can replicate in E. coli strains, such as pUC (e.g. pUC18 and pUC19 plasmids), pBR322, pSC101, p15a, pACYC, pET and pSC101 plasmids, and plasmids derived therefrom.

[0045] In a third aspect of the application, a composition comprising the above-mentioned transformant or culture thereof is provided.

[0046] In a fourth aspect of the application, a method for preparing L-isoleucine is provided, comprising culturing the above-mentioned transformant in the presence of ethylene glycol to obtain L-isoleucine.

[0047] In one embodiment of the application, the above-mentioned transformant is fermented in a culture medium containing ethylene glycol to obtain L-isoleucine.

[0048] In one embodiment of the application, the ethylene glycol accounts for 0.01%-20% (by weight) of the culture medium.

[0049] In an embodiment of the present application, the culture medium further comprises one or more of glucose, yeast powder, ammonium sulfate, magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, manganese sulfate pentahydrate, betaine, threonine, nicotine acid, VB1, and calcium carbonate.

[0050] In a fifth aspect of the present application, the use of the above-mentioned expression plasmid vector or expression plasmid vector combination, transformant or culture thereof, composition in the preparation of L-isoleucine is provided.

[0051] In a sixth aspect of the present application, a method for increasing the yield of L-isoleucine fermentation is provided, which comprises adding ethylene glycol to the culture medium, preferably the cumulative amount of ethylene glycol added is greater than 10 g / L, and / or introducing the above-mentioned first group of polynucleotides into the fermentation strain.

[0052] In a seventh aspect of the present application, a chassis strain is provided, which is an Escherichia coli strain or a Corynebacterium glutamicum strain, wherein the expression of one or both of the following genes is inhibited, weakened or eliminated: acetaldehyde / hydroxyacetone reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD.

[0053] In a further preferred embodiment of the present application, the metabolic byproduct production pathways of succinic acid, lactic acid, ethanol, acetic acid and formic acid and the L-isoleucine decomposition pathway in the chassis strain are inhibited or blocked.

[0054] In a further preferred embodiment of the present application, the expression of one, two or more of the following genes is inhibited, weakened or eliminated in the chassis strain: phosphotransacetylase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, acetolactate synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA.

[0055] Compared with the prior art, the present application has the following beneficial effects:

[0056] 1) The present application introduces ethylene glycol utilization genes, not only improves the glucose utilization rate, but also improves the yield of L-isoleucine; at the same time, by overexpressing or strengthening multiple L-isoleucine biosynthesis pathway related genes of different sources, knocking out or weakening L-isoleucine degradation pathway related genes, the efficient fermentation production of L-isoleucine is realized.

[0057] 2) Compared with the traditional chemical synthesis method, the present application provides an engineering strain for producing L-isoleucine, which has the advantages of high yield, no toxicity, no pollution, mild conditions, less environmental pollution, etc. Because food safety grade strains are used for fermentation, it has a wide application prospect in food, medicine and other industries. DETAILED DESCRIPTION

[0058] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0059] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0060] Materials and Methods

[0061] LB medium: 10 g / L peptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0062] Fermentation medium: glucose 30 g / L, yeast powder 5 g / L, ammonium sulfate 8 g / L, magnesium sulfate 2 mM, calcium chloride 0.1 mM, ethylene glycol 10 g / L, potassium dihydrogen phosphate 0.8 g / L, manganese sulfate pentahydrate 0.02 g / L, threonine 15 g / L, betaine 1 g / L, nicotinic acid 0.01 g / L, VB1 0.005 g / L, IPTG 0.1 mM, calcium carbonate 10 g / L. The pH of the culture medium was adjusted to 7.3-7.4 with concentrated aqueous ammonia and sterilized at 115°C for 15 min.

[0063] Shake flask fermentation method: Pick a single colony from an activated strain grown on a LB plate (containing an appropriate concentration of antibiotics) at 37°C and inoculate it into LB liquid medium (containing an appropriate concentration of antibiotics). Incubate at 37°C for 12-16 hours at 220 rpm. Inoculate a 1% inoculum of the overnight LB culture into 25 ml of fermentation medium (containing 10 g / L CaCO3 as a pH stabilizer) containing appropriate concentrations of ampicillin and kanamycin. Seal the flask with a breathable membrane and incubate at 30°C, 220 rpm. When the OD600 reaches 1, IPTG (final concentration 0.2 mM) is added to induce plasmid expression. Fermentation is terminated after 30 hours and sampling is performed.

[0064] Determination of L-isoleucine, glucose, and ethylene glycol (EG) concentrations: All standards were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). One mL of fermentation broth was centrifuged at 10,000 rpm for 5 minutes to remove bacterial cells. The resulting filtrate was filtered through a 0.22 μm pore size membrane. The sample filtrate was diluted to an appropriate multiple, and the concentrations of the aforementioned products in the sample were determined using high-performance liquid chromatography (HPLC).

[0065] Glucose and ethylene glycol (EG) determination conditions: high performance liquid chromatograph is Shimadzu Nexera LC-40, and the chromatographic column is Bio-Rad Aminex HPX-87H 300x7.8mm. The column oven is set to 40℃; the detector is RID differential refractive index detector (detector is set to constant temperature 40 degrees). The mobile phase is 5mM sulfuric acid solution, and the flow rate is 0.6ml / min.

[0066] L-isoleucine determination conditions: high performance liquid chromatograph is Shimadzu LC-40D; the chromatographic column is Welch Ultimate AQ-C18, 4.6x250mm; the column oven is set to 40℃; the detector is ultraviolet detector (detector is set to constant temperature 40℃), and the detection wavelength is 338nm; the mobile phase is sodium acetate aqueous solution and methanol, and gradient elution.

[0067] The information of the enzyme involved in the present application is as follows:

[0068] In the context of the present application, the enzyme mentioned includes mutants maintaining enzyme activity, which have an amino acid sequence having at least 98% or at least 99% sequence identity with the amino acid sequence represented by the protein accession number in Table 1.

[0069] Table 1 Enzyme species involved in the present application

[0070]

[0071] The biological material constructed in the present application is as shown in Table 2 below:

[0072] Table 2 Biological material constructed in the present application

[0073]

[0074]

[0075] Example 1 Construction of L-isoleucine synthesis chassis strain

[0076] Using E. coli Keio Knockout Collection library 1(Horizon Discovery, CO, USA) was used to prepare phages. The DNA target gene deletion fragment with kanamycin (Kan) resistance was introduced into the starting strain Escherichia coli BW25113 using the P1 phage transfection method. The fragment was plated on an LB plate containing 50 mg / L kanamycin and cultured overnight at 37°C to obtain a Kan-resistant deletion strain. The plasmid pCP20 was then transformed into the above-mentioned Kan-resistant transformants, plated on an LB plate containing 100 mg / L ampicillin, and cultured at 30°C for 24 hours. The correct transformants in which the kanamycin-resistance gene Kan was removed from the target gene were identified by PCR, thereby obtaining a non-resistant deletion strain. The correct transformants with the Kan gene removed were then streaked as single colonies on LB plates and cultured at 37 or 42°C to ultimately obtain a non-resistant Escherichia coli strain in which the pCP20 plasmid was removed. By repeating this process, the BW25113-based deletion strain Ile00 (ldhAΔptaΔpoxBΔadhEΔpflBΔmgsAΔfrdA) was constructed, in which the synthesis pathways of metabolic byproducts succinate, lactate, ethanol, acetate and formate, as well as the partial decomposition pathway of L-isoleucine were blocked and knocked out to avoid waste of carbon source and reduce the decomposition of L-isoleucine.

[0077] Based on the Ile00 strain, the yqhD and ycdW genes were knocked out in combination to obtain the Ile01 (Ile00ΔyqhD), Ile02 (Ile00ΔycdW) and Ile03 (Ile00ΔyqhDΔycdW) strains.

[0078] Example 2 Construction of recombinant plasmids pZE-ec.gldA, pZE-fucO, pZE-aldA, pZE-fucO-aldA, pZE-ec.gldA-aldA, pZE-kp.gldA-aldA and pZA-ilvAIHDCEygaZH

[0079] The kp.gldA gene was cloned from the Klebsiella pneumoniae genome by PCR; the fucO, ec.gldA, and aldA genes were cloned from the Escherichia coli BW25113 genome and assembled using Gibson Assembly. 2 The DNA fragments obtained above were combined in different ways and ligated into plasmid pZElac 3 After the lac promoter was inserted, the following recombinant plasmids were obtained (for details on the combination, see the Materials and Methods section): pZE-ec.gldA, pZE-aldA, pZE-fucO, pZE-ec.gldA-aldA, pZE-kp.gldA-aldA, and pZE-fucO-aldA.

[0080] ilvDCEygaZH and ilvAIH fragments were amplified from plasmids pZE-ilvDCmEygaZH and pZA-ilvAIH (plasmids derived from Chinese patent publication No. CN114410701A) by PCR, respectively, and the gene fragments were ligated to the pZA lac3 vector PCR fragment by using a recombinant cloning kit in one step to obtain the recombinant plasmid pZA-ilvAIHDCEygaZH (pILE).

[0081] The above-mentioned recombinant plasmid was transformed into the Ile00-03 strain, and the details of the combination of the plasmid and the strain are shown in the Materials and Methods section.

[0082] Example 3 Shake flask fermentation of strains Ile04, Ile05 and Ile06

[0083] With the Ile04 strain as a control, shake flask fermentation was performed, and the fermentation medium components and fermentation conditions are shown in the Materials and Methods section. The fermentation data after 30 hours of fermentation are shown in Table 3, and the results show that the overexpression of the first gene fucO or gldA in the ethylene glycol (EG) metabolic pathway slightly increases the L-isoleucine yield of Ile05 and Ile06 relative to the control strain (Ile04 without overexpression of the EG metabolic pathway-related gene).

[0084] Table 3 L-isoleucine shake flask fermentation results

[0085]

[0086] Example 4 Shake flask fermentation of strains Ile04, Ile07, Ile08 and Ile09

[0087] With the Ile04 strain as a control, shake flask fermentation was performed, and the fermentation medium components and fermentation conditions are shown in the Materials and Methods section. The fermentation data after 30 hours of fermentation are shown in Table 4. The results show that the introduction of aldA based on the overexpression of gldA / fucO significantly increases the consumption of EG; at the same time, compared with the control strain, the strain overexpressing gldA / fucO-aldA significantly increases the L-isoleucine yield, and the strain overexpressing ec.gldA (Ile09) has a significantly higher L-isoleucine yield than the strain overexpressing fucO (Ile08). In addition, the overexpression of aldA gene alone (Ile07) also slightly increases the consumption of ethylene glycol and the production of L-isoleucine.

[0088] Table 4 L-isoleucine shake flask fermentation results

[0089]

[0090] Example 5 Effect of yqhD and / or ycdW gene deletion on L-isoleucine fermentation

[0091] To explore the effect of YqhD and YcdW, the byproduct enzymes of EG utilization pathway, the Ile09 strain was used as control and the fermentation was carried out in shake flask. The fermentation medium composition and fermentation conditions were described in the Materials and Methods section. The fermentation data after 30 hours of fermentation were shown in Table 5. The results showed that the deletion of YqhD and / or YcdW increased the consumption of threonine, and the glycol utilization rate and L-isoleucine production of the double deletion strain were improved compared with the control group. In summary, the deletion of yqhD and ycdW genes improved the ability of the strain to utilize glycol to some extent, and significantly improved the production of L-isoleucine.

[0092] Table 5 L-isoleucine fermentation results in shake flask

[0093]

[0094] Example 6 Effect of gldA from other sources on L-isoleucine fermentation

[0095] The Ile12 strain was used as control, and the strain Ile13 with Klebsiella pneumoniae kp.gldA gene was used to carry out shake flask fermentation. The fermentation medium composition and fermentation conditions were described in the Materials and Methods section. The fermentation data after 30 hours of fermentation were shown in Table 6.

[0096] Table 6 L-isoleucine fermentation results in shake flask

[0097] Strain EG pathway overexpressed genes EG consumption (g / L) L-isoleucine production (g / L) Ile12 ec. gldA aldA 5.87 8.97 Ile13 kp. gldA aldA 6.23 9.32

[0098] REFERENCES

[0099] 1. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2006, 2 (1), 2006.0008.

[0100] 2. Gibson, D. G.; Young, L.; Chuang, R.-Y.; Venter, J. C; Hutchison, C. A.; Smith, H. O., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 2009, 6 (5), 343-345.

[0101] 3. Zhang, K.; Li, H.; Cho, K. M.; Liao, J. C, Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine. Proceedings of the National Academy of Sciences 2010, 107 (14), 6234-6239.

[0102] Finally, it is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments of the application will be apparent to those of ordinary skill in the art upon reviewing the above description. The scope of the application should, therefore, be determined not with reference to the above description, but instead with reference to the appended claims, along with their full scope of equivalents.

Claims

1. An expression plasmid vector or combination of expression plasmid vectors, characterized in that: The vector or combination comprises: a first set of polynucleotides comprising at least one polynucleotide encoding an enzyme that facilitates the utilization of ethylene glycol; and a second set of polynucleotides comprising at least one polynucleotide encoding an enzyme of the L-isoleucine acid synthesis pathway; and a backbone plasmid that is capable of autonomous replication in a host cell.

2. The expression plasmid vector or combination of expression plasmid vectors according to claim 1, characterized in that The first set of polynucleotides comprises a) a gene encoding a glycerol dehydrogenase gldA or a mutant thereof, or a lactaldehyde reductase fucO or a mutant thereof; and / or b) a gene encoding a lactaldehyde dehydrogenase (aldA) or a mutant thereof; Preferably, the glycerol dehydrogenase is a glycerol dehydrogenase or a propanediol / glycerol bifunctional dehydrogenase derived from the genus Escherichia; more preferably, a glycerol dehydrogenase derived from Escherichia coli AAC43051, or OSL28309.1 derived from Escherichia albertii, or a glycerol dehydrogenase ABR79620 derived from Klebsiella pneumoniae, or a glycerol dehydrogenase ABB68416.1 or ABB63725.1 derived from Shigella such as Shigella boydii or Shigella dysenteriae, or a glycerol dehydrogenase VTQ29411.1 derived from Streptococcal pneumoniae, or a glycerol dehydrogenase AAA22477 derived from Bacillus stearothermophilus, or a glycerol dehydrogenase such as CAB15083.2 derived from Bacillus subtilis, or a glycerol dehydrogenase AAC44426 derived from Pseudomonas putida, or a glycerol dehydrogenase AAA83520.1 derived from Clostridium saccharobutylicum, or a glycerol dehydrogenase AAK79593.1 derived from Clostridium acetobutylicum, or a glycerol dehydrogenase CNU06578.1 derived from Salmonella, or an alcohol dehydrogenase SJM54452.1 derived from Corynebacterium glutamicum, or an alcohol dehydrogenase AAW60096.1 derived from Gluconobacter oxydans, or a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 derived from Parasutterella excrementihominis, or a natural or artificial mutant of the same species enzyme; Preferably, the glycerol dehydrogenase is a glycerol dehydrogenase or a propanediol / glycerol bifunctional dehydrogenase derived from the genus Escherichia; more preferably, a glycerol dehydrogenase derived from Escherichia coli AAC43051, or OSL28309.1 derived from Escherichia albertii, or a glycerol dehydrogenase ABR79620 derived from Klebsiella pneumoniae, or a glycerol dehydrogenase ABB68416.1 or ABB63725.1 derived from Shigella such as Shigella boydii or Shigella dysenteriae, or a glycerol dehydrogenase VTQ29411.1 derived from Streptococcal pneumoniae, or a glycerol dehydrogenase AAA22477 derived from Bacillus stearothermophilus, or a glycerol dehydrogenase such as CAB15083.2 derived from Bacillus subtilis, or a glycerol dehydrogenase AAC44426 derived from Pseudomonas putida, or a glycerol dehydrogenase AAA83520.1 derived from Clostridium saccharobutylicum, or a glycerol dehydrogenase AAK79593.1 derived from Clostridium acetobutylicum, or a glycerol dehydrogenase CNU06578.1 derived from Salmonella, or an alcohol dehydrogenase SJM54452.1 derived from Corynebacterium glutamicum, or an alcohol dehydrogenase AAW60096.1 derived from Gluconobacter oxydans, or a propanediol / glycerol bifunctional dehydrogenase MTU07760.1 derived from Parasutterella excrementihominis, or a natural or artificial mutant of the same species enzyme; Preferably, the glycerol dehydrogenase gldA from Escherichia coli is the enzyme with protein sequence accession number AAC43051 (ec.gldA); preferably, the glycerol dehydrogenase gldA from Klebsiella pneumoniae is the enzyme with protein sequence accession number ABR79620 (kp.gldA); or an enzyme that maintains the above-mentioned enzyme activity and has an amino acid sequence that is at least 96% or 97% or 98%, or at least 99% identical to the amino acid sequence represented by protein accession number AAC43051 or ABR79620; Preferably, the lactal reductase fucO is the enzyme with protein sequence accession number AAA23825, or an enzyme that maintains the above-mentioned enzyme activity and has an amino acid sequence that is at least 96% or 97% or 98%, or at least 99% identical to the amino acid sequence represented by protein accession number AAA23825; Preferably, the second set of polynucleotides encodes at least one enzyme of at least one L-isoleucine synthesis pathway and / or transport pathway; Preferably, the L-isoleucine synthesis pathway comprises the pathway from L-threonine to L-isoleucine in KEGG entry M00570; Preferably, the L-isoleucine synthesis pathway comprises the pathway from 2-oxobutanoate to L-isoleucine in KEGG entry M00019; Preferably, the second set of polynucleotides encodes at least one enzyme, including: 1) L-isoleucine synthesis pathway, preferably at least one of threonine deaminase ilvA, acetohydroxy acid synthase ilvI, acetohydroxy acid synthase ilvH, dihydroxy-acid dehydratase ilvD, acetohydroxy acid reductoisomerase ilvC, branched-chain amino acid transaminase; and / or 2) L-isoleucine transport pathway, preferably branched-chain amino acid efflux protein (Z subunit) ygaZ, branched-chain amino acid efflux protein (H subunit) ygaH; Preferably, the threonine deaminase ilvA is a mutant of the enzyme with protein sequence accession number CAA28577 derived from Escherichia coli, with mutation sites L447F and L451A; Preferably, the acetohydroxy acid synthase ilvH is a mutant of the enzyme with protein sequence accession number CAA25756 derived from Escherichia coli, with mutation sites G14D and S17F; Preferably, the acetohydroxy acid reductoisomerase ilvC is a mutant of the enzyme with protein sequence accession number AAC76779 derived from Escherichia coli, with mutation sites L67E, R68F and K75E; Preferably, the polynucleotides in the first and second sets of polynucleotides are homologous or heterologous to the host cell; Preferably, the polynucleotides in the first and second sets of polynucleotides comprise codon optimization for the host cell; Preferably, the polynucleotides in the first to second sets of polynucleotides are operably linked to a promoter, which is homologous or heterologous to the host cell.

3. A transformant characterized in that: The expression plasmid vector or the combination of expression plasmid vectors of claim 1 or 2.

4. A transformant according to claim 3, characterized in that: The transformant is a host cell that has been altered by introducing one or more expression plasmid vectors into the host cell, wherein the one or more expression plasmid vectors are the same or different; Preferably, the transformant is obtained by introducing the expression plasmid vector into a host cell that exhibits competence for the expression plasmid vector by transformation; Preferably, the transformant is a mutant host cell, and the expression plasmid vector or the combination of expression plasmid vectors is integrated into the chromosome of the host cell; More preferably, the mutant host cell comprises a first set of polynucleotides and a second set of polynucleotides integrated into the chromosome of the host cell.

5. A transformant according to claim 3, characterized in that: The expression of one or both of the following genes in the transformant is inhibited, attenuated or eliminated: glyoxylate / hydroxy pyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD; Preferably, the production pathways of metabolic byproducts succinic acid, lactic acid, ethanol, acetic acid and formic acid and the L-isoleucine catabolic pathway in the transformant are inhibited or blocked; Preferably, the expression of one, two or more of the following genes in the transformant is inhibited, attenuated or eliminated: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA. Preferably, the transformant is selected from bacteria or fungi.

6. A composition characterized in that: The culture of the transformant of any one of claims 3-5.

7. A process for the preparation of L-isoleucine, characterized by: The use of the transformant of any one of claims 3-5 in the preparation of L-isoleucine, wherein the transformant is cultured in the presence of ethylene glycol. Preferably, the transformant is fermented in a culture medium containing ethylene glycol to obtain L-isoleucine. Preferably, the weight percentage of ethylene glycol in the culture medium is 0.01%-20%.

8. The use of the expression plasmid vector or the combination of expression plasmid vectors of claim 1 or 2, the transformant of any one of claims 3-5 or the culture thereof, the composition of claim 6 in the preparation of L-isoleucine.

9. A method of increasing the yield of L-isoleucine fermentation, characterized by: The method comprises adding ethylene glycol to the culture medium, preferably, the cumulative amount of ethylene glycol added is greater than 10 g / L, and / or introducing the first set of polynucleotides in claim 1 or 2 into the fermentation strain.

10. A chassis strain characterized in that: The chassis strain is an Escherichia coli strain or a Corynebacterium glutamicum strain, and the expression of one or both of the following genes in the chassis strain is inhibited, attenuated or eliminated: glyoxylate / hydroxy pyruvate reductase ycdW or / and NADPH-dependent aldehyde reductase yqhD; Preferably, the production pathway of metabolic byproduct acetic acid and the L-isoleucine catabolic pathway in the chassis strain are inhibited or blocked; Preferably, the expression of one, two or more of the following genes in the chassis strain is inhibited, attenuated or eliminated: phosphate acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, methylglyoxal synthase mgsA, D-lactate dehydrogenase ldhA, fumarate reductase flavoprotein subunit frdA.

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