A genetically engineered bacterium for producing L-leucine and its applications

Abstract

This invention relates to a genetically engineered strain for producing L-leucine and its applications, belonging to the field of metabolic engineering. Using E. coli W3110 as the starting strain, this invention utilizes systemic metabolic engineering techniques to enhance the supply of acetyl-CoA and reducing power, increase the L-leucine synthesis flux, strengthen its efflux system, and dynamically weaken the TCA cycle, ultimately constructing a high-yielding L-leucine strain. In a 5L fermenter, fed-batch fermentation achieved an L-leucine yield of 85.6 g / L after 34 hours, with a conversion rate of 36.6% and a fermentation intensity of 2.52 g / L / h. The fermentation process employed in this invention is simple, easy to control, and has low production costs, facilitating its promotion and application in industrial production. Compared with existing technologies, the engineered strain and fermentation process obtained in this invention exhibit no nutritional deficiencies, a short fermentation cycle, and high L-leucine yield and conversion rate, representing the highest reported indicators to date.

Description

Technical fields:

[0001] This invention relates to a genetically engineered bacterium that produces L-leucine and its applications, belonging to the field of metabolic engineering. Background technology:

[0002] L-Leucine is one of the eight essential amino acids, possessing excellent physiological properties, leading to increasing market demand in feed, pharmaceuticals, and chemicals. Currently, L-leucine production primarily involves hair hydrolysis and bio-fermentation. Extraction methods suffer from limitations such as restricted raw material sources, high production costs, and environmental pollution. In contrast, bio-fermentation offers advantages such as large-scale production, high yield, and lower cost. Currently, industrial production strains for L-leucine are mainly obtained through mutagenesis, which suffers from nutritional deficiencies, slow growth, and unstable genetic traits, resulting in long fermentation cycles, unstable fermentation performance, and low yield and conversion rates. *Escherichia coli*, with its well-defined metabolic background and rapid growth, is a commonly used industrial microorganism. Summary of the Invention:

[0003] To overcome the shortcomings of existing L-leucine-producing strains, such as slow growth, unstable performance, and nutritional deficiencies, this invention provides a genetically engineered strain that produces L-leucine with fast growth, no nutritional deficiencies, and high synthesis efficiency, as well as a method for directly fermenting L-leucine using this strain.

[0004] One of the technical solutions adopted by the present invention to solve the above-mentioned technical problems is: a high-yielding L-leucine strain, wherein the strain is based on Escherichia coli W3110, the lactose operon repressor protein encoding gene lacI is knocked out in the genome, and the acetylhydroxyl synthase encoding gene ilvBN that relieves feedback inhibition is overexpressed in the genome. M The plasmid overexpression was used to relieve the feedback inhibition of the isopropyl malate synthase encoding gene leuA. M The genome expresses the following genes: leuB (encoding β-isopropylmalate dehydrogenase) and leuCD (encoding α-isopropylmalate isomerase); ilvC (encoding hydroxy acid reductase); bcd (encoding Bacillus subtilis leucine dehydrogenase); ilvE (encoding branched-chain aminotransferase); pntAB (encoding pyridine nucleotide transhydrogenase); coaA (encoding ubiquitin kinase); rocG (encoding glutamate dehydrogenase); and fxpk (encoding phosphoryl ketonease); ackA (encoding acetate kinase); poxB (encoding pyruvate dehydrogenase); and ldhA (encoding lactate dehydrogenase). Furthermore, the promoter of gltA (encoding citrate synthase) is replaced with a leucine-weakened P… leuAThe promoter weakens the transcription of gltA when intracellular L-leucine accumulates, causing more pyruvate and acetyl-CoA to flow to L-leucine synthesis rather than the TCA cycle; by overexpressing the L-leucine transporter encoding gene yeaS on the genome, a high-yielding L-leucine strain was finally obtained.

[0005] Furthermore, the lactose operon repressor protein encoding gene lacI is from Escherichia coli W3110, ProteinID: BAE76127.1 in NCBI;

[0006] Furthermore, the acetylhydroxyl synthase encoding gene ilvBN that relieves feedback inhibition... M It has been published in Chinese invention patent ZL201910484362.5;

[0007] Furthermore, the method for relieving the L-leucine feedback inhibition of isopropyl malate synthase encoding the gene leuA... M It has been published in Chinese invention patent ZL 201910820591.X;

[0008] Furthermore, the β-isopropylmalate dehydrogenase encoding gene leuB is derived from Escherichia coli W3110, ProteinID: BAB96642.1 in NCBI;

[0009] Furthermore, the α-isopropylmalate isomerase encoding gene leuCD is derived from Escherichia coli W3110, with Protein IDs BAB96641.2-BAB96640.1 in NCBI.

[0010] Furthermore, the hydroxy acid reductase encoding gene ilvC is derived from Escherichia coli W3110, Protein ID: BAE77523.1 in NCBI;

[0011] Furthermore, the gene encoding the leucine dehydrogenase, bcd, is derived from Bacillus subtilis 168, Protein ID: NP_390288.1 in NCBI;

[0012] Furthermore, the branched-chain amino acid aminotransferase encoding gene ilvE is from Escherichia coli W3110, NCBI Protein ID: BAE77527.1;

[0013] Furthermore, the pyridine nucleotide transhydrogenase gene pntAB is derived from Escherichia coli W3110, with Protein IDs BAA15342.1-BAA15336.1 in NCBI.

[0014] Furthermore, the ubiquitin kinase encoding gene coaA is derived from Escherichia coli W3110, Protein ID: BAE77341.1 in NCBI;

[0015] Furthermore, the glutamate dehydrogenase encoding gene rocG is derived from Bacillus subtilis 168, Protein ID: NP_391659.2 in NCBI;

[0016] Furthermore, the phosphoryl ketolase encoding gene fxpk is derived from Bifidobacterium adolescentis ATCC 15703, ProteinID:WP_011743105.1 in NCBI;

[0017] Furthermore, the acetate kinase encoding gene ackA is derived from Escherichia coli W3110, with Protein ID BAA16135.1 in NCBI;

[0018] Furthermore, the pyruvate dehydrogenase encoding gene poxB is derived from Escherichia coli W3110, with Protein ID BAA35585.1 in NCBI;

[0019] Furthermore, the lactate dehydrogenase encoding gene ldhA is derived from Escherichia coli W3110, with Protein ID BAA14990.1 in NCBI;

[0020] Furthermore, the citrate synthase encoding gene gltA is derived from Escherichia coli W3110, ProteinID: BAA35384.2 in NCBI;

[0021] Furthermore, the promoter P leuA The leuA promoter is derived from Escherichia coli W3110, ProteinID: BAB96643.2 in NCBI;

[0022] Furthermore, the L-leucine transporter encoding gene yeaS is derived from Escherichia coli W3110, with Protein ID BAA15593.1 in NCBI;

[0023] The second technical solution provided by this invention is the application of the strain described in technical solution one in the production of L-leucine; particularly its application in the fermentation production of L-leucine, the specific method of which is as follows:

[0024] Inoculate the seed culture into the fermentation medium at an inoculum rate of 5-10% for fermentation culture, with an aeration rate of 2-5 m³ / h. 3 / h, stirring speed 300-900rpm, dissolved oxygen maintained at 20-50%, pH maintained at 6.8-7.2, culture temperature 32-37℃, fermentation cycle 30-36h, and residual sugar concentration maintained at 0.1-0.5% (W / V) during fermentation;

[0025] At the end of fermentation, the concentration of L-leucine in the fermentation broth reached 72.7-85.6 g / L.

[0026] Beneficial effects:

[0027] (1) This invention uses E. coli W3110 as the starting strain and employs systems metabolic engineering to improve the supply of acetyl-CoA and reducing power, enhance the synthesis flux of L-leucine, strengthen its efflux system, and dynamically weaken the TCA cycle, ultimately constructing a high-yield L-leucine strain. In a 5L fermenter, fed-batch fermentation was carried out, and the L-leucine yield reached 85.6 g / L after 34 h, with a conversion rate of 36.6% and a fermentation intensity of 2.52 g / L / h.

[0028] (2) The fermentation process used in this invention is simple, easy to control, and has low production costs, which is conducive to the promotion and application of industrial production. Compared with the prior art, the engineered strains and fermentation process obtained in this invention have no nutritional deficiencies, a short fermentation cycle, and high L-leucine yield and conversion rate, which are the highest reported indicators to date. Attached image description:

[0029] Figure 1 Example 14: Results of shake-flask fermentation of L-leucine-engineered bacteria;

[0030] Figure 2 Example 15: Fermentation process curve of L-leucine genetically engineered bacterium leu12 in a 5L fermenter. Detailed implementation method:

[0031] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this patent and are not intended to limit the scope of the invention.

[0032] The gene sequences involved in the embodiments of the present invention can be encoded and synthesized according to the sequence information corresponding to the Protein ID number in NCBI provided in the foregoing invention content section, or amplified using the microbial genome of the corresponding source as a template;

[0033] To relieve the feedback inhibition of L-leucine on isopropyl malate synthase encoding gene leuA MIt has been disclosed in Chinese invention patent ZL201910820591.X (specifically, SEQ ID NO.2 of the patent application text, from which leuA can be synthesized). M );

[0034] The gene encoding ilvBN, which relieves feedback inhibition of acetylhydroxyl synthase. M The sequence ilvBN has been published in Chinese invention patent ZL201910484362.5 (specifically, SEQ ID NO.2 of the patent application text, from which ilvBN can be synthesized). M ).

[0035] The method for detecting substances during the fermentation process of this invention is as follows:

[0036] (1) L-Leucine Detection Method

[0037] L-leucine was quantitatively analyzed using high-performance liquid chromatography (HPLC). The sample was derivatized with 2,4-dinitrofluorobenzene to make it detectable by the HPLC signal analyzer; the specific procedure is as follows:

[0038] Sample derivatization method: After centrifuging 1 ml of fermentation broth at 13000×g for 3 min, the supernatant was collected and diluted 10 times with deionized water. The fermentation broth was then derivatized with 0.8% (V / V) 2,4-dinitrofluorobenzene (the supernatant was passed through a 0.22 μm organic membrane for later use. 200 μL of derivatization buffer, 10 μL of the membrane-passed supernatant, and 300 μL of derivatizing agent were added to a 1.5 mL EP tube and shaken well. The tube was then placed in a 65℃ water bath in the dark for 60 min. After cooling, 690 μL of volume-adjusting buffer was added to a final volume of 1.2 mL, mixed, and passed through a membrane). The sample was filtered into the sample cell. 50% acetonitrile and 4.1 g / L sodium acetate (after ultrasonic filtration) were used as the organic and inorganic phases, respectively. The column temperature was adjusted to 33℃. When the baseline was 0 and the pressure line was horizontal, the determination of the sample was started. The detection conditions were: Agilent AAA (4.6 mm × 150 mm, 5-Micron), acetonitrile / sodium acetate binary gradient elution, flow rate 1 mL / min, column temperature 33 ℃, and detection wavelength 360 nm.

[0039] (2) Detection of bacterial cell concentration and other products

[0040] OD of bacterial cultures was measured using a spectrophotometer. 600 Cell growth was monitored. Glucose concentration during fermentation was detected using an SBA biosensor instrument (SBA-40C; Shandong Academy of Sciences Institute of Biology, Jinan).

[0041] The primer sequences used in the embodiments of this invention are shown in the table below:

[0042]

[0043]

[0044]

[0045] The genotypes of the strains in the embodiments of this invention are shown in the table below.

[0046] E. coli W3110ΔlacI E. coli W3110 knockout lacI leu1 <![CDATA[E.coli W3110ΔlacI yncI::P trc -ilvBN M -T trc <!-- 6 -->]]> leu2 <![CDATA[leu1 carrying pTrc99aΔlacI-leuA M BCD]]> leu3 <![CDATA[leu2 yeeL::P trc -ilvC-T trc ]]> leu4 <![CDATA[leu3 ycgH::P trc -bcd-T trc ]]> leu5 leu4ΔilvE leu6 <![CDATA[leu5 yjgX::P trc -pntAB-T trc ]]> leu7 <![CDATA[leu6 yjiP::P trc -coaA-T trc ]]> leu8 <![CDATA[leu7 yghX::P trc -rocG-T trc ]]> leu9 <![CDATA[leu8 gapC::P trc -fxpk-T trc ]]> leu10 leu9ΔackAΔpoxBΔldhA leu11 <![CDATA[leu10 P gltA ::P leuA ]]> leu12 <![CDATA[leu11 yjiT::P trc -yeaS-T trc ]]>

[0047] It should be noted that the gene editing order can be adjusted according to actual needs during the construction of the strain of the present invention, and the order of editing will not affect the final strain's ability to produce L-leucine.

[0048] The present invention will be further explained and illustrated below through specific embodiments.

[0049] Example 1: Construction of lacI knockout strain E. coli W3110ΔlacI

[0050] (1) Overlapping segment U lacI -D lacI Construction

[0051] Using the genome of wild-type Escherichia coli W3110 as a template, the upstream and downstream homologous arms of lacI were amplified using primers lac-1 / lac-2 and lac-3 / lac-4, respectively. Then, the fusion fragment U of the upstream and downstream homologous arms of lacI was obtained by overlap PCR. lacI -D lacI .

[0052] (2) Construction of pGRB-lacI plasmid

[0053] Based on the lacI sequence, 20bp forward and reverse sequences pGRB-lacI-S / pGRB-lacI-A of gRNA were designed and synthesized. After annealing, the two sequences were ligated into plasmid pGRB using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.). The recombinant plasmid pGRB-lacI was obtained by transformation into E. coli DH5α, screening on LB solid medium containing 100 μg / mL ampicillin, and sequencing.

[0054] (3) Construction of lacI knockout strain E. coli W3110ΔlacI

[0055] The recombinant plasmid pGRB-lacI and the fusion fragment U lacI -D lacI Electroporation was performed into competent E. coli W3110 cells containing the pREDcas9 plasmid. After recovery, the cells were plated on LB agar containing 100 μg / mL spectinomycin and ampicillin and incubated overnight at 32°C. The next day, colony PCR was performed using primers lac-1 / lac-4 to identify positive transformants. The transformants were activated, and arabinose was added to a final concentration of 0.2 mmol / L. The cells were then incubated overnight at 32°C with shaking to induce the loss of pGRB-lacI. The cells were then incubated overnight at 42°C with shaking to induce the loss of the pREDcas9 plasmid, yielding strain E. coli W3110ΔlacI.

[0056] Example 2: Construction of L-leucine engineered strain leu1

[0057] (1) Overlapping segment U yncI -P trc -ilvBN M -T trc -D yncI Construction

[0058] Using the genome of *E. coli* W3110 as a template, the upstream and downstream homologous arms of yncI were amplified using primers U-yncI-S / U-yncI-A and D-yncI-S / D-yncI-A, respectively; and artificially synthesized ilvBN was used. M Using the fragment as a template, primer ilvBN was used. M -S and ilvBN M -A amplification includes P trc promoter, T trc Terminator and ilvBN M gene segment P trc -ilvBN M -T trc (P trc Promoters and T trc The terminator has been designed into primer ilvBN M -S and ilvBN M -A (in the middle). After PCR product recovery, the product was obtained by overlap PCR using primers U-yncI-S / D-yncI-A, containing the upstream and downstream homologous arms of yncI and P. trc -ilvBN M -T trc fusion fragment U yncI -P trc -ilvBN M -T trc -D yncI .

[0059] (2) Construction of pGRB-yncI plasmid

[0060] Based on the yncI sequence, 20bp forward and reverse sequences pGRB-yncI-S and pGRB-yncI-A of gRNA were designed and synthesized. The recombinant plasmid pGRB-yncI was constructed using the same method as step (2) in Example 1.

[0061] (3) Construction of L-leucine engineered strain leu1

[0062] The recombinant plasmid pGRB-yncI and the fusion fragment U yncI -P trc -ilvBN M -T trc -D yncI Transformed into competent E. coli W3110ΔlacI cells containing the pREDcas9 plasmid, positive transformants were identified using primers U-yncI-S / D-yncI-A. The plasmid was then lost using the same method as in step (3) of Example 1 to obtain strain leu1.

[0063] Example 3: Construction of L-leucine engineered strain leu2

[0064] (1) Plasmid pTrc99aΔlacI-leuA M BCD Construction

[0065] Primers Ptrc-1 and Ptrc-2 were designed based on the lacI gene sequence in the pTrc99a plasmid. Using these primers, linear pTrc99aΔlacI with the lacI gene knocked out was amplified using the pTrc99a plasmid as a template. The artificially synthesized operon leuA was then used. M BCD is the template (the gene sequence in this operon is leuA). M The leuB ribosome binding site sequence (AAGGAAACCGTGTG, leuB, leuCD) was used with primer leuA M BCD-S / leuA M BCD-A amplification yielded leuA M BCD. Linear pTrc99aΔlacI and leuA M The PCR product of BCD was recovered and ligated using the ClonExpress II One Step Cloning Kit, then transformed into E. coli DH5α competent cells. Cells were selected using LB solid medium containing 100 μg / mL ampicillin and primer leuA was used. M BCD-S / leuA MThe recombinant plasmid pTrc99aΔlacI-leuA was obtained after BCD-A colony PCR identification. M BCD. (2) Construction of L-leucine engineered strain leu2

[0066] pTrc99aΔlacI-leuA M BCD was transformed into strain leu1, and after resuscitation, it was plated on LB solid medium containing 100 μg / mL ampicillin and incubated overnight at 37°C. The next day, primer leuA was used... M BCD-S / leuA M BCD-A was used for colony PCR identification, and positive transformants were screened to obtain strain leu2.

[0067] Example 4: Construction of L-leucine engineered strain leu3

[0068] (1) Overlapping segment U yeeL -P trc -ilvC-T trc -D yeeL Construction

[0069] Using the genome of E. coli W3110 as a template, the upstream homologous arm, downstream homologous arm, and the part containing P were amplified using primers U-yeeL-S / U-yeeL-A, D-yeeL-S / D-yeeL-A, and ilvC-S / ilvC-A, respectively. trc promoter, T trc Terminator and P of ilvC gene trc -ilvC-T trc Fragment. After PCR product recovery, overlap PCR was performed using primers U-yeeL-S / D-yeeL-A to obtain fragments containing the upstream and downstream homologous arms of yeeL and P. trc -ilvC-T trc fusion fragment U yeeL -P trc -ilvC-T trc -D yeeL (2) Construction of pGRB-yeeL plasmid

[0070] Based on the yeeL sequence, 20bp forward and reverse sequences pGRB-yeeL-S and pGRB-yeeL-A of gRNA were designed and synthesized. The recombinant plasmid pGRB-yeeL was constructed using the same method as step (2) in Example 1.

[0071] (3) Construction of L-leucine engineered strain leu3

[0072] The recombinant plasmid pGRB-yeeL and the fusion fragment U yeeL-P trc -ilvC-T trc -D yeeL Transformed into competent leu2 cells containing the pREDcas9 plasmid, positive transformants were identified using primers U-yeeL-S / D-yeeL-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu3.

[0073] Example 5: Construction of L-leucine engineered strain leu4

[0074] (1) Overlapping segment U ycgH -P trc -bcd-T trc -D ycgH Construction

[0075] Using the genome of *E. coli* W3110 as a template, the upstream and downstream homologous arms of ycgH were amplified using primers U-ycgH-S / U-ycgH-A and D-ycgH-S / D-ycgH-A, respectively. Using the genome of *Bacillus subtilis* 168 as a template, primers bcd-A / D-ycgH-S were used to amplify the portion containing P... trc promoter, T trc Terminator and P of the BCD gene trc -bcd-T trc Fragment. After PCR product recovery, the fragment was obtained by overlap PCR using primers U-ycgH-S / D-ycgH-A, containing the upstream and downstream homologous arms of ycgH and P. trc -bcd-T trc fusion fragment U ycgH -P trc -bcd-T trc -D ycgH .

[0076] (2) Construction of pGRB-ycgH plasmid

[0077] Based on the ycgH sequence, 20bp forward and reverse sequences pGRB-ycgH-S and pGRB-ycgH-A of gRNA were designed and synthesized. The recombinant plasmid pGRB-ycgH was constructed using the same method as step (2) in Example 1.

[0078] (3) Construction of L-leucine engineered strain leu4

[0079] The recombinant plasmid pGRB-ycgH and the fusion fragment U ycgH -P trc -bcd-T trc -D ycgHTransformed into competent leu3 cells containing the pREDcas9 plasmid, positive transformants were identified using primers U-ycgH-S / D-ycgH-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu4.

[0080] Example 6: Construction of L-leucine engineered strain leu5

[0081] (1) Overlapping segment U ilvE -D ilvE Construction

[0082] Using the genome of wild-type Escherichia coli W3110 as a template, the upstream and downstream homologous arms of ilvE were amplified using primers U-ilvE-S / U-ilvE-A and D-ilvE-S / D-ilvE-A, respectively. Then, the fusion fragment U of the upstream and downstream homologous arms of ilvE was obtained by overlap PCR. ilvE -D ilvE .

[0083] (2) Construction of pGRB-ilvE plasmid

[0084] Based on the ilvE sequence, 20bp forward and reverse sequences pGRB-ilvE-S and pGRB-ilvE-A of gRNA were designed and synthesized. The recombinant plasmid pGRB-ilvE was constructed using the same method as step (2) in Example 1.

[0085] (3) Construction of L-leucine engineered strain leu5

[0086] The recombinant plasmid pGRB-ilvE and the fusion fragment U ilvE -D ilvE Electroporation was performed into leu4 competent cells containing the pREDcas9 plasmid, and positive transformants were identified using primers U-ilvE-S / D-ilvE-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu5.

[0087] Example 7: Construction of L-leucine engineered strain leu6

[0088] (1) Overlapping segment U yjgX -P trc -pntAB-T trc -D yjgX Construction

[0089] Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of yjgX were amplified using primers U-yjgX-S / U-yjgX-A, D-yjgX-S / D-yjgX-A, and pntAB-S / pntAB-A, respectively, as well as the arm containing P.trc promoter, T trc Terminator and fragment P of the pntAB gene trc -pntAB-T trc After PCR product recovery, overlap PCR was performed using primers U-yjgX-S / D-yjgX-A to obtain the product containing the upstream and downstream homologous arms of yjgX and P. trc -pntAB-T trc fusion fragment U yjgX -P trc -pntAB-T trc -D yjgX .

[0090] (2) Construction of pGRB-yjgX plasmid

[0091] Based on the yjgX sequence, 20bp forward and reverse sequences pGRB-yjgX-S and pGRB-yjgX-A of gRNA were designed and synthesized. The recombinant plasmid pGRB-yjgX was constructed using the same method as step (2) in Example 1.

[0092] (3) Construction of L-leucine engineered strain leu6

[0093] The recombinant plasmid pGRB-yjgX and the fusion fragment U yjgX -P trc -pntAB-T trc -D yjgX Transformed into competent leu5 cells containing the pREDcas9 plasmid, positive transformants were identified using primers U-yjgX-S / D-yjgX-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu6.

[0094] Example 8: Construction of L-leucine engineered strain leu7

[0095] (1) Overlapping segment U yjiP -P trc -coaA-T trc -D yjiP Construction

[0096] Using the genome of E. coli W3110 as a template, primers U-yjiP-S / U-yjiP-A, D-yjiP-S / D-yjiP-A, and coaA-S / coaA-A were used to amplify the upstream and downstream homologous arms of yjiP, as well as the arm containing P. trc promoter, T trc Terminator and coaA gene P trc -coaA-T trcFragment. After PCR product recovery, overlap PCR was performed using primers U-yjiP-S / D-yjiP-A to obtain fragments containing the upstream and downstream homologous arms of yjiP and P. trc -coaA-T trc fusion fragment U yjiP -P trc -coaA-T trc -D yjiP (2) Construction of pGRB-yjiP plasmid

[0097] Based on the yjiP sequence, 20bp forward and reverse sequences pGRB-yjiP-S and pGRB-yjiP of gRNA were designed and synthesized. The recombinant plasmid pGRB-yjiP was constructed using the same method as step (2) in Example 1.

[0098] (3) Construction of L-leucine engineered strain leu7

[0099] The recombinant plasmid pGRB-yjiP and the fusion fragment U yjiP -P trc -coaA-T trc -D yjiP Transformed into competent leu6 cells containing the pREDcas9 plasmid, positive transformants were identified using primers U-yjiP-S / D-yjiP-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu7.

[0100] Example 9: Construction of L-leucine engineered strain leu8

[0101] (1) Overlapping segment U yghX -P trc -rocG-T trc -D yghX Construction

[0102] Using the genome of *E. coli* W3110 as a template, the upstream and downstream homologous arms of *yghX* were amplified using primers U-yghX-S / U-yghX-A and D-yghX-S / D-yghX-A, respectively. Using the genome of *Bacillus subtilis* 168 as a template, primers rocG-A / D-rocG-S were used to amplify the portion containing P... trc promoter, T trc Terminator and P of the rocG gene trc -rocG-T trc Fragment. After PCR product recovery, overlap PCR was performed using primers U-yghX-S / D-yghX-A to obtain fragments containing the upstream and downstream homologous arms of yghX and P. trc -rocG-T trcfusion fragment U yghX -P trc -rocG-T trc -D yghX .

[0103] (2) Construction of pGRB-yghX plasmid

[0104] Based on the yghX sequence, 20bp forward and reverse sequences pGRB-yghX-S and pGRB-yghX-A of gRNA were designed and synthesized. The recombinant plasmid pGRB-yghX was constructed using the same method as step (2) in Example 1.

[0105] (3) Construction of L-leucine engineered strain leu8

[0106] The recombinant plasmid pGRB-yghX and the fusion fragment U yghX -P trc -rocG-T trc -D yghX Transformed into competent leu7 cells containing the pREDcas9 plasmid, positive transformants were identified using primers U-yghX-S / D-yghX-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu8.

[0107] Example 10: Construction of L-leucine engineered strain leu9

[0108] (1) Overlapping segment U gapC -P trc -fxpk-T trc -D gapC Construction

[0109] Using the genome of *Escherichia coli* W3110 as a template, the upstream and downstream homologous arms of gapC were amplified using primers U-gapC-S / U-gapC-A and D-gapC-S / D-gapC-A, respectively. Using the genome of *Bifidobacterium adolescentis* ATCC 15703 as a template, primers fxpk-A / fxpk-S were used to amplify the portion containing P... trc promoter, T trc Terminator and gene fragment P of fxpk-gene trc -gapC-T trc After PCR product recovery, overlap PCR was performed using primers U-gapC-S / D-gapC-A to obtain the product containing the upstream and downstream homologous arms of gapC and P. trc -gapC-T trc fusion fragment U gapC -P trc-fxpk-T trc -D gapC (2) Construction of pGRB-gapC plasmid

[0110] Based on the gapC sequence, 20bp forward and reverse sequences pGRB-gapC-S and pGRB-gapC-A of gRNA were designed and synthesized. The recombinant plasmid pGRB-gapC was constructed using the same method as step (2) in Example 1.

[0111] (3) Construction of L-leucine engineered strain leu9

[0112] The recombinant plasmid pGRB-gapC and the fusion fragment U gapC -P trc -fxpk-T trc -D gapC Transformed into competent leu8 cells containing the pREDcas9 plasmid, positive transformants were identified using primers U-gapC-S / D-gapC-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu9.

[0113] Example 11: Construction of L-leucine engineered strain leu10 leu9

[0114] (1) Overlapping segment U ackA -D ackA U poxB -D poxB and U ldhA -D ldhA Construction

[0115] Using the genome of wild-type Escherichia coli W3110 as a template, the upstream and downstream homologous arms of ackA, poxB, and ldhA were amplified using primers U-ackA-S / U-ackA-A, D-ackA-S / D-ackA-A, U-poxB-S / U-poxB-A, D-poxB-S / D-poxB-A, U-ldhA-S / U-ldhA-A, and D-ldhA-S / D-ldhA-A, respectively. Then, overlap PCR was used to obtain the fusion fragment U of the upstream and downstream homologous arms. ackA -D ackA U poxB -D poxB and U ldhA -D ldhA .

[0116] (2) Construction of pGRB-ackA, pGRB-poxB and pGRB-ldhA plasmids

[0117] Based on the ackA, poxB, and ldhA sequences, 20bp forward and reverse sequences pGRB-ackA-S / pGRB-ackA-A, pGRB-poxB-S / pGRB-poxB-A, and pGRB-ldhA-S / pGRB-ldhA-A were designed and synthesized. Recombinant plasmids pGRB-ackA, pGRB-poxB, and pGRB-ldhA were constructed using the same method as step (2) in Example 1.

[0118] (3) Construction of L-leucine engineered strain leu10

[0119] The recombinant plasmid pGRB-ackA and the fusion fragment U ackA -D ackA Electroporation was performed into Leu9 competent cells containing the pREDcas9 plasmid, and positive transformants were identified using primers U-ackA-S / D-ackA-A. The plasmid was then lost using the same method as in step (3) of Example 1 to obtain strain leu9ΔackA. The recombinant plasmid pGRB-poxB and the fusion fragment U... poxB -D poxB Electroporation was performed into leu9ΔackA competent cells containing the pREDcas9 plasmid, and positive transformants were identified using primers U-poxB-S / D-poxB-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain the strain leu9ΔackAΔpoxB. The recombinant plasmid pGRB-ldhA and the fusion fragment U... ldhA -D ldhA Electroporation was performed into leu9ΔackAΔpoxB competent cells containing the pREDcas9 plasmid, and positive transformants were identified using primers U-ldhA-S / D-ldhA-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu10.

[0120] Example 12: Construction of L-leucine engineered strain leu11

[0121] (1) Overlapping segment U gltA -P leuA -D gltA Construction

[0122] Using the genome of Escherichia coli W3110 as a template, primers U-gltA-S / U-gltA-A, D-gltA-S / D-gltA-A, and P were used respectively. leuA -S / P leuA -A amplifies the upstream and downstream homologous arms of the gltA promoter and the P gene promoter of the leuA gene. leuAAfter PCR product recovery, overlap PCR was performed using primers U-gltA-S / D-gltA-A to obtain the homologous arms upstream and downstream of the gltA gene promoter, P... leuA fusion fragment U gltA -P leuA -D gltA .

[0123] (2) Construction of pGRB-gltA plasmid

[0124] Based on the promoter sequence of the gltA gene, 20bp forward and reverse sequences pGRB-P of gRNA were designed and synthesized. gltA -S and pGRB-P gltA -A, construct the recombinant plasmid pGRB-P using the same method as step (2) in Example 1. gltA .

[0125] (3) Construction of L-leucine engineered strain leu11

[0126] The recombinant plasmid pGRB-P gltA and fusion fragment U gltA -P leuA -D gltA Transformed into competent leu10 cells containing the pREDcas9 plasmid, positive transformants were identified using primers U-gltA-S / D-gltA-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu11.

[0127] Example 13: Construction of L-leucine engineered strain leu12

[0128] (1) Overlapping segment U yjiT -P trc -yeaS-T trc -D yjiT Construction

[0129] Using the genome of *E. coli* W3110 as a template, the upstream and downstream homologous arms of the yjiT gene, as well as the arm containing P, were amplified using primers U-yjiT-S and U-yjiT-A, D-yjiT-S / D-yjiT-A, and yeaS-S / yeaS-A, respectively. trc promoter, T trc Terminator and fragment P of the yeaS gene trc -yeaS-T trc After PCR product recovery, overlap PCR was performed using primers U-yjiT-S / D-yjiT-A to obtain the product containing the upstream and downstream homologous arms of yjiT and P. trc -yeaS-T trc fusion fragment UyjiT -P trc -yeaS-T trc -D yjiT .

[0130] (2) Construction of pGRB-yjiT plasmid

[0131] Based on the yjiT sequence, 20bp forward and reverse sequences pGRB-yjiT-S and pGRB-yjiT-A of gRNA were designed and synthesized. The recombinant plasmid pGRB-yjiT was constructed using the same method as step (2) in Example 1.

[0132] (3) Construction of L-leucine engineered strain leu12

[0133] The recombinant plasmid pGRB-yjiT and the fusion fragment U yjiT -P trc -yeaS-T trc -D yjiT Transformed into competent cells of leu11 containing the pREDcas9 plasmid, positive transformants were identified using primers U-yjiT-S / D-yjiT-A. The plasmid was then lost using the same method as step (3) in Example 1 to obtain strain leu12.

[0134] Example 14: Shake-flask fermentation of L-leucine engineered bacteria

[0135] (1) Seed culture

[0136] L-leucine-engineered bacteria leu1, leu2, leu3, leu4, leu5, leu6, leu7, leu8, leu9, leu10, leu11, and leu12 were inoculated onto LB agar slants, with E. coli W3110ΔlacI as a control, and cultured at 37°C for 12 h. They were then inoculated into 30 mL of seed culture medium and cultured at 37°C with shaking at 220 rpm for 6-8 h.

[0137] (2) Fermentation culture

[0138] Inoculate the fermentation medium with 1% inoculum and culture at 35°C and 220 rpm for 20 h using a shaker. During fermentation, supplement with 80% glucose 2-3 times as needed to maintain the residual sugar concentration at 0.1-0.5%, adding 1 mL each time, and adjust the pH to approximately 7 with ammonia.

[0139] (3) Detection of L-leucine in fermentation broth

[0140] After 20 hours of fermentation, the fermentation broth was centrifuged at 8000×g for 10 minutes, and the supernatant was taken and diluted 10 times with deionized water. The fermentation broth was then derivatized with 0.8% (V / V) 2,4-dinitrofluorobenzene, and the L-leucine content was determined by high performance liquid chromatography. The L-leucine yields of strains leu1, leu2, leu3, leu4, leu5, leu6, leu7, leu8, leu9, leu10, leu11, and leu12 reached 0.5 g / L, 7.6 g / L, 9.1 g / L, 11.8 g / L, 13.2 g / L, 14.7 g / L, 16.4 g / L, 17.8 g / L, 22.3 g / L, 24.6 g / L, 32.5 g / L, and 36.8 g / L, respectively. The control strain E. coli W3110ΔlacI showed no L-leucine synthesis (e.g., ...). Figure 1 (As shown).

[0141] (4) Culture medium

[0142] The seed culture medium consisted of: 20 g / L glucose, 10 g / L yeast extract, 6 g / L peptone, 1.2 g / L KH₂PO₄, 1 g / L MgSO₄·7H₂O, 10 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·7H₂O, and 1.3 mg / L MgSO₄·7H₂O. B1 0.3 mg / LV H 20 mL / L phenol red, the remainder being water, pH 7.0-7.2, autoclaved at 115°C for 15 min.

[0143] The fermentation medium consisted of: 20 g / L glucose, 2 g / L yeast extract, 4 g / L peptone, 2 g / L KH₂PO₄, 1.8 g / L MgSO₄·7H₂O, 1 g / L sodium citrate dihydrate, 10 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·7H₂O, and 0.8 mg / L MgSO₄·7H₂O. B1 0.2mg / LV H 20 mL / L phenol red, the remainder being water, pH 7.0-7.2, autoclaved at 115°C for 15 min.

[0144] Example 15: Fermentation of L-leucine engineered bacteria in a 5L fermenter

[0145] (1) Seed culture

[0146] Using an inoculation loop, inoculate 3-5 strains of L-leucine engineered bacteria (leu1, leu2, leu3, leu4, leu5, leu6, leu7, leu8, leu9, leu10, leu11, and leu12) activated on fresh LB slant medium into a 5L fermenter containing 2.5L of seed culture medium. Adjust the pH of the fermentation broth to 6.8-7.2 by adding 25% ammonia water, maintain dissolved oxygen at 20-40%, and aerate at 2-4 m³ / h. 3 / h, stirring speed 200-800rpm, incubate at 37℃ for 6h.

[0147] (2) Fermentation culture

[0148] The seed culture from step (1) was inoculated at a rate of 10% into a 5L fermenter containing 3L of fermentation medium for fermentation culture at a temperature of 35℃ and an aeration rate of 2-4m³ / h. 3 Stirring speed: 300-900 rpm, dissolved oxygen maintained at 30-40%, add 80% glucose solution to maintain residual sugar concentration at 0.1-0.5%, add 25% ammonia to adjust the pH of fermentation broth to 6.8-7.2. Ferment each strain until its maximum L-leucine yield is reached.

[0149] (3) Detection of L-leucine in fermentation broth

[0150] After centrifuging the fermentation broth at 8000×g for 10 min, the supernatant was collected and diluted 10 times with deionized water. The fermentation broth was then derivatized using 0.8% (v / v) 2,4-dinitrofluorobenzene, and the L-leucine content was determined by high-performance liquid chromatography (HPLC). The maximum L-leucine yield and fermentation time for each strain are shown in Table 1.

[0151] Table 1. Fermentation parameters of L-leucine engineered strains

[0152]

[0153]

[0154] The engineered strain leu12, after 34 hours of fermentation, achieved an L-leucine yield of 85.6 g / L, a conversion rate of 36.6%, and a production intensity of 2.52 g / L / h. These figures represent the highest reported L-leucine yield, conversion rate, and production intensity to date, while the fermentation cycle is the shortest reported to date.

[0155] (4) Culture medium

[0156] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 2 g / L peptone, 2 g / L KH₂PO₄, 1.5 g / L MgSO₄·7H₂O, 10 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·7H₂O, and 1.3 mg / L V. B1 0.3 mg / LV H 8 mL / L corn steep liquor, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0157] The fermentation medium consisted of: 10 g / L glucose, 3 g / L yeast extract, 1 g / L glutamic acid, 3 g / L KH₂PO₄, 1.8 g / L MgSO₄·7H₂O, 2 g / L sodium citrate dihydrate, 10 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·7H₂O, and 2 mg / L L V. B1 0.2mg / LV H 8 mL / L corn steep liquor, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0158] Example 16: Fermentation of L-leucine engineered bacteria leu12 in a 5L fermenter

[0159] (1) Seed culture

[0160] Same as step (1) in Example 15

[0161] (2) Fermentation culture

[0162] The seed culture from step (1) was inoculated at a rate of 5% into a 5L fermenter containing 3L of fermentation medium for fermentation culture at a fermentation temperature of 32℃ and an aeration rate of 2-3m³ / h. 3 Stirring speed: 300-700 rpm, dissolved oxygen maintained at 25-40%, add 80% glucose solution to maintain residual sugar concentration at 0.1-0.5%, add 25% ammonia to adjust the pH of fermentation broth to 6.8-7.2. Ferment for 30 hours.

[0163] (3) Detection of L-leucine in fermentation broth

[0164] Same as step (3) in Example 15. After 30 hours of fermentation, the L-leucine engineered strain leu12 produced 72.7 g / L of L-leucine, with a conversion rate of 32.3% and a production intensity of 2.42 g / L / h.

[0165] (4) Culture medium

[0166] Same as step (4) in Example 15.

[0167] Example 17: Fermentation of L-leucine engineered bacteria leu12 in a 5L fermenter

[0168] (1) Seed culture

[0169] Same as step (1) in Example 15

[0170] (2) Fermentation culture

[0171] The seed culture from step (1) was inoculated at an 8% inoculum into a 5L fermenter containing 3L of fermentation medium for fermentation culture at a fermentation temperature of 37℃ and an aeration rate of 3-5m³ / h. 3 Stirring speed: 400-900 rpm, dissolved oxygen maintained at 40-50%, add 80% glucose solution to maintain residual sugar concentration at 0.1-0.5%, add 25% ammonia to adjust the pH of fermentation broth to 6.8-7.2. Ferment for 36 hours.

[0172] (3) Detection of L-leucine in fermentation broth

[0173] Same as step (3) in Example 15. After 36 hours of fermentation, the L-leucine engineered strain leu12 produced 79.3 g / L of L-leucine, with a conversion rate of 36.2% and a production intensity of 2.20 g / L / h.

[0174] (4) Culture medium

[0175] Same as step (4) in Example 15.

[0176] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A high-yield L-leucine strain, characterized in that, The strain is Escherichia coli (Escherichia coli) Escherichia coli W3110 was the starting strain, and the gene encoding the lactose operon repressor protein was knocked out in the genome. lacI Overexpression of the gene encoding acetylhydroxyl synthase, which relieves feedback inhibition, on the genome ilvBN M Plasmid overexpression was used to relieve the feedback inhibition of the isopropyl malate synthase encoding gene by L-leucine. leuA M β-Isopropylmalate dehydrogenase encoding gene leuB α-Isopropylmalate isomerase encoding gene leuCD Overexpression of the gene encoding hydroxy acid reductase on the genome ilvC Bacillus subtilis ( Bacillus subtilis The gene encoding leucine dehydrogenase bcd Knockout of the gene encoding branched-chain amino acid aminotransferase ilvE Genes that overexpress pyridine nucleotide transhydrogenase in the genome pntAB ubiquitin kinase encoding gene coaA Glutamate dehydrogenase encoding gene rocG and the gene encoding phosphoryl ketolase fxpk Knockout of the gene encoding acetate kinase ackA pyruvate dehydrogenase encoding gene poxB lactate dehydrogenase encoding gene ldhA Based on this, the gene encoding citrate synthase in the genome was identified. gltA The promoter is replaced with a P-type promoter weakened by leucine. leuA Promoter; overexpression of the L-leucine transporter encoding gene on the genome yeaS Ultimately, a high-yield strain of L-leucine was obtained; The lactose operon repressor protein encoding gene lacI From Escherichia coli W3110; The gene encoding acetylhydroxyl synthase that relieves feedback inhibition ilvBN M It has been published in Chinese invention patent ZL201910484362.5; The gene encoding L-leucine feedback inhibition of isopropyl malate synthase was removed. leuA M It has been published in Chinese invention patent ZL 201910820591.X; The gene encoding β-isopropylmalate dehydrogenase leuB From Escherichia coli W3110; The α-isopropylmalate isomerase encoding gene leuCD From Escherichia coli W3110; The hydroxy acid reductase encoding gene ilvC From Escherichia coli W3110; The gene encoding the leucine dehydrogenase bcd From Bacillus subtilis 168; The branched-chain amino acid aminotransferase encoding gene ilvE From Escherichia coli W3110; The gene for the pyridine nucleotide transhydrogenase pntAB From Escherichia coli W3110; The ubiquitin kinase encoding gene coaA From Escherichia coli W3110; The gene encoding glutamate dehydrogenase rocG From Bacillus subtilis 168; The phosphoryl ketonease encoding gene fxpk From Bifidobacterium adolescentis ( Bifidobacterium adolescentis ATCC 15703; The acetate kinase encoding gene ackA From Escherichia coli W3110; The pyruvate dehydrogenase encoding gene poxB From Escherichia coli W3110; The lactate dehydrogenase encoding gene ldhA From Escherichia coli W3110; The citrate synthase encoding gene gltA From Escherichia coli W3110; The promoter P leuA for leuA The promoter is derived from Escherichia coli W3110; The L-leucine transporter gene yeaS From Escherichia coli W3110.

2. The high-yield L-leucine strain as described in claim 1, characterized in that: The lactose operon repressor protein encoding gene lacI From Escherichia coli W3110, Protein ID in NCBI: BAE76127.1; The gene encoding acetylhydroxyl synthase that relieves feedback inhibition ilvBN M The sequence is as shown in SEQ ID NO.2 of Chinese invention patent ZL201910484362.5; The gene encoding L-leucine feedback inhibition of isopropyl malate synthase was removed. leuA M The sequence is as shown in SEQ ID NO.2 of Chinese invention patent ZL 201910820591.X; The gene encoding β-isopropylmalate dehydrogenase leuB From Escherichia coli W3110, Protein ID in NCBI: BAB96642.1; The α-isopropylmalate isomerase encoding gene leuCD From Escherichia coli W3110, Protein IDs in NCBI: BAB96641.2 and BAB96640.1; The hydroxy acid reductase encoding gene ilvC From Escherichia coli W3110, Protein ID in NCBI: BAE77523.1; The gene encoding the leucine dehydrogenase bcd From Bacillus subtilis 168, Protein ID in NCBI: NP_390288.1; The branched-chain amino acid aminotransferase encoding gene ilvE From Escherichia coli W3110, NCBI Protein ID: BAE77527.1; The gene for the pyridine nucleotide transhydrogenase pntAB From Escherichia coli W3110, Protein IDs in NCBI: BAA15342.1 and BAA15336.1; The ubiquitin kinase encoding gene coaA From Escherichia coli W3110, Protein ID in NCBI: BAE77341.1; The gene encoding glutamate dehydrogenase rocG From Bacillus subtilis 168, Protein ID in NCBI: NP_391659.2; The phosphoryl ketonease encoding gene fxpk From Bifidobacterium adolescentis ATCC 15703, Protein ID in NCBI: WP_011743105.1; The acetate kinase encoding gene ackA From Escherichia coli W3110, Protein ID in NCBI: BAA16135.1; The pyruvate dehydrogenase encoding gene poxB From Escherichia coli W3110, Protein ID in NCBI: BAA35585.1; The lactate dehydrogenase encoding gene ldhA From Escherichia coli W3110, Protein ID in NCBI: BAA14990.1; The citrate synthase encoding gene gltA From Escherichia coli W3110, Protein ID in NCBI: BAA35384.2; The promoter P leuA for leuA The promoter is derived from *E. coli* W3110, Protein ID in NCBI: BAB96643.2; The L-leucine transporter gene yeaS From Escherichia coli W3110, Protein ID in NCBI: BAA15593.

1.

3. The use of the strain described in claim 1 or 2 in the production of L-leucine.

4. The application as described in claim 3, characterized in that, The application of L-leucine in fermentation production is described in the following methods: Inoculate the seed culture into the fermentation medium at an inoculum rate of 5-10% for fermentation culture, with an aeration rate of 2-5 m³ / h. 3 The stirring speed is 300-900 rpm, the dissolved oxygen is maintained at 20-50%, the pH is maintained at 6.8-7.2, the culture temperature is 32-37℃, the fermentation cycle is 30-36h, and the residual sugar concentration (W / V) is maintained at 0.1-0.5% during the fermentation process.

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