Method for improving l-isoleucine yield of escherichia coli based on methyl malonic acid efflux and engineering strain

Abstract

This invention relates to the field of agricultural biotechnology, specifically to a method for increasing the synthesis of L-isoleucine based on methylmalate efflux. This invention determines that knocking out the gene encoding the carboxylic acid transporter TtdT effectively reduces the efflux of methylmalate by *E. coli*, allowing more methylmalate to remain intracellularly, thereby increasing the intracellular conversion rate of methylmalate and ultimately improving the efficiency of L-isoleucine synthesis via the methylmalate pathway.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for increasing the production of L-isoleucine by Escherichia coli based on methylmalic acid efflux. Background Technology

[0002] Isoleucine is one of the three essential branched-chain amino acids, and microbial fermentation is currently the primary method for large-scale production of L-isoleucine. Besides the threonine pathway, microorganisms have evolved several alternative biosynthetic pathways for L-isoleucine. Among these, the methylmalate pathway (also known as the pyruvate pathway) is the most common. Compared to the threonine pathway, the methylmalate pathway is considered a more efficient biosynthetic pathway for L-isoleucine due to its simpler synthetic route and simpler feedback regulation mechanism.

[0003] Wild-type *E. coli* strains do not contain L-isoleucine produced via the methylmalate pathway. Through artificial modification, the highest L-isoleucine yield achieved by engineered strains of *E. coli* metabolized via the methylmalate pathway in current research is 56.6 g / L. However, a large accumulation of methylmalate occurs in the fermentation broth of this engineered strain, although this can be mitigated by expressing a methylmalate uptake protein gene. dcuD The strain's ability to utilize methylmalic acid was improved, but the utilization rate was still low, limiting the strain's production capacity of L-isoleucine.

[0004] Carboxylic acid transporters in *E. coli* are divided into several families, including DcuAB, DctA, and CitT. Their selectivity for transport substrates and the direction of transport (absorption or efflux) vary under different physiological conditions. For example, the transporter DcuA can exhibit uptake, exchange (or antiport), and efflux functions depending on the substrate and physiological conditions. For instance, Janausch et al. found that... DcuA While possessing succinate (Succ) uptake capabilities, Debora Trichez et al. discovered that DcuA is an important malate efflux protein in *E. coli*. Current techniques have also revealed that DcuA has the function of independently uptaken aspartate (L-Asp), and can exchange aspartate with fumarate (Fum), or aspartate with succinate (Succ). This indicates that the selectivity of such transport proteins for transport substrates and the direction of transport may vary under different physiological conditions.

[0005] CN 118853722 B discloses a method and engineered strain for increasing the yield of L-isoleucine synthesis based on the methylmalate pathway, and identifies the carboxylic acid transport proteins DcuD, DauA, DcuAIt possesses the function of a methylmalic acid (intermediate product) absorption protein, which can effectively relieve the inhibitory effect of glucose on methylmalic acid utilization during fermentation of engineered strains and significantly improve the L-isoleucine synthesis capacity based on the methylmalic acid pathway.

[0006] CN 120424843 A uses overexpression of the methylmalate uptake protein gene dcuD This improves the utilization efficiency of methylmalic acid.

[0007] Furthermore, according to current reports, TtdT was initially thought to be a tartaric acid-succinate antiporter, and the protein has diverse functions.

[0008] Therefore, studying the methylmalic acid efflux mechanism in Escherichia coli, blocking or weakening methylmalic acid efflux, and improving the utilization efficiency of intracellular methylmalic acid are of great significance for improving the yield and conversion rate of L-isoleucine based on the methylmalic acid pathway, and also provide an important reference for elucidating the efficient synthesis mechanism of the methylmalic acid pathway.

[0009] However, after knocking out certain transporter genes in *E. coli*, other alternative transporter genes are expressed to adapt to changes in intracellular metabolites. These newly expressed proteins also cause the uptake or efflux of carboxylic acid substrates such as methylmalate. Therefore, it is currently difficult to predict whether regulation based on methylmalate transporters can increase the production of L-isoleucine. Summary of the Invention

[0010] The purpose of this invention is to provide a method for increasing the production of L-isoleucine based on the regulation of methylmalate transporter protein.

[0011] Another object of the present invention is to provide an engineered strain of Escherichia coli that improves the synthesis of L-isoleucine based on methylmalic acid efflux.

[0012] Another object of this application is to provide a method for producing L-isoleucine by fermentation.

[0013] The method for increasing L-isoleucine synthesis in *Escherichia coli* based on methylmalate efflux according to the present invention comprises the following steps:

[0014] In an engineered strain of *E. coli* that synthesizes L-isoleucine via the methylmalate pathway, the gene encoding the carboxylic acid transporter TtdT was knocked out, wherein the amino acid sequence of the carboxylic acid transporter TtdT is shown in SEQ ID NO: 1.

[0015] According to the present invention, the method for increasing the L-isoleucine synthesis yield of Escherichia coli based on methylmalate efflux is wherein the coding gene of the carboxylic acid transporter TtdT is knocked out by inserting inactivation or knocking out part or all of the gene sequence of the coding gene of the carboxylic acid transporter TtdT, or by knocking out the promoter.

[0016] The method for increasing L-isoleucine synthesis in *Escherichia coli* based on methylmalate efflux according to the present invention, wherein the engineered *Escherichia coli* strain synthesizing L-isoleucine via the methylmalate pathway has the following characteristics:

[0017] Knockout of branched-chain amino acid uptake protein gene brnQ, livJ, livK, and phosphoacetyltransferase gene ackA ;

[0018] Overexpression of branched-chain amino acid exoprotein genes on the genome ygaZ , ygaH and regulatory protein genes lrp ;

[0019] Overexpression of the methylmalate synthase high-activity mutant gene on the genome cimA3.7 Isopropyl malate isomerase gene GsleuCD 3-Isopropylmalate dehydrogenase gene AfleuB , and leucine dehydrogenase gene LsleuDH Gene;

[0020] Overexpression of the methylmalate uptake protein gene on the genome dcuD ;

[0021] Overexpression of bifunctional phosphatosterolase genes on the genome Bafxpk and the phosphotransacetase gene of E. coli itself pta .

[0022] The *E. coli* engineered strain of the present invention, which improves the synthesis of L-isoleucine based on methylmalate efflux, has the following characteristics:

[0023] An engineered strain of *E. coli* that synthesizes L-isoleucine via the methylmalate pathway; and

[0024] The gene encoding the carboxylic acid transporter TtdT was knocked out, and the amino acid sequence of the carboxylic acid transporter TtdT is shown in SEQ ID NO: 1.

[0025] The *E. coli* engineered strain of the present invention, which improves the synthesis of L-isoleucine based on methylmalate efflux, wherein the *E. coli* engineered strain that synthesizes L-isoleucine via the methylmalate pathway has the following characteristics:

[0026] Knockout of branched-chain amino acid uptake protein gene brnQ, livJ, livK, and phosphoacetyltransferase gene ackA ;

[0027] Overexpression of branched-chain amino acid exoprotein genes on the genome ygaZ , ygaH and regulatory protein genes lrp ;

[0028] Overexpression of the methylmalate synthase high-activity mutant gene on the genome cimA3.7 Isopropyl malate isomerase gene GsleuCD 3-Isopropylmalate dehydrogenase gene AfleuB , and leucine dehydrogenase gene LsleuDH Gene;

[0029] Overexpression of the methylmalate uptake protein gene on the genome dcuD ;

[0030] Overexpression of bifunctional phosphatosterolase genes on the genome Bafxpk and the phosphotransacetase gene of E. coli itself pta .

[0031] According to the present invention, a method for preparing L-isoleucine by fermentation includes the step of fermenting the above-mentioned engineered strain of *Escherichia coli* with increased L-isoleucine production based on methylmalic acid efflux in a fermenter.

[0032] Advantages of the technical solution of the present invention:

[0033] Engineered *E. coli* strains that produce L-isoleucine via the methylmalate pathway (also known as the pyruvate pathway) produce large amounts of methylmalate intracellularly during fermentation. This intracellular methylmalate is secreted extracellularly by the strain's efficient efflux system, leading to a reduced conversion efficiency of intracellular methylmalate to L-isoleucine and ultimately a decrease in L-isoleucine production. Although expressing a methylmalate uptake protein gene... dcuD While extracellular methylmalic acid can be reabsorbed into the cell to some extent, the overall utilization rate of methylmalic acid remains low. This invention determines that knocking out the gene encoding the carboxylic acid transporter TtdT can effectively reduce the efflux of methylmalic acid in E. coli, allowing more methylmalic acid to remain inside the cell, thereby increasing the intracellular conversion rate of methylmalic acid and ultimately improving the efficiency of L-isoleucine synthesis based on the methylmalic acid pathway.

[0034] The conclusions of CN 118853722 B and CN 120424843 A are consistent, suggesting that DcuD has the function of a methylmalate-absorbing protein.

[0035] After knocking out certain transporter genes in E. coli, other alternative transporter genes will be expressed to adapt to changes in intracellular metabolites. The newly expressed proteins will also cause the absorption or efflux of carboxylic acid substrates such as methylmalic acid. Therefore, although CN 118853722 B confirmed that DcuA has the function of methylmalic acid absorption protein, this application found that knocking out the transporter genes DcuA and CitT on the basis of TtdT actually reduced the production of L-isoleucine. Attached Figure Description

[0036] Figure 1 A schematic diagram showing the knockout of nine carboxylic acid transporter genes in an L-isoleucine-producing strain based on the methylmalate pathway is displayed.

[0037] Figure 2 The results of methylmalic acid production in a 3 L fermenter are shown for the starting strain ILE-13 and engineered strain CAT1-CAT9 provided in Example 1 of this invention.

[0038] Figure 3 The results show the yield of L-isoleucine in a 3 L fermenter of the starting strain ILE-13 and the engineered strain CAT1-CAT9 provided in Example 1 of the present invention.

[0039] Figure 4 The results of methylmalic acid production in a 3L fermenter are shown for the starting strain ILE-13 and the engineered strains CAT10-CAT12 provided in Example 2 of this invention.

[0040] Figure 5 The results show the yield of L-isoleucine in a 3L fermenter of the starting strain ILE-13 and the engineered strains CAT10-CAT12 provided in Example 2 of the present invention. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below through specific embodiments, but this does not limit the scope or implementation of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0043] Table 1. List of engineered Escherichia coli strains constructed in various embodiments of the present invention.

[0044] .

[0045] The following embodiments involve the following sequences:

[0046] SEQ ID NO: 1: TtdT

[0047] MKPSTEWWRYLAPLAVIAIIALLPVPAGLENHTWLYFAVFTGVIVGLILEPVPGAVVAMVGISIIAILSPWLLFSPEQLAQPGFKFTAKSLSWAVSGFSNSVIWLIFAAFMFGTGYEKTGLGRRIALILVKKMGHRTLFLGYAVMFSELILAPVTPSNSARGAGIIYPIIRNLPPLYQSQPNDSSSRSIGSYIMWMGIVADCVTSAIFLTAMAPNLLLIGLMKSASHATLSWGDWFLGMLPLSILLVLLVPWLAYVLYPPVLKSGDQVPRWAETELQAMGPLCSREKRMLGLMVGALVLWIFGGDYIDAAMVGYSVVALMLLLRIISWDDIVSNKAAWNVFFWLASLITLATGLNNTGFISWFGKLLAGSLSGYSPTMVMVALIVVFYLLRYFFASATAYTSALAPMMIAAALAMPEIPLPVFCLMVGAAIGLGSILTPYATGPSPIYYGSGYLPTADYWRLGAIFGLIFLVLLVITGLLWMPVVLL;

[0048] SEQ ID NO:2:DauA

[0049] MNKIFSSHVMPFRALIDACWKEKYTAARFTRDLIAGITVGIIAIPLAMALAIGSGVAPQYGLYTAAVAGIVIALTGGSRFSVSGPTAAFVVILYPVSQQFGLAGLLVATLLSGIFLILMGLARFGRLIEYIPVSVTLGFTSGIGITIGTMQIKDFLGLQMAHVPEHYLQKVGALFMALPTINVGDAAIGIVTLGILVFWPRLGIRLPGHLPALLAGCAVMGIVNLLGGHVATIGSQFHYVLADGSQGNGIPQLLPQLVLPWDLPNSEFTLTWDSIRTLLPAAFSMAMLGAIESLLCAVVLDGMTGTKHKANSELVGQGLGNIIAPFFGGITATAAIARSAANVRAGATSPISAVIHSILVILALLVLAPLLSWLPLSAMAALLLMVAWNMSEAHKVVDLLRHAPKDDIIVMLLCMSLTVLFDMVIAISVGIVLASLLFMRRIARMTRLAPVVVDVPDDVLVLRVIGPLFFAAAEGLFTDLESRLEGKRIVILKWDAVPVLDAGGLDAFQRFVKRLPEGCELRVCNVEFQPLRTMARAGIQPIPGRLAFFPNRRAAMADL;

[0050] SEQ ID NO:3:DcuA

[0051] MLVVELIIVLLAIFLGARLGGIGIGFAGGLGVLVLAAIGVKPGNIPFDVISIIMAVIAAISAMQVAGGLDYLVHQTEKLLRRNPKYITILAPIVTYFLTIFAGTGNISLATLPVIAEVAKEQGVKPCRPLSTAVVSAQIAITASPISAAVVYMSSVMEGHGISYLHLLSVVIPSTLLAVLVMSFLVTMLFNSKLSDDPIYRKRLEEGLVELRGEKQIEIKSGAKTSVWLFLLGVVGVVIYAIINSPSMGLVEKPLMNTTNAILIIMLSVATLTTVICKVDTDNILNSSTFKAGMSACICILGVAWLGDTFVSNNIDWIKDTAGEVIQGHPWLLAVIFFFASALLYSQAATAKALMPMALALNVSPLTAVASFAAVSGLFILPTYPTLVAAVQMDDTGTTRIGKFVFNHPFFIPGTLGVALAVCFGFVLGSFML;

[0052] SEQ ID NO:4:SatP

[0053] MGNTKLANPAPLGLMGFGMTTILLNLHNVGYFALDGIILAMGIFYGGIAQIFAGLLEYKKGNTFGLTAFTSYGSFWLTLVAILLMPKLGLTDAPNAQFLGVYLGLWGVFTLFMFFGTLKGARVLQFVFFSLTVLFALLAIGNIAGNAAIIHFAGWIGLICGASAIYLAMGEVLNEQFGRTVLPIGESH;

[0054] SEQ ID NO:5:DctA

[0055] MKTSLFKSLYFQVLTAIAIGILLGHFYPEIGEQMKPLGDGFVKLIKMIIAPVIFCTVVTGIAGMESMKAVGRTGAVALLYFEIVSTIALIIGLIIVNVVQPGAGMNVDPATLDAKAVAVYADQAKDQGIVAFIMDVIPASVIGAFASGNILQVLLFAVLFGFALHRLGSKGQLIFNVIESFSQVIFGIINMIMRLAPIGAFGAMAFTIGKYGVGTLVQLGQLIICFYITCILFVVLVLGSIAKATGFSIFKFIRYIREELLIVLGTSSSESALPRMLDKMEKLGCRKSVVGLVIPTGYSFNLDGTSIYLTMAAVFIAQATNSQMDIVHQITLLIVLLLSSKGAAGVTGSGFIVLAATLSAVGHLPVAGLALILILGIDRFMSEARALTNLVGNGVATIVVAKWVKELDHKKLDDVLNNRAPDGKTHELSS;

[0056] SEQ ID NO: 6: DcuC

[0057] MLTFIELLIGVVVVIVGVARYIIKGYSATGVLFVGGLLLLIISAIMGHKVLPSSQASTGYSATDIVEYVKILLMSRGGDLGMMIMMLCGFAAYMTHIGANDMVVKLASKPLQYINSPYLLMIAAYFVACLMSLAVSSATGLGVLLMATLFPVMVNVGISRGAAAICASPAAIILAPTSGDVVLAAQASEMSLIDFAFKTTLPISIAAIIGMAIAHFFWQRYLDKKEHISHEMLDVSEITTTAPAFYAILPFTPIIGVLIFDGKWPGQLHIITILVICMLIASILEFLRSFNTQKVFSGLEVAYRGMADAFANVVMLLVAAGVFAQGLSTIGFIQSLISIATSFGSASIILMLVLVILTMLAAVTTGSGNAPFYAFVEMIPKLAHSSGINPAYLTIPMQASNLGRTLSPVSGVVVAVAGMAKISPFEVVKRTSVPVLVGLVIVIVATELMVPGTAAAVTGK;

[0058] SEQ ID NO:7:CitT

[0059] MSLAKDNIWKLLAPLVVMGVMFLIPVPDGMPPQAWHYFAVFVAMIVGMILEPIPATAISFIAVTICVIGSNYLLFDAKELADPAFNAQKQALKWGLAGFSSTTVWLVFGAFIFALGYEVSGLGRRIALFLVKFMGKRTLTLGYAIVIIDILLAPFTPSNTARTGGTVFPVIKNLPPLFKSFPNDPSARRIGGYLMWMMVISTSLSSSMFVTGAAPNVLGLEFVSKIAGIQISWLQWFLCFLPVGVILLIIAPWLSYVLYKPEITHSEEVATWAGDELKTMGALTRREWTLIGLVLLSLGLWVFGSEVINATAVGLLAVSLMLALHVVPWKDITRYNSAWNTLVNLATLVVMANGLTRSGFIDWFAGTMSTHLEGFSPNATVIVLVLVFYFAHYLFASLSAHTATMLPVILAVGKGIPGVPMEQLCILLVLSIGIMGCLTPYATGPGVIIYGCGYVKSKDYWRLGAIFGVIYISMLLLVGWPILAMWN;

[0060] SEQ ID NO:8:DcuB

[0061] MLFTIQLIIILICLFYGARKGGIALGLLGGIGLVILVFVFHLQPGKPPVDVMLVIIAVVAASATLQASGGLDVMLQIAEKLLRRNPKYVSIVAPFVTCTLTILCGTGHVVYTILPIIYDVAIKNNIRPERPMAASSIGAQMGIIASPVSVAVVSLVAMLGNVTFDGRHLEFLDLLAITIPSTLIGILAIGIFSWFRGKDLDKDEEFQKFISVPENREYVYGDTATLLDKKLPKSNWLAMWIFLGAIAVVALLGADSDLRPSFGGKPLSMVLVIQMFMLLTGALIIILTKTNPASISKNEVFRSGMIAIVAVYGIAWMAETMFGAHMSEIQGVLGEMVKEYPWAYAIVLLLVSKFVNSQAAALAAIVPVALAIGVDPAYIVASAPACYGYYILPTYPSDLAAIQFDRSGTTHIGRFVINHSFILPGLIGVSVSCVFGWIFAAMYGFL;

[0062] SEQ ID NO:9:YbhI

[0063] MNKKSLWKLILILAIPCIIGFMPAPAGLSELAWVLFGIYLAAIVGLVIKPFPEPVVLLIAVAASMVVVGNLSDGAFKTTAVLSGYSSGTTWLVFSAFTLSAAFVTTGLGKRIAYLLIGKIGNTTLGLGYVTVFLDLVLAPATPSNTARAGGIVLPIINSVAVALGSEPEKSPRRVGHYLMMSIYMVTKTTSYMFFTAMAGNILALKMINDILHLQISWGGWALAAGLPGIIMLLVTPLVIYTMYPPEIKKVDNKTIAKAGLAELGPMKIREKMLLGVFVLALLGWIFSKSLGVDESTVAIVVMATMLLLGIVTWEDVVKNKGGWNTLIWYGGIIGLSSLLSKVKFFEWLAEVFKNNLAFDGHGNVAFFVIIFLSIIVRYFFASGSAYIVAMLPVFAMLANVSGAPLMLTALALLFSNSYGGMVTHYGGAAGPVIFGVGYNDIKSWWLVGAVLTILTFLVHITLGVWWWNMLIGWNML。

[0064] Example 1: Enhancing L-Isoleucine Synthesis in Escherichia coli via the Methylmalate Pathway by Knocking Out the Carboxylic Acid Transporter Gene

[0065] Previously, an engineered Escherichia coli strain ILE-13 (CN120424843 A) was constructed to produce L-isoleucine via the methylmalic acid pathway. When fermented in a 10 L fermenter, the L-isoleucine yield reached 56.6 g / L (the L-isoleucine yield was 50.7 g / L when using a 3 L fermenter).

[0066] The construction process of this strain is as follows:

[0067] Using Escherichia coli BW25113 as the starting strain, the branched-chain amino acid uptake protein gene was knocked out using the CRISPR-associated transposases gene editing method. brnQ , livJ , livK, Obtaining the genome brnQ , livJ , livK Gene knockout E. coli ILE-1 strain ；

[0068] Using ILE1 as the starting strain, gene editing was performed using the CRISPR-associated transposases method. livK Site-integrated branched-chain amino acid exoprotein gene ygaZH and regulatory protein genes lrp， Integration into the genome ygaZH and lrp The engineered strain ILE-2 of the expression cassette;

[0069] Derived from ILE-2, in yjip Site overexpression cimA3.7 , GsleuCD , AfleuB Genes, in gapC Site overexpression ilvIH* , ilvC* , ilvD , LsleuDH, dcuD Genes were used to ultimately obtain the Escherichia coli ILE-5 strain;

[0070] In Escherichia coli ILE-5 strain pflB , ldhA Each site integrates one copy of P araBAD - cimA3.7 - AfleuB Expression cassette was used to obtain strain ILE-8;

[0071] In Escherichia coli ILE-8 strainadhE , ycjV Each site integrates one copy of P araBAD - AfleuB Expression cassette was used to obtain strain ILE-9;

[0072] Knockout in Escherichia coli ILE-9 strain ackA Genes were used to reduce the synthesis of acetyl-CoA into acetic acid, resulting in the Escherichia coli ILE-12 strain.

[0073] In Escherichia coli strain ILE-12 ackA P site overexpression araBAD - Bafxpk - pta The module was used to obtain strain ILE-13.

[0074] Although the methylmalate uptake protein gene has been overexpressed in this ILE-13 strain dcuD This improved the intracellular utilization efficiency of methylmalic acid, but 31.2 g / L of methylmalic acid in the fermentation broth remained unutilized, affecting the synthesis of L-isoleucine based on the methylmalic acid pathway. Based on strain ILE-13, this invention knocked out nine genes encoding carboxylic acid transporters (…). Figure 1 ), discovered knockout TtdT The gene can significantly reduce the accumulation of methylmalic acid in the fermentation broth and increase the yield of L-isoleucine. The specific steps are as follows:

[0075] I. Construction of Escherichia coli engineered strain CAT1-CAT9 with carboxylic acid transporter gene knockout

[0076] Using L-isoleucine-producing strain ILE-13 as the starting strain, the coding genes for nine carboxylic acid transport proteins, namely DauA, DcuA, SatP, TtdT, DctA, DcuC, CitT, DcuB, and YbhI, were knocked out using the CRISPR-associated transposases gene editing method. Specifically:

[0077] (1) Preparation of electrotransformation competent cells of ILE-13 strain

[0078] Take 0.3 mL of L-isoleucine-producing strain ILE-13 glycerol stock culture and inoculate it into 50 mL of fresh LB broth, then incubate overnight at 37 °C with shaking. Transfer the 1% inoculum to 100 mL of fresh, sterile LB broth and incubate at 37 °C with shaking until OD (dose-free ratio) is reached. 600The culture concentration was approximately 0.6. The cultured bacterial suspension was placed on ice for 15 min, then centrifuged at 6000 rpm for 8 min at 4 °C to collect the bacterial cells. The bacterial cells were resuspended in 100 mL of pre-cooled sterile water, centrifuged again under the same conditions to collect the bacterial strain, and resuspended in 40 mL of pre-cooled 10% glycerol. Finally, the bacterial cells were collected by centrifugation at 7000 rpm for 8 min at 4 °C, resuspended in 0.5 mL of pre-cooled 10% glycerol, aliquoted into 80 µL vials, and stored at -80 °C for later use.

[0079] (2) Electroporation and transformant screening of strain ILE-13

[0080] When using the CRISPR-associated transposases gene editing system to knock out genes, pTetQCas (containing...) is required. tniQ and cas876 (gene), pRE57-tns-Ter (containing) tnsABC The three plasmids are: the gene and its right end-terminator-left end sequence, and pUC-spacer (containing the spacer sequence of the target gene). The knockout plasmid... DauA The spacer sequence used in gene knockout is: GGAATATACTCAATCAGGCGACCAAAGCGTGC (SEQ ID NO:10); DcuA The spacer sequence used in gene knockout is: GCTGAGATTGGCGATGCGGTGATCGCAATCTG (SEQ ID NO:11); SatP The spacer sequence used in gene knockout is: AAGGAACTGTGCATTTGGCGCATCGGTCAGAC (SEQ ID NO:12); TtdT The spacer sequence used in gene knockout is: GCTGGCGGTCATCGCCATTATTGCTCTACTTC (SEQ ID NO:13); DctA The spacer sequence used in gene knockout is: AGGCTGCACGACGTTAACGATGATAAGACCAA (SEQ ID NO:14); DcuC The spacer sequence used in gene knockout is: GTGGCGCTGTAGCCTGTTGAAGCCTGGCTGGA (SEQ ID NO:15); CitT The spacer sequence used in gene knockout is: GCAATAAAACTGATCGCTGTTGCCGGAATTGG (SEQ ID NO:16); DcuBThe spacer sequence used in gene knockout is: GGACGGATGTTGTTCTTAATGGCGACGTCGTA (SEQ ID NO:17); YbhI The spacer sequence used in the gene is: TCATGGTGGTGGTCGGTAACTTATCCGACGG (SEQ ID NO:18).

[0081] First, 0.5 μg of plasmid pTetQCas and 0.5 μg of plasmid pRE57-tns-Ter were mixed and added to ILE-13 electroporation competent cells. After mixing, the mixture was transferred to a 1 mm electroporation cuvette, and both plasmids were transformed into ILE-13 cells using an electroporator. Then, 0.8 mL of fresh antibiotic-free LB broth was added, and the cells were incubated at 37 °C with shaking for 30 min to recover. After recovery, the cells were centrifuged at 6000 rpm for 3 min to collect the bacterial cells. The bacterial cells were then plated on LB agar plates containing 100 µg / mL ampicillin and 50 µg / mL streptomycin and incubated overnight at 37 °C. After colonies appeared, single clones were picked with a sterile toothpick for PCR verification, confirming that both plasmids had been successfully transformed into ILE-13 cells.

[0082] Subsequently, following the above-described methods for preparing electrotransformation competent cells, electrotransformation, and transformant screening, nine pUC-spacer plasmids carrying different spacer sequences were transformed into ILE-13 strain containing pTetQCas and pRE57-tns-Ter plasmids, respectively. Transformants were screened using LB plates containing 50 µg / mL kanamycin, 100 µg / mL ampicillin, and 50 µg / mL streptomycin. Cloning PCR confirmed that all three plasmids were successfully transformed into ILE-13 cells.

[0083] (3) Construction of engineered strains CAT1-CAT9

[0084] ILE-13 transformants containing the three plasmids pTetQCas, pRE57-tns-Ter, and pUC-spacer (containing spacer sequences of different target genes) were inoculated into LB liquid medium containing kanamycin, ampicillin, and streptomycin, and cultured at 37 °C with shaking at 200 r / min. 600When the expression level reached 0.6-0.8, 100 ng / mL of adehydrotetracycline was added for induction to induce the expression of proteins related to the CRISPR-associated transposases gene editing system. The culture was incubated overnight at 37 °C with shaking. The induced culture medium was diluted and plated onto LB agar plates containing the three antibiotics and adehydrotetracycline inducer. The plates were incubated statically at 37 °C for 14 h. Once single clones appeared on the plates, clonal PCR was performed to verify the gene knockout status. DauA , DcuA , SatP , TtdT , DctA, DcuC , CitT , DcuB , YbhI The PCR product band of the successfully knocked-out mutant strain was 841 bp larger than that of the wild-type strain. Positive clones were inoculated into LB liquid medium without antibiotics and inducers.

[0085] To discard the gene-editing plasmids in the engineered strains, positive clones (containing three gene-editing plasmids) were inoculated into 100 mL of LB liquid medium without antibiotics and inducers. The medium was passaged at 45 °C and 200 r / min, with serial dilutions of the culture medium after six passages. The culture was then plated on antibiotic-free LB plates and incubated overnight at 37 °C. After cloning, single clones were randomly picked with a sterile toothpick and spotted onto four different LB plates: antibiotic-free, containing kanamycin, ampicillin, or streptomycin. The single clones on each plate were matched one-to-one, and the plates were incubated overnight at 37 °C. Single clones that grew only on antibiotic-free LB plates and not on the other three antibiotic-containing plates were selected for further verification using clonal PCR. DauA , DcuA , SatP , TtdT , DctA, DcuC , CitT , DcuB , YbhI Gene knockout status: positive clones are CAT1-CAT9 engineered strains (Table 1).

[0086] II. Fermentation experiments with engineered strains CAT1-CAT9

[0087] The obtained CAT1-CAT9 engineered strains were fed-batch fermented in a 3 L fermenter. The specific method is as follows: CAT1-CAT9 strains were inoculated into 30 mL LB liquid medium and cultured at 37 ℃ and 200 r / min for 12 h to obtain the primary seed culture. A 1% inoculum was then transferred to a 1 L Erlenmeyer flask containing 300 mL of fermentation medium and cultured at 37 ℃ and 200 r / min for 12 h to obtain the secondary seed culture. The OD of the secondary seed culture was measured.600 Inoculate into a 3 L fermenter containing 1 L of fermentation medium, so that the initial OD... 600 Approximately 0.5. The fermentation medium was formulated as follows: 5 g / L yeast extract, 20 g / L glucose, 14 g / L KH2PO4, 4 g / L (NH4)2HPO4, 0.6 g / L MgSO4·7H2O, 2 g / L Na2SO4, 1.8 g / L citric acid, and trace elements (13 mg / L Zn(CH3COO)2·2H2O, 10 mg / L ferric citrate, 8.4 mg / L EDTA, 3 mg / L H3BO3, 2.5 mg / L Na2MoO4·2H2O, 2.5 mg / L CoCl2·6H2O, 1.5 mg / L CuCl2·2H2O, 0.9 mg / L VB1, 0.15 mg / L MnCl2·4H2O). Fermentation lasted 36 hours. During fermentation, the temperature was maintained at 37 °C, the pH was maintained at approximately 7.0 by automatic ammonia replenishment, and the dissolved oxygen level was controlled between 10% and 30% by motor speed coupling. After the initial 20 g / L glucose was consumed, a peristaltic pump was used to replenish the feed, keeping the glucose concentration in the fermentation broth below 5 g / L. The feed consisted of a 700 g / L glucose solution. Samples were taken every 3 hours. The concentration of L-isoleucine in the fermentation broth was separated and determined by high-performance liquid chromatography (HPLC) using the Shimadzu AJS-01 amino acid analysis method kit. The concentration of methylmalic acid in the fermentation broth was analyzed using a Shimadzu LC-20A HPLC system equipped with a Bio-Rad HPX-87H column. Figure 2 and Figure 3 As shown, compared with the original strain ILE-13, strain CAT2 (knockout) DcuA Gene), CAT4 (knockout) TtdT (gene) and CAT7 (knockout) CitT The accumulation of methylmalic acid in the fermentation broth decreased by 46.6%, 60.0%, and 38.0%, respectively, while the production of L-isoleucine increased by 9.3%, 46.8%, and 8.0%, respectively, indicating that... DcuA , TtdT and CitT Gene knockout helps reduce methylmalate efflux and increase L-isoleucine production. Specifically, knockout... ​ The engineered strain CAT4, obtained from the gene, achieved the best L-isoleucine yield of 75.6 g / L after 34 hours of fermentation.

[0088] Example 2: Effects of combinatorial knockout of carboxylic acid transporter genes on L-isoleucine production

[0089] Due to knocking ​ ,​ and ​ Individual carboxylic acid transporter genes have shown positive effects on increasing L-isoleucine production via the methylmalate pathway, therefore, genes including... ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ Nine carboxylic acid transporter genes, including [list of genes], were divided into three groups for combined knockout. The grouping is as follows: ​ , ​ , ​ Gene; ​ , ​ , ​ Gene; ​ , ​ , ​ Genes. Three genes were simultaneously knocked out to determine the effect of combined gene knockout on L-isoleucine production. The specific steps are as follows:

[0090] 2.1 Construction of Escherichia coli engineered strain CAT10-CAT12 with combined knockout of carboxylic acid transporter gene

[0091] Starting with L-isoleucine-producing strain ILE-13, the strain was knocked out using CRISPR-associated transposases gene editing. ​ , ​ , ​ Gene knockout (triple knockout); ​ , ​ , ​ Gene knockout (triple knockout); ​ , ​ , ​ The three gene knockouts are as follows:

[0092] (1) Preparation of electrotransformation competent cells of ILE-13 strain

[0093] Competent cells were prepared according to the electroconversion competent cell preparation method described in Example 1.

[0094] (2) Electroporation and transformant screening of strain ILE-13

[0095] The electroconversion and converter screening method described in Example 1 shall be followed.

[0096] (3) Construction of engineered strain CAT10-CAT12

[0097] The engineered strain construction method described in Example 1 was followed, and the strain ILE-13 was knocked out. ​ ,​ and ​ The strain obtained after gene sequencing was CAT10; the gene was knocked out in the ILE-13 strain. ​ , ​ and ​ The strain obtained after gene sequencing was CAT11; in the ILE-13 strain, the gene was knocked out. ​ , ​ and ​ The strain obtained after gene sequencing was CAT12 (Table 1).

[0098] 2.2 Fermentation experiments of engineered strains CAT10-CAT12

[0099] The obtained CAT10-CAT12 engineered strains were subjected to fed-batch fermentation in a 3 L fermenter. The specific method is as follows:

[0100] The fermentation medium formulation is the same as that described in Example 1.

[0101] The fermentation method is the same as that described in Example 1.

[0102] The method for detecting fermentation products is the same as that described in Example 1.

[0103] like ​ and ​ As shown, compared with the starting strain ILE-13, only the CAT12 strain showed a significant increase in L-isoleucine production, while methylmalic acid accumulation was significantly reduced; however, the CAT12 strain (with DcuA knockout) showed a different result. ​ and ​ The L-isoleucine production of the gene reached 69.4 g / L, a decrease of 8.2% compared to CAT4, indicating that in ​ Knock out on the basis ​ and ​ The gene is unfavorable for the production of L-isoleucine.

[0104] The above results indicate that the method for regulating methylmalate efflux proteins provided by the present invention can significantly improve the utilization efficiency of methylmalate and the synthesis capacity of L-isoleucine based on the methylmalate pathway.

[0105] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for increasing the production of L-isoleucine in *Escherichia coli* based on methylmalate efflux, characterized in that, The method includes the following steps: In an engineered *E. coli* strain that synthesizes L-isoleucine via the methylmalate pathway, the gene encoding the carboxylic acid transporter TtdT was knocked out. The amino acid sequence of the carboxylic acid transporter TtdT is shown in SEQ ID NO:

1. The engineered Escherichia coli strain that synthesizes L-isoleucine via the methylmalic acid pathway has the following characteristics: Knockout of branched-chain amino acid uptake protein gene brnQ, livJ, livK, and phosphoacetyltransferase gene ackA ; Overexpression of branched chain amino acid exporter genes on the genome ygaZ , ygaH and regulator genes lrp ; Overexpression of the methylmalate synthase-high-activity mutant gene in the genome cimA3.7 Isopropyl malate isomerase gene GsleuCD 3-Isopropylmalate dehydrogenase gene AfleuB , and leucine dehydrogenase gene LsleuDH Gene; Overexpression of a methylmalate uptake protein gene on the genome dcuD ; Overexpression of bifunctional phosphatidyl alcoholase gene on the genome Bafxpk and the phosphotransacetase gene of E. coli itself pta .

2. The method for increasing L-isoleucine synthesis in *Escherichia coli* based on methylmalate efflux according to claim 1, characterized in that, The coding gene for the carboxylic acid transporter TtdT can be inactivated or knocked out by inserting part or all of its gene sequence, or by knocking out the promoter to knock out the coding gene for the carboxylic acid transporter TtdT.

3. An engineered *Escherichia coli* strain with increased L-isoleucine synthesis based on methylmalic acid efflux, characterized in that... The engineered strain of Escherichia coli has the following characteristics: An engineered strain of *E. coli* that synthesizes L-isoleucine via the methylmalate pathway; and The gene encoding the carboxylic acid transporter TtdT was knocked out, and the amino acid sequence of the carboxylic acid transporter TtdT is shown in SEQ ID NO:

1. The engineered Escherichia coli strain that synthesizes L-isoleucine via the methylmalic acid pathway has the following characteristics: Knockout branched-chain amino acid absorption protein genes brnQ, livJ, livK, and phosphotransacetylase genes ackA ; Overexpression of branched-chain amino acid exoprotein genes on the genome ygaZ , ygaH and regulatory protein genes lrp ; Overexpression of the methylmalate synthase-high-activity mutant gene in the genome cimA3.7 Isopropyl malate isomerase gene GsleuCD 3-Isopropylmalate dehydrogenase gene AfleuB , and leucine dehydrogenase gene LsleuDH Gene; Overexpression of a methylmalate uptake protein gene on the genome dcuD ; Overexpression of bifunctional phosphoketolase gene on the genome Bafxpk and the phosphotransacetylase gene of e. coli itself pta .

4. A method for preparing L-isoleucine by fermentation, characterized in that, The method includes the step of fermenting the engineered Escherichia coli strain of claim 3, which has improved L-isoleucine synthesis based on methylmalic acid efflux, in a fermenter.

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