WO2024011666A1 - 一种l-高丝氨酸高产菌株及其构建方法和应用 - Google Patents

一种l-高丝氨酸高产菌株及其构建方法和应用 Download PDF

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WO2024011666A1
WO2024011666A1 PCT/CN2022/108501 CN2022108501W WO2024011666A1 WO 2024011666 A1 WO2024011666 A1 WO 2024011666A1 CN 2022108501 W CN2022108501 W CN 2022108501W WO 2024011666 A1 WO2024011666 A1 WO 2024011666A1
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fadd
seq
homoserine
nucleotide sequence
fadr
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张莎莎
史鲁秋
苏桂珍
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南京盛德生物科技研究院有限公司
南京盛德百泰生物科技有限公司
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Definitions

  • the invention belongs to the field of bioengineering technology, and specifically relates to an L-homoserine high-yield strain and its construction method and application.
  • L-homoserine is a naturally occurring non-essential amino acid and is a non-protein amino acid. It is a prerequisite for the synthesis of important high-accessory compounds such as L-methionine. L-homoserine and its derivatives have important application prospects in pharmacology, physiology and other aspects as pharmaceutical intermediates.
  • microbial fermentation methods at home and abroad have many advantages such as low cost, mild conditions, and less environmental pollution. In recent years, they have become the preferred process for producing various amino acids.
  • glucose is used as the substrate, and there is insufficient reducing power and energy (Glucose+2HCO 3 - +2NH + +2ATP+4NADPH ⁇ 2HS).
  • Glucose+2HCO 3 - +2NH + +2ATP+4NADPH ⁇ 2HS Part of the glucose needs to be consumed to provide reducing power, which will cause the sugar to Acid conversion rate decreases.
  • Fatty acids have received more and more attention as a carbon source.
  • the reduction degree of fatty acids is higher than that of glucose.
  • Using fatty acids as a carbon source can not only provide a carbon skeleton, but also provide a large amount of reducing power and energy.
  • palmitic acid 1 molecule When palmitic acid is converted into 4 molecules of L-homoserine, it is accompanied by 11 molecules of FADH and 11 molecules of NADH (C 16: 0 +4NH 4 +5ATP+8NADPH ⁇ 4HS+11FADH+11NADH), eliminating the 8 molecules of NADPH and 11 molecules necessary for the reaction. 5 molecules of ATP, there is still a lot of reducing power and energy left.
  • the main purpose of the present invention is to provide an L-homoserine high-producing strain and its construction method and application in view of the above problems.
  • the present invention constructs a strain with high L-homoserine production through genetic engineering, and uses glucose + fatty acid dual substrates as carbon sources to synthesize L-homoserine, resulting in higher L-homoserine yield, higher sugar-acid conversion rate, and further reducing The production cost is reduced, the production process is more green and environmentally friendly, and the market competitive advantage is obvious.
  • One aspect of the invention provides an L-homoserine high-producing strain, which is Escherichia coli (Escherichia coli), strain number 13-XA, and was deposited in the China Microbial Culture Collection on June 16, 2022. Committee General Microbiology Center, deposit number: CGMCC No. 25099.
  • Escherichia coli Escherichia coli
  • strain number 13-XA was deposited in the China Microbial Culture Collection on June 16, 2022. Committee General Microbiology Center, deposit number: CGMCC No. 25099.
  • the engineered strain 13-XA is one or more genes related to fatty acid metabolism on the chromosomal DNA that have been knocked out or weakened, and/or the promoter has been replaced to enhance; one or more genes related to the L-homoserine metabolic pathway is knocked out or weakened, and/or one or more genes related to the L-homoserine metabolic pathway are overexpressed or enhanced, and/or one or more genes related to the L-homoserine metabolic pathway are mutated.
  • fadR DNA-binding transcription dual regulator gene
  • fadD ligase gene
  • Another aspect of the invention also provides a method for constructing a strain for efficient fermentation production of L-homoserine, including:
  • Plasmid insert the aspartokinase/homoserine dehydrogenase 1 gene thrA (S345F) that relieves feedback inhibition between the NcoI and EcoRI sites of the plasmid vector pXB1k to obtain a recombinant vector, named pXA;
  • the mutant E. coli ST11 is recorded in patent 202011270812. ::asd, ⁇ poxB::aspA, ⁇ iclR, ⁇ lysA, ⁇ metA, ⁇ thrB;
  • the genotype of the mutant E. coli ST13 is E.coli ST11 ⁇ fadR, ⁇ P fadD ::P CPA1 ;
  • the aspartokinase/homoserine dehydrogenase 1 gene thrA (S345F) is derived from E. coli K-12 MG1655.
  • the construction steps of the recombinant vector plasmid pXA are as follows:
  • PCR amplification was performed using primers thrA-F and S345F-R, S345F-F and thrA-R to obtain the aspartokinase/homoserine dehydrogenase 1 gene that relieves feedback inhibition.
  • the recombinant vector plasmid pXA is obtained by replacing the fragment between the NcoI and EcoRI sites of the pXB1k vector with the aspartokinase/homoserine dehydrogenase 1 gene that relieves feedback inhibition; the core of the pXB1k vector
  • the nucleotide sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the aspartokinase/homoserine dehydrogenase 1 gene that relieves feedback inhibition is shown in SEQ ID NO.2.
  • the construction steps of the mutant E. coli ST13 are as follows:
  • pTargetF As the template, use primers pTarget-fadR-F and pTarget-fadR-R, pTarget-fadDp-F and pTarget-fadDp-R to perform PCR amplification respectively, and use DpnI methylation to amplify the fragments After digestion with enzyme, transform the E. coli Fast-T1 competent cells, screen the positive clones on LB plates containing streptomycin, and verify them with primer pTarget-cexu-F sequencing. After the sequencing is correct, they will be named pTarget-fadR and pTarget-fadDp respectively;
  • R, fadD-down500-F and fadD-down500-R were PCR amplified to obtain three fragments respectively.
  • primers were used to PCR amplify fadD-up500-F and fadD-down500-R.
  • step (3) Pick positive clones from the plate in step (3), prepare electroporated competent cells, mix them with pTarget-fadR plasmid and targeting fragment ⁇ fadR, place them in an electroporation cup for electroporation, add LB liquid culture medium for recovery at 30°C, and apply Spread on LB plates containing kanamycin and streptomycin, culture at 30°C, screen positive clones, and use primers fadR-up700-F and fadR-down700-R to perform PCR amplification, and sequence the amplified fragments to verify the screening. Positive clones emerged;
  • it also includes the step of preparing electrotransfection-competent cells: transforming the pCas plasmid into E. coli ST11 using a chemical transformation method, culturing it on an LB plate containing kanamycin at 30°C to select positive clones, and inoculating the positive clones in a solution containing 2g/L After culturing in LB liquid medium containing arabinose at 30°C until the OD 600 is about 0.6, prepare electroporated competent cells.
  • the nucleotide sequence of the forward primer pTarget-fadR-F described in step (1) is as shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer pTarget-fadR-R is as shown in SEQ ID Shown in NO.10; the nucleotide sequence of the forward primer pTarget-fadDp-F is shown in SEQ ID NO.11, and the nucleotide sequence of the reverse primer pTarget-fadDp-R is shown in SEQ ID NO. Shown in 12;
  • the PCR amplification system is: 5X SF Buffer 10ul, dNTP Mix (10mM each) 1ul, template pTargetF 20ng, primers (10uM) 2ul each, Phanta Super-Fidelity DNA Polymerase 1ul, distilled water 34ul, the total volume is 50ul;
  • the PCR amplification conditions are: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 1.5 minutes (30 cycles); extension at 72°C for 10 minutes (1 cycle).
  • the nucleotide sequence of the forward primer fadR-up500-F in step (2) is as shown in SEQ ID NO.19, and the nucleotide sequence of the reverse primer fadR-up500-R is as shown in SEQ ID Shown in NO.20; the nucleotide sequence of the forward primer fadR-down500-F is shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer fadR-down500-R is shown in SEQ ID NO.
  • nucleotide sequence of the forward primer fadD-up500-F is shown in SEQ ID NO.13
  • nucleotide sequence of the reverse primer fadD-up500-R is shown in SEQ ID NO.14 shown
  • nucleotide sequence of the forward primer CPA1-fadD-F is shown in SEQ ID NO.15
  • nucleotide sequence of the reverse primer CPA1-fadD-R is shown in SEQ ID NO.16
  • the nucleotide sequence of the forward primer fadD-down500-F is shown in SEQ ID NO.17
  • nucleotide sequence of the reverse primer fadD-down500-R is shown in SEQ ID NO.18;
  • the PCR amplification system is: 5X SF Buffer 10ul, dNTP Mix (10mM each) 1ul, template 5-20ng, primers (10uM) 2ul each, Phanta Super-Fidelity DNA Polymerase 1ul, distilled water 34ul, the total volume is 50ul;
  • the PCR amplification conditions are: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 0.5-2 minutes (30 seconds/kb) (30 cycles) ; Extension at 72°C for 10 minutes (1 cycle).
  • the nucleotide sequence of the forward primer fadR-up700-F described in step (3) is as shown in SEQ ID NO23
  • the nucleotide sequence of the reverse primer fadR-down700-R is as shown in SEQ ID NO. 24
  • the nucleotide sequence of the forward primer fadD-up700-F in step (5) is shown in SEQ ID NO.25
  • the nucleotide sequence of the reverse primer fadD-down700-R is shown in SEQ ID Shown in NO.26.
  • Another aspect of the present invention also provides the use of an L-homoserine high-producing strain for preparing L-homoserine.
  • the application is to inoculate the activated high-efficiency fermentation-producing L-homoserine strain into a fermentation medium and use a biological fermentation method to prepare L-homoserine.
  • the method includes:
  • the temperature is 37°C
  • the initial air flux is 2vvm
  • the stirring speed is 300rpm
  • the dissolved oxygen concentration at this time is set to 100%
  • the air flux is adjusted until 3vvm during the bacterial growth process
  • the stirring speed is controlled by correlating the DO value.
  • the dissolved oxygen concentration is always greater than 30%.
  • the fermentation medium is composed of: citric acid 1-5g/L, potassium dihydrogen phosphate 1-20g/L, nitrogen source 1-5g/L, polyether defoaming agent 150uL/L, glucose 5-5g/L. 30g/L, MgSO 4 ⁇ 7H 2 O 0.3-1g/L, VB1 5-10mg/L, lysine 0.1-1g/L, methionine 0.1-1g/L, isoleucine 0.1-1g/ L, threonine 0.1-1g/L, trace inorganic salt I 1-10mL/L, pH 7.0 ⁇ 0.5;
  • composition of the feeding medium is: glucose 100-800g/L, MgSO 4 ⁇ 7H 2 O 1-5g/L, lysine 1-10g/L, methionine 1-10g/L, isoleucine Acid 1-10g/L, threonine 1-10g/L, palmitic acid 2-5g/L, trace inorganic salt II 1-10mL/L.
  • the composition of the trace inorganic salt I in the fermentation medium is: EDTA 840 mg/L, CoCl 2 ⁇ 6H 2 O 250 mg/L, MnCl 2 ⁇ 4H 2 O 1500 mg/L, CuCl 2 ⁇ 2H 2 O 150 mg /L, H 3 BO 3 300mg/L, Na 2 MoO 4 ⁇ 2H 2 O 250mg/L, Zn(CH 3 COO) 2 ⁇ 2H 2 O 1300mg/L, iron citrate 10g/L;
  • the nitrogen source is selected One or more of ammonium chloride, ammonium acetate, ammonium sulfate and ammonium phosphate;
  • composition of the trace inorganic salt II in the feed medium is: EDTA 1300mg/L, CoCl 2 ⁇ 6H 2 O 400mg/L, MnCl 2 ⁇ 4H 2 O 2350mg/L, CuCl 2 ⁇ 2H 2 O 250mg/L , H 3 BO 3 500mg/L, Na 2 MoO 4 ⁇ 2H 2 O 400mg/L, Zn(CH 3 COO) 2 ⁇ 2H 2 O 1600mg/L, ferric citrate 4g/L.
  • the present invention has at least the following advantages: the present invention constructs a mutant Escherichia coli recombinant engineering strain 13-XA that can efficiently produce L-homoserine.
  • the present invention obtains an engineering strain with high L-homoserine production through genetic engineering, and adopts glucose + palmitic acid culture to further reduce production costs, make the production process more green and environmentally friendly, and have obvious market competitive advantages.
  • the double-substrate biological production process adopted by the present invention replaces the traditional petrochemical process, uses renewable bio-based raw materials as raw materials to replace non-renewable petrochemical raw materials, and realizes energy conservation, emission reduction, clean production, green environmental protection, and circular economy. Big industry. Through continuous optimization and upgrading of bacterial strains and manufacturing processes, the products have better quality and lower costs, and have great market prospects.
  • Figure 1 shows the physical map of pXB1k
  • Figure 2 shows the change curve of L-homoserine production with time during the fermentation process of E. coli 13-XA.
  • the sequencing verification process involved is completed by a third-party testing organization, which is Suzhou Jinweizhi Biotechnology Co., Ltd.
  • E. coli K12 is recorded in the document "Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H: Construction of Escherichia coli K- 12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006, 2: 2006.0008.” is a non-pathogenic bacterium with a clear genetic background, short generation time, easy cultivation and low-cost medium raw materials.
  • GenBank Accession of the whole genome sequence of E. coli K12 is U00096.3 (GI: 545778205, update date is AUG 01, 2014, version is 3). The public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only used to replicate the present invention. It is used for related experiments and cannot be used for other purposes.
  • 1 molecule of glucose generates 2 molecules of phosphoenolpyruvate through the glycolysis pathway. It combines with 2 molecules of CO2 under the action of carboxylase to generate 2 molecules of oxaloacetate. The oxaloacetate is converted into 2 molecules of aspartate under the action of transaminase aspC.
  • Acid or converted into 2 molecules of fumaric acid through the reverse TCA cycle, which is converted into 2 molecules of aspartic acid under the action of ammonia lyase aspA, and aspartate is further converted into 2 molecules under the action of bifunctional aspartokinase
  • Aspartate phosphate aspartate phosphate is converted into aspartate semialdehyde by the action of aspartate semialdehyde dehydrogenase, and further converted into 2 molecules of homoserine under the action of bifunctional aspartate kinase.
  • 1 molecule of glucose can be converted into 2 molecules of homoserine, but the reducing power and energy of this pathway are insufficient, and part of the glucose needs to be consumed to provide reducing power and energy.
  • fatty acids can also be used by bacteria to convert them into the nutrients they need.
  • the process of fatty acid oxidation provides a large amount of reducing power.
  • the complete oxidation of 1 molecule of palmitic acid can provide 16 molecules of NADH.
  • the invention added a small amount of palmitic acid to provide reducing power during the fermentation process, thereby greatly increasing the yield of homoserine to 154g/L.
  • the coding sequence of the aspartokinase/homoserine dehydrogenase 1 gene that relieves feedback inhibition is shown in SEQ ID NO. 2.
  • the coding sequence of the DNA-binding transcription dual regulator gene (fadR) is shown as Gene ID: 948652 (composed of 720 nucleotides), encoding the DNA-binding transcription dual regulator gene shown as Acession number NP_415705 (composed of 239 amino acid residues) regulatory factors;
  • the coding sequence of the long-chain fatty acid coenzyme a ligase gene is shown in Gene ID: 946327 (composed of 1686 nucleotides), encoding the long-chain fatty acid shown in Acession number NP_416319 (composed of 561 amino acid residues) Coenzyme A ligase.
  • the nucleotide sequence of the pXB1k vector in the following examples is shown in SEQ ID NO.1, including the following fragments: (1) araC-araBAD-MCS fragment (containing arabinose-inducible promoter, multiple cloning site); (2) ) MCS-TrrnB fragment (containing multiple cloning site and terminator TrrnB); (3) p15A replication origin site fragment; (4) kanamycin resistance gene Kan fragment.
  • the pXB1k vector map is shown in Figure 1.
  • Example 1 Construction of recombinant plasmid pXA expressing aspartokinase/homoserine dehydrogenase 1 that relieves feedback inhibition
  • PCR amplification was performed using primers thrA-F and S345F-R, S345F-F and thrA-R to obtain the aspartokinase/homoserine dehydrogenase 1 gene that relieves feedback inhibition. 2 fragments thrA-1 and thrA-2. After the pXB1k vector was double-digested with NcoI and EcoRI, a large fragment of the vector was recovered, about 3450 bp.
  • the recovered thrA-1 and thrA-2 gene fragments and the large fragment of the vector were analyzed using the Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6: 343-345.) for ligation, and the ligation product was transformed into Fast-T1 competent cells (Nanjing Novizan Biotech Technology Co., Ltd., product catalog C505), coated LB solid plates containing kanamycin.
  • Gibson method Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6: 343-345.
  • the primer sequences are as follows:
  • the promoter that starts the transcription of the aspartokinase/homoserine dehydrogenase 1 gene that releases feedback inhibition is pBAD Promoter.
  • the E. coli mutant constructed in this example is ST11, and its construction method can be found in Chinese patent application CN202011270812.X.
  • Escherichia coli mutant ST13 was developed using CRISPR technology (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Appl Environ Microbiol 2015,81:2506 -2514.)
  • the mutant of E. coli ST11 obtained by knocking out the DNA-binding transcription repressor gene (fadR) of E. coli ST11 and strengthening the promoter of the long-chain fatty acid coenzyme a ligase gene (fadD) is referred to in this application as ST13, its genotype is E.coli ST11 ⁇ fadR CPA1-fadD.
  • the E. coli mutant ST13 is obtained by knocking out the DNA-binding transcription repressor gene (fadR) of E. coli ST11 and strengthening the promoter of the long-chain fatty acid coenzyme a ligase gene (fadD) E. coli mutant (ST13 for short).
  • the specific construction steps of E. coli mutant ST11 are as follows:
  • the PCR amplification system is: 5X SF Buffer 10ul, dNTP Mix (10mM each) 1ul, template pTargetF 20ng, primers (10uM) 2ul each, Phanta Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd., product catalog: P501)1ul, distilled water 34ul, the total volume is 50ul.
  • Amplification conditions are: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, extension at 72°C for 1.5 minutes (30 cycles); extension at 72°C for 10 minutes (1 cycle) .
  • primer pairs fadR-up500-F and fadR-up500-R, fadR-down500-F and fadR-down500-R to perform PCR amplification respectively, and obtain fragments of approximately 500bp and 500bp respectively; the mixture of the two fragments is Template, use primers fadR-up500-F and fadR-down500-R to perform PCR amplification, and obtain a ⁇ fadR targeting fragment with a size of approximately 1000 bp.
  • the PCR amplification system is: 5X SF Buffer 10ul, dNTP Mix (10mM each) 1ul, template 5-20ng, primers (10uM) 2ul each, Phanta Super-Fidelity DNA Polymerase (Nanjing Novozan Biotechnology Co., Ltd., product catalog It is P501) 1ul, distilled water 34ul, the total volume is 50ul.
  • Amplification conditions are: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, extension at 72°C for 0.5-2 minutes (30 seconds/kb) (30 cycles); 72°C Stretch for 10 minutes (1 cycle).
  • the targeting fragments fadD::CPA1-fadD and ⁇ fadR were recovered respectively.
  • the targeting fragment contains a 500bp upstream homology arm, a replacement gene expression cassette and a 500bp downstream homology arm from upstream to downstream.
  • the primer sequences used are as follows:
  • Electrotransformation Mix 200ng pTarget-fadR plasmid, 400ng targeting fragment ⁇ fadR and 100 ⁇ l electrotransformed competent cells prepared in step (1), place them in a 2mm electroporation cup, electroshock at 2.5kV, and add 1ml LB liquid culture medium After recovery at 30°C, spread on LB plates containing kanamycin and streptomycin (kanamycin concentration is 50ug/ml, streptomycin concentration is 50ug/ml), culture at 30°C, and screen positive clones.
  • Use primers to perform PCR amplification of fadD-up700-F and fadD-down700-R, and sequence the amplified fragments for verification.
  • the PCR amplification system is: Green Taq Mix 10ul (Nanjing Novozan Biotechnology Co., Ltd., product catalog: P131), 0.8ul each primer (10uM), 8.4ul distilled water, 0.2ul template bacterial solution, the total volume is 20ul; the PCR amplification conditions are: pre-denaturation at 95°C for 3 minutes (1 cycle); denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 1-5 minutes (60 seconds/kb) (30 cycle); extend at 72°C for 5 minutes (1 cycle).
  • Eliminate the pTarget plasmid Inoculate the positive clones verified by sequencing in LB liquid medium containing 0.1mM IPTG and kanamycin overnight at 30°C to eliminate the pTarget plasmid. The strain after overnight culture was streaked on an LB solid plate containing kanamycin and cultured at 30°C overnight to obtain an E. coli mutant ST11 ⁇ fadR containing pCas plasmid, named ST12.
  • step (5) prepare electroporated competent cells, mix with pTarget-fadDp plasmid and fadD::CPA1-fadD targeting fragment, repeat steps (4)-(5), The primer pairs fadD-up700-F and fadD-down700-R were sequenced and verified, and the E. coli mutant ST11 ⁇ fadRCPA1-fadD containing pCas plasmid was obtained, named ST13.
  • Eliminate pCas plasmid Inoculate the E. coli mutant ST11 ⁇ fadRCPA1-fadD (ST13) containing pCas plasmid that has been verified by sequencing in LB liquid medium and culture it overnight at 37°C to eliminate pCas plasmid. The strain after overnight culture was streaked on an LB solid plate and cultured overnight at 37°C to obtain the plasmid-free E. coli mutant ST11 ⁇ fadRCPA1-fadD, referred to as ST13.
  • the primer sequences used for verification and sequencing are as follows:
  • the expression vector pXA constructed in Example 1 was transformed into the E. coli mutant ST13 by chemical transformation, and positive clones were screened on the LB plate containing kanamycin (the concentration of kanamycin is 50ug/ml). The obtained clones The strain was named 13-XA.
  • composition of the fermentation medium is: citric acid 1.7g/L, potassium dihydrogen phosphate 14g/L, diammonium hydrogenphosphate 4g/L, polyether defoaming agent 150uL/L, glucose 20g/L, MgSO 4 ⁇ 7H 2 O 0.6g/L, VB1 9mg/L, lysine 0.4g/L, methionine 0.2g/L, isoleucine 0.2g/L, threonine 0.3g/L, trace inorganic salt I 10mL/ L, pH 7.0.
  • the composition of trace inorganic salt I is: EDTA 840mg/L, CoCl 2 ⁇ 6H 2 O 250mg/L, MnCl 2 ⁇ 4H 2 O 1500mg/L, CuCl 2 ⁇ 2H 2 O 150mg/L, H 3 BO 3 300mg/L, Na 2 MoO 4 ⁇ 2H 2 O 250mg/L, Zn(CH 3 COO) 2 ⁇ 2H 2 O 1300mg/L, iron citrate 10g/L.
  • the fed-batch medium composition is: glucose 600g/L, MgSO 4 ⁇ 7H 2 O 2g/L, lysine 4g/L, methionine 2g/L, isoleucine 2g/L, threonine 3g/L, palmitic acid 5g/L, trace inorganic salt II 10mL/L.
  • the composition of trace inorganic salt II is: EDTA 1300mg/L, CoCl 2 ⁇ 6H 2 O 400mg/L, MnCl 2 ⁇ 4H 2 O 2350mg/L, CuCl 2 ⁇ 2H 2 O 250mg/L, H 3 BO 3 500mg/L, Na 2 MoO 4 ⁇ 2H 2 O 400mg/L, Zn(CH 3 COO) 2 ⁇ 2H 2 O 1600mg/L, iron citrate 4g/L.
  • Fatty acid 2g/L was added 4 hours after induction, and 2g/L was added every 4 hours.
  • the component content of the fermentation medium can be replaced with any value within the following range: citric acid 1-5g/L, potassium dihydrogen phosphate 1-20g/L, nitrogen source 1 -5g/L, glucose 5-30g/L, MgSO 4 ⁇ 7H 2 O 0.3-1g/L, VB1 5-10mg/L, lysine 0.1-1g/L, methionine 0.1-1g/L, Isoleucine 0.1-1g/L, threonine 0.1-1g/L, trace inorganic salt I 1-10mL/L, pH 7.0 ⁇ 0.5.
  • the nitrogen source is an inorganic nitrogen-containing compound, which can be selected from one or more of ammonium chloride, ammonium acetate, ammonium sulfate and ammonium phosphate.
  • the trace amount of inorganic salt is selected from one or more of soluble iron salts, cobalt salts, copper salts, zinc salts, manganese salts and molybdates.
  • the component content of the fed-batch culture medium can be replaced with any value within the following range: glucose 100-800g/L, MgSO 4 ⁇ 7H 2 O 1-5g/L, lysine 1-10g/L, methyl sulfide Acid 1-10g/L, isoleucine 1-10g/L, threonine 1-10g/L, palmitic acid 2-5g/L, trace inorganic salt II 1-10mL/L.
  • the fatty acid is selected from one or more types of palmitic acid, oleic acid, lauric acid, and soybean oil, and the amount and timing of addition can be adjusted based on experience.
  • Seed liquid culture 100 mL of liquid LB was placed in a 250 mL Erlenmeyer flask and sterilized at 121°C for 20 min. After cooling, add the glycerol bacteria 13-A stored at -80°C.
  • the culture temperature is 37°C
  • the shaker speed is 200rpm
  • the culture is carried out for 6-8 hours for inoculation of the fermentation medium.
  • Those skilled in the art can adjust the above conditions to a certain extent according to actual conditions without affecting the realization of the purpose of the present invention.
  • This embodiment only provides a specific implementation solution.
  • the culture conditions can be replaced with any value within the following range: the culture temperature is 25-42°C, and the shaker rotation speed is 100-300 rpm.
  • the volume of the fermentation culture medium in the 5L fermentor is 2.5L.
  • the inoculation amount is 5% (V/V)
  • the initial concentration of glucose is 20g/ L.
  • the temperature is 37°C
  • the initial air flux is 2vvm
  • the stirring speed is 300rpm
  • the dissolved oxygen concentration at this time is set to 100%
  • the air flux is adjusted until 3vvm during the bacterial growth process
  • the stirring speed is controlled by correlating the DO value. Dissolved oxygen concentration is always greater than 30%.
  • Ammonia is used to control the pH at 7.0 during the fermentation process.
  • L-homoserine Use Agilent-1200 high performance liquid chromatography to measure the components in the fermentation broth.
  • the detection method of L-homoserine is: after the sample is appropriately diluted, it is derivatized with 2,4-dinitrofluorobenzene (DNFB).
  • DNFB 2,4-dinitrofluorobenzene
  • To 100uL sample add 50uL 10g/L DNFB acetonitrile solution and 100uL 0.5M NaHCO 3 solution, and mix thoroughly. , react in the dark at 60°C for 1 hour. After cooling, add 750uL 0.01M KH 2 PO 4 solution, mix well, filter with a 0.22um filter membrane and perform high-performance liquid chromatography detection.
  • the chromatographic column is ZORBAX Eclipse XDB-C18 column (4.6 ⁇ 150mm, 5um; Agilent), the column temperature is 30°C, the mobile phase is 35% acetonitrile formic acid (one thousandth) aqueous solution, the flow rate is 1mL/min, and the detection wavelength is 360nm. .
  • Patent No. CN201710953111.8 knocks out the thrB gene of E. coli K-12 MG1655 strain, overexpresses the rhtA gene, knocks out the thrL gene, mutates the thrA gene, and expresses thrA*, ppc, aspA, and pntA in multiple copies on the chromosomal DNA.

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Abstract

一种通过代谢工程手段改造的重组大肠杆菌及其用于生产L-高丝氨酸的方法，该菌株为大肠埃希氏菌(Escherichia coli)，株号13-XA，于2022年6月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为：CGMCC No.25099。其染色体DNA上一个或多个与脂肪酸代谢相关的基因被敲除或弱化，和/或启动子被替换以增强；一个或多个与L-高丝氨酸代谢途径相关的基因被敲除或弱化，并且/或者一个或多个与L-高丝氨酸代谢途径相关的基因被过表达或增强，并且/或者一个或多个与L-高丝氨酸代谢途径相关的基因被突变。L-高丝氨酸生产菌株，具有产量高、成本低、条件温和、环境污染少等诸多优点，具有广阔的应用前景。

Description

一种L-高丝氨酸高产菌株及其构建方法和应用 技术领域
本发明属于生物工程技术领域，具体涉及一种L-高丝氨酸高产菌株及其构建方法和应用。
背景技术
L-高丝氨酸是一种天然存在的非必需氨基酸，属于非蛋白氨基酸，是合成L-甲硫氨酸等重要高附件化合物的前提。基于L-高丝氨酸及其衍生物作为医药中间体在药物学、生理学等方面有重要应用前景。
目前，国内外微生物发酵法，具有成本低、条件温和、环境污染少等诸多优点，近年来已经成为生产各类氨基酸的首选工艺。但是，微生物发酵法中仅以葡萄糖为底物存在还原力和能量不足的情况(Glucose+2HCO 3 -+2NH ++2ATP+4NADPH→2HS)，需要消耗部分葡萄糖来提供还原力，这样会使糖酸转化率降低。
脂肪酸作为一种碳源受到越来越多的关注，脂肪酸还原度较葡萄糖高，以脂肪酸作为碳源不仅能够提供碳骨架，还能够提供大量的还原力和能量，以棕榈酸为例，1分子棕榈酸转化为4分子L-高丝氨酸的同时伴随着11分子的FADH和11分子NADH(C 16：0+4NH 4+5ATP+8NADPH→4HS+11FADH+11NADH)，除去反应必须的8分子NADPH和5分子ATP，还有大量的还原力和能量剩余。
鉴于此，我们可以使用同时使用葡萄糖和棕榈酸两种碳源用于L-高丝氨酸的合成，通过添加少量的棕榈酸或其他脂肪酸来提供还原力，从而最大限度的保证葡萄糖转化为L-高丝氨酸的糖酸转化率。
发明内容
本发明的主要目的在于：针对上述问题，提供一种L-高丝氨酸高产菌株及其构建方法和应用。本发明通过基因工程构建高产L-高丝氨酸的菌株，并以葡萄糖+脂肪酸双底物为碳源合成L-高丝氨酸，使得L-高丝氨酸的产量更高，糖酸转化率更高，进一步降低了生产成本，生产过程更加绿色环保，市场竞争优势明显。
本发明的目的及解决其技术问题是采用以下技术方案来实现的。
本发明的一个方面提供了一种L-高丝氨酸高产菌株，该菌株为大肠埃希氏菌(Escherichia coli)，株号13-XA，于2022年6月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为：CGMCC No.25099。
该工程菌株13-XA是染色体DNA上一个或多个与脂肪酸代谢相关的基因被敲除或弱化，和/或启动子被替换以增强；一个或多个与L-高丝氨酸代谢途径相关的基因被敲除或弱化，并且/或者一个或多个与L-高丝氨酸代谢途径相关的基因被过表达或增强，并且/或者一个或多个与L-高丝氨酸代谢途径相关的基因被突变。通过依次敲除突变型大肠杆菌ST11基因组中DNA结合转录双调节因子基因(fadR)并强化长链脂肪酸辅酶a连接酶基因(fadD)启动子，得到突变型大肠杆菌，得到宿主菌株，然后在宿主菌株中过表达解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因thrA(S345F)而得到的。
本发明的目的及解决其技术问题还通过采用以下技术方案来实现。
本发明的另一方面还提供了一种高效发酵生产L-高丝氨酸菌株的构建方法，包括：
宿主菌株的构建：敲除突变型大肠杆菌ST11基因组中DNA结合转录双调节因子基因(fadR)得到突变型大肠杆菌，命名为ST12，并在此基础上强化长链脂肪酸辅酶a连接酶基因(fadD)启动子，得到突变型大肠杆菌，命名为ST13；
质粒的构建：将解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因thrA(S345F)插入质粒载体pXB1k的NcoI和EcoRI位点间得到重组载体，命名为pXA；
工程菌株的构建：分别将上述重组载体质粒pXA导入上述突变型大肠杆菌ST12和ST13中，分别即得到重组工程菌株，命名为12-XA和13-XA；
其中，所述突变型大肠杆菌ST11记载于专利202011270812.X中，其基因型为E.coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB；
所述突变型大肠杆菌ST13基因型为E.coli ST11ΔfadR,ΔP fadD::P CPA1；
所述天冬氨酸激酶/高丝氨酸脱氢酶1基因thrA(S345F)源自大肠杆菌K-12 MG1655。
优选地，所述重组载体质粒pXA的构建步骤如下：
以大肠杆菌K12的基因组DNA为模板，用引物thrA-F和S345F-R、S345F-F和thrA-R进行PCR扩增得到解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因的2个片段thrA-1和thrA-2；所述正向引物thrA-F的核苷酸序列如SEQ ID NO.3所示，所述反向引物S345F-R的核苷酸序列如SEQ ID NO.4所示；所述正向引物S345F-F的核苷酸序列如SEQ ID NO.5所示，所述反向引物thrA-R的核苷酸序列如SEQ ID NO.6所示；将pXB1k载体用NcoI和EcoRI双酶切后，回收载体大片段，将上述得到的thrA-1和thrA-2基因片段与载体大片段用Gibson方法进行连接，将产物转化感受态细胞，涂布含链霉素的LB固体平板，37℃过夜，挑取单克隆提取质粒，设计一对引物pBAD-F和pBAD-R进行PCR验证，筛选出正确构建的重组载体质粒pXA；所述正向引物pBAD-F的核苷酸序列如SEQ ID NO.7所示，所述反向引物pBAD-R的核苷酸序列如SEQ ID NO.8所示。
优选地，所述重组载体质粒pXA是将pXB1k载体的NcoI和EcoRI位点间的片段替换为解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因得到的；所述pXB1k载体的核苷酸序列如SEQ ID NO.1所示，所述解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因的核苷酸序列如SEQ ID NO.2所示。
优选地，所述突变型大肠杆菌ST13的构建步骤如下：
(1)以pTargetF为模板，分别使用引物对pTarget-fadR-F和pTarget-fadR-R，pTarget-fadDp-F和pTarget-fadDp-R进行PCR扩增，将扩增得到的片段用DpnI甲基化酶消化后转化大肠杆菌Fast-T1感受态，在含链霉素的LB平板上筛选阳性克隆，并用引物pTarget-cexu-F测序验证，测序正确后分别命名为pTarget-fadR，pTarget-fadDp；
(2)分别用引物对fadR-up500-F和fadR-up500-R，fadR-down500-F和fadR-down500-R进行PCR扩增，分别得到两个片段，以两个片段的混合物为模板，用引物对fadR-up500-F和fadR-down500-R进行PCR扩增，得到ΔfadR打靶片段；分别用引物对fadD-up500-F和fadD-up500-R，CPA1-fadD-F和CPA1-fadD-R，fadD-down500-F和fadD-down500-R进行PCR扩增，分别得到三个片段，以三个片段的混合物为模板，用引物对fadD-up500-F和fadD-down500-R进行PCR扩增，得到ΔP fadD::P CPA1打靶片段；将得到的打靶片段ΔfadR，ΔP fadD::P CPA1分别回收；
(3)将大肠杆菌突变体ST11制备化转感受态细胞，并转化pCas质粒，涂布在含卡那霉素的LB平板上，30℃培养，筛选阳性克隆；
(4)从步骤(3)的平板上挑取阳性克隆，制备电转感受态细胞，与pTarget-fadR质粒和打靶片段ΔfadR混合，置于电转杯中电击，加入LB液体培养基30℃复苏后涂布在含卡那霉素和链霉素的LB平板上，30℃培养，筛选阳性克隆，并用引物对fadR-up700-F和fadR-down700-R进行PCR扩增，将扩增片段测序验证筛选出阳性克隆；
(5)将上述得到的阳性克隆接种在含有IPTG和卡那霉素的LB液体培养基中30℃培养过夜以消除pTarget-fadR质粒，过夜培养后的菌株在含有卡那霉素的LB固体平板上划线，30℃培养过夜，得到含有pCas质粒的大肠杆菌突变体ST11ΔfadR，命名为ST12；
(6)将含有pCas质粒的大肠杆菌突变体ST12制备电转感受态细胞，与pTarget-fadDp质粒和ΔP fadD::P CPA1打靶片段混合，重复上述步骤(4)-(5)，并用引物对fadD-up700-F和fadD-down700-R测序验证，得到含有pCas质粒的大肠杆菌突变体ST11ΔfadR,ΔP fadD::P CPA1，命名为ST13。
(7)将测序验证正确的含有pCas质粒的大肠杆菌突变体ST11ΔfadR,ΔP fadD::P CPA1(ST13)接种在LB液体培养基中，37℃培养过夜以消除pCas质粒，将过夜培养后的菌株在LB固体平板上划线，37℃培养过夜培养，得到不含质粒的大肠杆菌突变体ST11ΔfadR,ΔP fadD::P CPA1，简称ST13。
优选地，还包括制备电转感受态细胞的步骤：将pCas质粒利用化学转化法转化大肠杆菌ST11，在含卡那霉素的LB平板上30℃培养筛选阳性克隆，阳性克隆接种在含2g/L阿拉伯糖的LB液体培养基中30℃培养至OD 600约为0.6后，制备电转感受态细胞。
优选地，步骤(1)中所述正向引物pTarget-fadR-F的核苷酸序列如SEQ ID NO.9所示，所述反向引物pTarget-fadR-R的核苷酸序列如SEQ ID NO.10所示；所述正向引物pTarget-fadDp-F的核苷酸序列如SEQ ID NO.11所示，所述反向引物pTarget-fadDp-R的核苷酸序列如SEQ ID NO.12所示；
所述PCR扩增体系为：5X SF Buffer 10ul、dNTP Mix(10mM each)1ul、模板pTargetF 20ng、引物(10uM)各2ul、Phanta Super-Fidelity DNA Polymerase 1ul、蒸馏水34ul，总体积为50ul；
所述PCR扩增条件为：95℃预变性2分钟(1个循环)；95℃变性10秒、55℃退火20秒、72℃延伸1.5分钟(30个循环)；72℃延伸10分钟(1个循环)。
优选地，步骤(2)中所述正向引物fadR-up500-F的核苷酸序列如SEQ ID NO.19所示，所述反向引物fadR-up500-R的核苷酸序列如SEQ ID NO.20所示；所述正向引物fadR-down500-F的核苷酸序列如SEQ ID NO.21所示，所述反向引物fadR-down500-R的核苷酸序列如SEQ ID NO.22所示；所述正向引物fadD-up500-F的核苷酸序列如SEQ ID NO.13所示，所述反向引物fadD-up500-R的核苷酸序列如SEQ ID NO.14所示；所述正向引物CPA1-fadD-F的核苷酸序列如SEQ ID NO.15所示，所述反向引物CPA1-fadD-R的核苷酸序列如SEQ ID NO.16所示；所述正向引物fadD-down500-F的核苷酸序列如SEQ ID NO.17所示，所述反向引物fadD-down500-R的核苷酸序列如SEQ ID NO.18所示；
所述PCR扩增体系为：5X SF Buffer 10ul、dNTP Mix(10mM each)1ul、模板5-20ng、引物(10uM)各2ul、Phanta Super-Fidelity DNA Polymerase 1ul、蒸馏水34ul，总体积为50ul；
所述PCR扩增条件为：95℃预变性2分钟(1个循环)；95℃变性10秒、55℃退火20秒、72℃延伸0.5-2分钟(30秒/kb)(30个循环)；72℃延伸10分钟(1个循环)。
优选地，步骤(3)中所述正向引物fadR-up700-F的核苷酸序列如SEQ ID NO23所示，所述反向引物fadR-down700-R的核苷酸序列如SEQ ID NO.24所示；步骤(5)所述正向引物fadD-up700-F的核苷酸序列如SEQ ID NO.25所示，所述反向引物fadD-down700-R的核苷酸序列如SEQ ID NO.26所示。
本发明的目的及解决其技术问题还通过采用以下技术方案来实现。
本发明的另一个方面还提供了一种L-高丝氨酸高产菌株的应用，用于制备L-高丝氨酸。
优选地，该应用是将活化后的高效发酵生产L-高丝氨酸菌株接种于发酵培养基中并采用生物发酵法制备L-高丝氨酸，所述方法包括：
温度为37℃，初始空气通量为2vvm，搅拌转速300rpm，此时的溶解氧浓度设定为100％，菌体生长过程中调节空气通量直至3vvm，同时将搅拌转速与DO值关联来控制溶解氧浓度始终大于30％，当初始葡萄糖消耗完时，开启补料培养基，发酵过程采用氨水控制pH在7.0，当菌体密度达到600nm的吸光度(OD 600)为30时，加入终浓度2g/L的L-阿拉伯糖诱导蛋白表达，诱导4h后加入终浓度2g/L的棕榈酸，之后每隔4h补加2g/L棕榈酸，直至发酵结束，当补料培养基耗尽即结束发酵。
优选地，所述发酵培养基组成为：柠檬酸1-5g/L，磷酸二氢钾1-20g/L，氮源1-5g/L，聚醚类消泡剂150uL/L，葡萄糖5-30g/L，MgSO 4·7H 2O 0.3-1g/L，VB1 5-10mg/L，赖氨酸0.1-1g/L，甲硫氨酸0.1-1g/L，异亮氨酸0.1-1g/L，苏氨酸0.1-1g/L，微量无机盐I 1-10mL/L，pH 7.0±0.5；
所述补料培养基组成为：葡萄糖100-800g/L，MgSO 4·7H 2O 1-5g/L，赖氨酸1-10g/L，甲硫氨酸1-10g/L，异亮氨酸1-10g/L，苏氨酸1-10g/L，棕榈酸2-5g/L，微量无机盐II 1-10mL/L。
优选地，所述发酵培养基中所述微量无机盐I组成为：EDTA 840mg/L，CoCl 2·6H 2O 250mg/L，MnCl 2·4H 2O 1500mg/L，CuCl 2·2H 2O 150mg/L，H 3BO 3 300mg/L，Na 2MoO 4·2H 2O 250mg/L，Zn(CH 3COO) 2·2H 2O 1300mg/L，柠檬酸铁10g/L；所述氮源选自氯化铵、乙酸铵、硫酸铵和磷酸铵中的一种或多种；
所述补料培养基中所述微量无机盐II组成为：EDTA 1300mg/L，CoCl 2·6H 2O 400mg/L，MnCl 2·4H 2O 2350mg/L，CuCl 2·2H 2O 250mg/L，H 3BO 3 500mg/L，Na 2MoO 4·2H 2O 400mg/L，Zn(CH 3COO) 2·2H 2O 1600mg/L，柠檬酸铁4g/L。
借由上述技术方案，本发明至少具有下列优点：本发明构建了一种能够高效生产L-高丝氨酸的突变型大肠杆菌重组工程菌株13-XA。
本发明通过基因工程改造，获得高产L-高丝氨酸的工程菌株，同时采用葡萄糖+棕榈酸培养，进一步降低了生产成本，生产过程更加绿色环保，市场竞争优势明显。本发明所采用的双底物生物法生产工艺替代了传统的石化工艺，以可再生的生物基为原料替代了不可再生的石化原料，实现了节能减排、清洁生产、绿色环保、循环经济的大产业。通过对菌株和制造工艺的不断优化升级，使得产品的品质更好、成本更低，具有很大的市场前景。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并结合附图详细说明如后。
附图说明
图1为pXB1k的物理图谱；
图2为大肠杆菌13-XA的发酵过程中L-高丝氨酸产量随时间变化曲线。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面将结合本发明实施例及附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
本发明的实施例中所使用的实验方法如无特殊说明，均为常规方法。
本发明的实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明的实施例中的定量实验，均设置三次重复实验，结果取平均值。
在本发明的实施例中，如无特殊说明，所涉及的测序验证过程均由第三方检测机构完成，该机构为苏州金唯智生物科技有限公司。
本发明的实施例中，大肠杆菌K12记载于文献“Baba T,Ara T,Hasegawa M,Takai Y,Okumura Y,Baba M,Datsenko KA,Tomita M,Wanner BL,Mori H:Construction of Escherichia coli K-12 in-frame,single-gene knockout mutants:the Keio collection.Mol Syst Biol 2006,2:2006.0008.”中，是一株非病原菌，遗传背景清楚，世代时间短，容易培养且培养基原料低廉。大肠杆菌K12的全基因组序列的GenBank Accession为U00096.3(GI：545778205，update date是AUG 01，2014，version是3)，公众可从中国科学院微生物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
1分子葡萄糖经糖酵解途径生成2分子磷酸烯醇式丙酮酸，在羧化酶作用下结合2分子CO 2生成2分子草酰乙酸，草酰乙酸在转氨酶aspC作用下转化为2分子天冬氨酸，或者经反向TCA循环转化为2分子富马酸，在氨裂解酶aspA作用下转化为2分子天冬氨酸，天冬氨酸进一步在双功能天冬氨酸激酶作用下转化为天冬氨酸磷酸，天冬氨酸磷酸经天冬氨酸半醛脱氢酶作用转化为天冬氨酸半醛，进一步在双功能天冬氨酸激酶作用下转化为2分子高丝氨酸。经该途径，1分子葡萄糖可转化为2分子高丝氨酸，但该途径还原力和能量不足，需消耗部分葡萄糖提供还原力和能量。
脂肪酸作为还原性较高的碳源同样可以被菌体利用转化为自身所需的营养物质，同时脂肪酸氧化的过程提供大量还原力，经计算，1分子棕榈酸彻底氧化可提供16分子 NADH，本发明为了提高高丝氨酸的产量和糖酸转化率，在发酵过程中添加少量棕榈酸来提供还原力，使高丝氨酸产量大幅提高至154g/L。
在本发明的实施例中，解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因的编码序列如SEQ ID NO.2所示。DNA结合转录双调节因子基因(fadR)的编码序列如Gene ID:948652(由720个核苷酸组成)所示，编码Acession号NP_415705(由239个氨基酸残基组成)所示的DNA结合转录双调节因子；
长链脂肪酸辅酶a连接酶基因(fadD)的编码序列如Gene ID:946327(由1686个核苷酸组成)所示，编码Acession号NP_416319(由561个氨基酸残基组成)所示的长链脂肪酸辅酶a连接酶。
下述实施例中pXB1k载体的核苷酸序列如SEQ ID NO.1所示，包括如下片段：(1)araC-araBAD-MCS片段(含阿拉伯糖诱导启动子、多克隆位点)；(2)MCS-TrrnB片段(含多克隆位点、终止子TrrnB)；(3)p15A复制起始位点片段；(4)卡那霉素抗性基因Kan片段。pXB1k载体图谱如图1所示。
SEQ ID NO.1：
Figure PCTCN2022108501-appb-000001
Figure PCTCN2022108501-appb-000002
SEQ ID NO.2：
Figure PCTCN2022108501-appb-000003
Figure PCTCN2022108501-appb-000004
实施例1构建表达解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1的重组质粒pXA
以大肠杆菌K12的基因组DNA为模板，用引物thrA-F和S345F-R、S345F-F和thrA-R进行PCR扩增得到解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因的2个片段thrA-1和thrA-2。将pXB1k载体用NcoI和EcoRI双酶切后，回收载体大片段，约 3450bp，将回收的thrA-1和thrA-2基因片段与载体大片段用Gibson方法(Gibson DG,Young L,Chuang RY,Venter JC,Hutchison CA,3rd,Smith HO:Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods 2009,6:343-345.)进行连接，连接产物转化Fast-T1感受态细胞(南京诺维赞生物科技股份有限公司，产品目录为C505)，涂布含卡那霉素的LB固体平板。37℃过夜，挑取单克隆提取质粒，设计一对引物(pBAD-F和pBAD-R)进行PCR验证，正确的克隆送测序。将pXB1k载体的NcoI和EcoRI位点间的片段替换为SEQ ID NO.2所示的解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因得到的重组载体命名为pXA。引物序列如下：
thrA-F SEQ ID NO.35’-ggctaacaggaggaattaaccatgcgagtgttgaagttcgg-3’
S345F-R SEQ ID NO.4 5’-agcaccacgaaaatacgggcgcgtgacatc-3’
S345F-F SEQ ID NO.55’-gcccgtattttcgtggtgctgattacgcaatc-3’
thrA-R SEQ ID NO.65’-gctgcagaccgagctcaccgaattctcagactcctaacttccatg-3’
pBAD-F SEQ ID NO.75’-cggcgtcacactttgctatg-3’
pBAD-R SEQ ID NO.8 5’-cgtttcacttctgagttcggc-3’
在所述解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因表达盒中，启动所述解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因转录的启动子是pBAD启动子。
实施例2构建大肠杆菌突变体ST11
本实施例构建的大肠杆菌突变体为ST11，其构建方法可见中国专利申请CN202011270812.X。
实施例3构建大肠杆菌突变体ST13
大肠杆菌突变体ST13是利用CRISPR技术(Jiang Y,Chen B,Duan C,Sun B,Yang J,Yang S:Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Appl Environ Microbiol 2015,81:2506-2514.)敲除大肠杆菌ST11的DNA结合转录抑制因子基因(fadR)并强化长链脂肪酸辅酶a连接酶基因(fadD)启动子从而获得的大肠杆菌ST11的突变体，在本申请中简称为ST13，其基因型为E.coli ST11ΔfadR CPA1-fadD。
具体而言，在该实施例中，所述大肠杆菌突变体ST13是将大肠杆菌ST11的DNA结合转录抑制因子基因(fadR)敲除并强化长链脂肪酸辅酶a连接酶基因(fadD)启动子得到的大肠杆菌突变体(简称ST13)。大肠杆菌突变体ST11的具体构建步骤如下：
(1)制备电转感受态细胞：将pCas质粒(Jiang Y,Chen B,Duan C,Sun B,Yang J,Yang S:Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Appl Environ Microbiol 2015,81:2506-2514.)利用化学转化法转化大肠杆菌ST11，在含卡那霉素的LB平板(卡那霉素浓度为50ug/ml)上30℃培养筛选阳性克隆，阳性克隆接种在含2g/L阿拉伯糖的LB液体培养基中30℃培养至OD600约为0.6后，制备电转感受态细胞。
(2)构建pTarget质粒：利用网站https://crispy.secondarymetabolites.org选取敲除位点的N20，设计引物构建pTarget质粒。以pTargetF(Jiang Y,Chen B,Duan C,Sun B,Yang J,Yang S:Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Appl Environ Microbiol 2015,81:2506-2514.)为模板，分别用引物对pTarget-fadDp-F和pTarget-fadDp-R，pTarget-fadR-F和pTarget-fadR-R进行PCR扩增，分别扩增得到大小约2100bp的片段。
PCR扩增体系为：5X SF Buffer 10ul、dNTP Mix(10mM each)1ul、模板pTargetF 20ng、引物(10uM)各2ul、Phanta Super-Fidelity DNA Polymerase(南京诺维赞生物科技股份有限公司，产品目录为P501)1ul、蒸馏水34ul，总体积为50ul。扩增条件为：95℃预变性2分钟(1个循环)；95℃变性10秒、55℃退火20秒、72℃延伸1.5分钟(30个循环)；72℃延伸10分钟(1个循环)。用DpnI甲基化酶反应约3h后，直接利用化学转化法转化大肠杆菌Fast-T1感受态，在含链霉素的LB平板(链霉素浓度为50ug/ml)上筛选阳性克隆，并用引物pTarget-cexu-F测序验证。测序正确后分别命名为pTarget-fadD，pTarget-fadR所用引物序列如下(下划线所示为N20的序列)：
pTarget-fadDp-F SEQ ID NO.9
5’-ACGACGAACACGCATTTTAGGTTTTAGAGCTAGAAATAGC-3’
pTarget-fadDp-R SEQ ID NO.10
5’-CTAAAATGCGTGTTCGTCGTACTAGTATTATACCTAGGAC-3’
pTarget-fadR-F SEQ ID NO.11
5’-GCTGGCTACCGCTAATGAAGGTTTTAGAGCTAGAAATAGC-3’
pTarget-fadR-R SEQ ID NO.12
5’-CTTCATTAGCGGTAGCCAGCACTAGTATTATACCTAGGAC-3’
(3)扩增打靶片段：分别用引物对fadD-up500-F和fadD-up500-R，CPA1-fadD-F和CPA1-fadD-R，fadD-down500-F和fadD-down500-R进行PCR扩增，分别得到大小约为500bp，1700bp和500bp的片段；以三个片段的混合物为模板，用引物对fadD-up500-F和fadD-down500-R进行PCR扩增，得到大小约为2700bp的fadD::CPA1-fadD打靶片段。
分别用引物对fadR-up500-F和fadR-up500-R，fadR-down500-F和fadR-down500-R进行PCR扩增，分别得到大小约为500bp和500bp的片段；以两个片段的混合物为模板，用引物对fadR-up500-F和fadR-down500-R进行PCR扩增，得到大小约为1000bp的ΔfadR打靶片段。
PCR扩增体系为：5X SF Buffer 10ul、dNTP Mix(10mM each)1ul、模板5-20ng、引物(10uM)各2ul、Phanta Super-Fidelity DNA Polymerase(南京诺维赞生物科技股份有限公司，产品目录为P501)1ul、蒸馏水34ul，总体积为50ul。扩增条件为：95℃预变性2分钟(1个循环)；95℃变性10秒、55℃退火20秒、72℃延伸0.5-2分钟(30秒/kb)(30个循环)；72℃延伸10分钟(1个循环)。
将打靶片段fadD::CPA1-fadD，ΔfadR分别回收。打靶片段从上游到下游依次包含500bp上游同源臂，替换基因表达盒和500bp下游同源臂。所用引物序列如下：
fadD-up500-F SEQ ID NO.13 5’-attaaaggcagcagtcccac-3’
fadD-up500-R SEQ ID NO.14 5’-TATAAGGAGGgctgtttctttttctttaaaaac-3’
CPA1-fadD-F SEQ ID NO.155’-aagaaacagcCCTCCTTATAACTTCGTATAATG-3’
CPA1-fadD-R SEQ ID NO.16 5’-ccttcttcatGATATCTCCTTCGTAAAAGATC-3’
fadD-down500-F SEQ ID NO.17 5’-AGGAGATATCatgaagaaggtttggcttaac-3’
fadD-down500-R SEQ ID NO.18 5’-tcggcaccaaacgcttgatg-3’
fadR-up500-F SEQ ID NO.19 5’-acttcaagatttgccgccac-3’
fadR-up500-R SEQ ID NO.205’-gaatggctaacatagtgagatttccataacac-3’
fadR-down500-F SEQ ID NO.21 5’-tctcactatgttagccattcaggggcgata-3’
fadR-down500-R SEQ ID NO.225’-gatatcgccggttccgactg-3’
(4)电转化：将200ng pTarget-fadR质粒，400ng打靶片段ΔfadR与100μl步骤(1)制备的电转化感受态细胞混合，置于2mm的电转杯中，2.5kV电击，加入1ml LB液体培养基30℃复苏后涂布在含卡那霉素和链霉素的LB平板上(卡那霉素浓度为50ug/ml，链霉素浓度为50ug/ml)，30℃培养，筛选阳性克隆。用引物对fadD-up700-F和fadD-down700-R进行PCR扩增，将扩增片段测序验证。所述PCR扩增体系为：Green Taq Mix 10ul(南京诺维赞生物科技股份有限公司，产品目录为P131)、引物(10uM)各0.8ul、蒸馏水8.4ul、模板菌液0.2ul，总体积为20ul；所述PCR扩增条件为：95℃预变性3分钟(1个循环)；95℃变性15秒、55℃退火15秒、72℃延伸1-5分钟(60秒/kb)(30个循环)；72℃延伸5分钟(1个循环)。
(5)消除pTarget质粒：将测序验证正确的阳性克隆接种在含有0.1mM IPTG和卡那霉素的LB液体培养基中30℃培养过夜以消除pTarget质粒。过夜培养后的菌株在含有卡那霉素的LB固体平板上划线，30℃培养过夜，得到含有pCas质粒的大肠杆菌突变体ST11ΔfadR，命名为ST12。
(6)从步骤(5)的平板上挑取单克隆，制备电转感受态细胞，与pTarget-fadDp质粒和fadD::CPA1-fadD打靶片段混合，重复步骤(4)-(5)的步骤，并用引物对fadD-up700-F和fadD-down700-R测序验证，得到含有pCas质粒的大肠杆菌突变体ST11ΔfadRCPA1-fadD，命名为ST13。
(7)消除pCas质粒：将测序验证正确的含有pCas质粒的大肠杆菌突变体ST11ΔfadRCPA1-fadD(ST13)接种在LB液体培养基中，37℃培养过夜以消除pCas质粒。将过夜培养后的菌株在LB固体平板上划线，37℃培养过夜培养，得到不含质粒的大肠杆菌突变体ST11ΔfadRCPA1-fadD，简称ST13。
用于验证和测序的引物序列如下：
fadR-up700-F SEQ ID NO.23 5’-tgtcttcggtacgggaagag-3’
fadR-down700-R SEQ ID NO.24 5’-ggcactacaccatccttaac-3’
fadD-up700-F SEQ ID NO.25 5’-taaaacggtggcggtggaac-3’
fadD-down700-R SEQ ID NO.26 5’-gtcgcgttaacctgttccag-3’
实施例4构建高产L-高丝氨酸的工程菌株13-XA
将实施例1中构建的表达载体pXA用化学转化法转化大肠杆菌突变体ST13，在含卡那霉素的LB平板(卡那霉素的浓度为50ug/ml)上筛选阳性克隆，得到的克隆菌株命名为13-XA。
实施例5菌株13-XA的高密度发酵
发酵培养基组成为：柠檬酸1.7g/L，磷酸二氢钾14g/L，磷酸氢二铵4g/L，聚醚类消泡剂150uL/L，葡萄糖20g/L，MgSO 4·7H 2O 0.6g/L，VB1 9mg/L，赖氨酸0.4g/L，甲硫氨酸0.2g/L，异亮氨酸0.2g/L，苏氨酸0.3g/L，微量无机盐I 10mL/L，pH 7.0。微量无机盐I组成为：EDTA 840mg/L，CoCl 2·6H 2O 250mg/L，MnCl 2·4H 2O 1500mg/L，CuCl 2·2H 2O 150mg/L，H 3BO 3 300mg/L，Na 2MoO 4·2H 2O 250mg/L，Zn(CH 3COO) 2·2H 2O 1300mg/L，柠檬酸铁10g/L。补料流加培养基组成为：葡萄糖600g/L，MgSO 4·7H 2O 2g/L，赖氨酸4g/L，甲硫氨酸2g/L，异亮氨酸2g/L，苏氨酸3g/L，棕榈酸5g/L，微量无机盐II 10mL/L。微量无机盐II组成为：EDTA 1300mg/L，CoCl 2·6H 2O 400mg/L，MnCl 2·4H 2O 2350mg/L，CuCl 2·2H 2O 250mg/L，H 3BO 3 500mg/L，Na 2MoO 4·2H 2O 400mg/L，Zn(CH 3COO) 2·2H 2O 1600mg/L，柠檬酸铁4g/L。诱导4h后添加脂肪酸2g/L，并每隔4h补加2g/L。本领域技术人员可根据实际情况对上述各成分进行一定幅度的调整，本实施例仅提供一种具体实现方案。作为本实施例可替换的实施方式，所述发酵培养基包括的成分含量可替换为以下范围内的任意值：柠檬酸1-5g/L，磷酸二氢钾1-20g/L，氮源1-5g/L，葡萄糖5-30g/L，MgSO 4·7H 2O 0.3-1g/L，VB1 5-10mg/L，赖氨酸0.1-1g/L，甲硫氨酸0.1-1g/L，异亮氨酸0.1-1g/L，苏氨酸0.1-1g/L，微量无机盐I 1-10mL/L，pH 7.0±0.5。
所述氮源为无机含氮化合物，可选自氯化铵、乙酸铵、硫酸铵和磷酸铵中的一种或多种。所述微量无机盐选自可溶性的铁盐、钴盐、铜盐、锌盐、锰盐和钼酸盐中的一种或多种。
所述流加培养基包括的成分含量可替换为以下范围内的任意值：葡萄糖100-800g/L，MgSO 4·7H 2O 1-5g/L，赖氨酸1-10g/L，甲硫氨酸1-10g/L，异亮氨酸1-10g/L，苏氨酸1-10g/L，棕榈酸2-5g/L，微量无机盐II 1-10mL/L。
所述脂肪酸选自棕榈酸、油酸、月桂酸、大豆油中的一种或多种，添加量及添加时机可根据经验进行调整。
种子液培养：250mL三角瓶中装液LB为100mL，121℃灭菌20min。冷却后接入-80℃保藏的甘油菌13-A，培养温度为37℃，摇床转速200rpm，培养6-8h，用于发酵培养基接种。本领域技术人员可根据实际情况对上述条件进行一定幅度的调整，并不影响本发明目的的实现。本实施例仅提供一种具体实现方案，作为本实施例可替换的实施方式，所述培养条件可替换为以下范围内的任意值：培养温度为25-42℃，摇床转速100-300rpm。
发酵罐接种：作为本实施例优选的实施方式，5L发酵罐发酵培养基体积为2.5L，灭菌后接入上述种子液，接种量为5％(V/V)，葡萄糖初始浓度为20g/L。温度为37℃，初始空气通量为2vvm，搅拌转速300rpm，此时的溶解氧浓度设定为100％，菌体生长过程中调节空气通量直至3vvm，同时将搅拌转速与DO值关联来控制溶解氧浓度始终大于30％。当初始葡萄糖消耗完时，开启补料。发酵过程采用氨水控制pH在7.0。当菌体密度达到600nm的吸光度(OD600)为30时，加入终浓度2g/L的L-阿拉伯糖诱导蛋白表达，诱导4h后加入终浓度2g/L的棕榈酸，之后每隔4h补加2g/L棕榈酸，直至发酵结束，当补料培养基耗尽即结束发酵。本领域技术人员可根据实际情况对上述条件进行一定幅度的调整，并不影响本发明目的的实现。
分析方法：使用安捷伦(Agilent-1200)高效液相色谱仪对发酵液中的组分进行测定。L-高丝氨酸的检测方法为：样品适当稀释后用2,4-二硝基氟苯(DNFB)进行衍生，100uL样品加入50uL 10g/L DNFB乙腈溶液和100uL 0.5M NaHCO 3溶液，充分混匀，60℃避光反应1h，冷却后加入750uL 0.01M KH 2PO 4溶液，混匀，用0.22um滤膜过滤后进行高效液相色谱检测。色谱柱为ZORBAX Eclipse XDB-C18 column(4.6×150mm,5um；Agilent)，柱温为30℃，流动相为35％乙腈甲酸(千分之一)水溶液，流速为1mL/min，检测波长为360nm。
结果：如图2所示，转化液中，L-高丝氨酸的产量达154g/L，整个发酵阶段L-高丝氨酸转化率可达0.65g L-高丝氨酸/g葡萄糖。
对比实施例1
专利号CN201710953111.8通过将大肠杆菌K-12 MG1655菌株的thrB基因敲除，rhtA基因过表达、thrL基因敲除、thrA基因突变，并在染色体DNA上多拷贝表达thrA*、ppc、aspA、pntA和pntB(MG1655ΔthrB,rhtA23,ΔthrL,thrA*(G433R),ΔcadA::thrA*-ppc-aspA-pntAB,ΔyidJ::thrA*-ppc-aspA-pntAB,ΔatpC::thrA*-ppc-aspA-pntAB,ΔdacA::thrA*,ΔbcsB::thrA*,ΔmenH::aspC,ΔyddB::asd)，获得高产L-高丝氨酸的工程菌株Hom8，其发酵可得88.1g/L的L-高丝氨酸，明显低于本发明的菌株产L-高丝氨酸的产量。
以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案范围内，当可利用上述揭示的方法及技术内容作出些许的更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (13)

  1. 一种L-高丝氨酸高产菌株，其特征在于，该菌株为大肠埃希氏菌(Escherichia coli)，株号13-XA，于2022年6月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为：CGMCC No.25099。
  2. 一种如权利要求1所述的L-高丝氨酸高产菌株的构建方法，其特征在于，包括：
    宿主菌株的构建：敲除突变型大肠杆菌ST11基因组中DNA结合转录双调节因子基因(fadR)得到突变型大肠杆菌，命名为ST12，并在此基础上强化长链脂肪酸辅酶a连接酶基因(fadD)启动子，得到突变型大肠杆菌，命名为ST13；
    质粒的构建：将解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因thrA(S345F)插入质粒载体pXB1k的NcoI和EcoRI位点间得到重组载体，命名为pXA；
    工程菌株的构建：分别将上述重组载体质粒pXA导入上述突变型大肠杆菌ST12和ST13中，分别即得到重组工程菌株，命名为12-XA和13-XA；
    其中，所述突变型大肠杆菌ST11记载于专利202011270812.X中，其基因型为E.coli BW25113ΔptsG::glk,ΔgalR::zglf,ΔompT::ppc,ΔldhA::rhtA,ΔlpxM::rhtB,ΔpflB::asd,ΔpoxB::aspA,ΔiclR,ΔlysA,ΔmetA,ΔthrB；
    所述突变型大肠杆菌ST13基因型为E.coli ST11ΔfadR,ΔP fadD::P CPA1；
    所述天冬氨酸激酶/高丝氨酸脱氢酶1基因thrA(S345F)源自大肠杆菌K-12 MG1655。
  3. 根据权利要求2所述的L-高丝氨酸高产菌株的构建方法，其特征在于，所述重组载体质粒pXA的构建步骤如下：
    以大肠杆菌K12的基因组DNA为模板，用引物thrA-F和S345F-R、S345F-F和thrA-R进行PCR扩增得到解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因的2个片段thrA-1和thrA-2；所述正向引物thrA-F的核苷酸序列如SEQ ID NO.3所示，所述反向引物S345F-R的核苷酸序列如SEQ ID NO.4所示；所述正向引物S345F-F的核苷酸序列如SEQ ID NO.5所示，所述反向引物thrA-R的核苷酸序列如SEQ ID NO.6所示；将pXB1k载体用NcoI和EcoRI双酶切后，回收载体大片段，将上述得到的thrA-1和thrA-2基因片段与载体大片段用Gibson方法进行连接，将产物转化感受态细胞，涂布含链霉素的LB固体平板，37℃过夜，挑取单克隆提取质粒，设计一对引物pBAD-F和pBAD-R 进行PCR验证，筛选出正确构建的重组载体质粒pXA；所述正向引物pBAD-F的核苷酸序列如SEQ ID NO.7所示，所述反向引物pBAD-R的核苷酸序列如SEQ ID NO.8所示。
  4. 根据权利要求3所述的L-高丝氨酸高产菌株的构建方法，其特征在于，所述重组载体质粒pXA是将pXB1k载体的NcoI和EcoRI位点间的片段替换为解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因得到的；所述pXB1k载体的核苷酸序列如SEQ ID NO.1所示，所述解除反馈抑制的天冬氨酸激酶/高丝氨酸脱氢酶1基因的核苷酸序列如SEQ ID NO.2所示。
  5. 根据权利要求2所述的L-高丝氨酸高产菌株的构建方法，其特征在于，所述突变型大肠杆菌ST13的构建步骤如下：
    (1)以pTargetF为模板，分别使用引物对pTarget-fadR-F和pTarget-fadR-R，pTarget-fadDp-F和pTarget-fadDp-R进行PCR扩增，将扩增得到的片段用DpnI甲基化酶消化后转化大肠杆菌Fast-T1感受态，在含链霉素的LB平板上筛选阳性克隆，并用引物pTarget-cexu-F测序验证，测序正确后分别命名为pTarget-fadR，pTarget-fadDp；
    (2)分别用引物对fadR-up500-F和fadR-up500-R，fadR-down500-F和fadR-down500-R进行PCR扩增，分别得到两个片段，以两个片段的混合物为模板，用引物对fadR-up500-F和fadR-down500-R进行PCR扩增，得到ΔfadR打靶片段；分别用引物对fadD-up500-F和fadD-up500-R，CPA1-fadD-F和CPA1-fadD-R，fadD-down500-F和fadD-down500-R进行PCR扩增，分别得到三个片段，以三个片段的混合物为模板，用引物对fadD-up500-F和fadD-down500-R进行PCR扩增，得到ΔP fadD::P CPA1打靶片段；将得到的打靶片段ΔfadR，ΔP fadD::P CPA1分别回收；
    (3)将大肠杆菌突变体ST11制备化转感受态细胞，并转化pCas质粒，涂布在含卡那霉素的LB平板上，30℃培养，筛选阳性克隆；
    (4)从步骤(3)的平板上挑取阳性克隆，制备电转感受态细胞，与pTarget-fadR质粒和打靶片段ΔfadR混合，置于电转杯中电击，加入LB液体培养基30℃复苏后涂布在含卡那霉素和链霉素的LB平板上，30℃培养，筛选阳性克隆，并用引物对fadR-up700-F和fadR-down700-R进行PCR扩增，将扩增片段测序验证筛选出阳性克隆；
    (5)将上述得到的阳性克隆接种在含有IPTG和卡那霉素的LB液体培养基中30℃培养过夜以消除pTarget-fadR质粒，过夜培养后的菌株在含有卡那霉素的LB固体平板上划线，30℃培养过夜，得到含有pCas质粒的大肠杆菌突变体ST11ΔfadR，命名为ST12；
    (6)将含有pCas质粒的大肠杆菌突变体ST12制备电转感受态细胞，与pTarget-fadDp质粒和ΔP fadD::P CPA1打靶片段混合，重复上述步骤(4)-(5)，并用引物对fadD-up700-F和fadD-down700-R测序验证，得到含有pCas质粒的大肠杆菌突变体ST11ΔfadR,ΔP fadD::P CPA1，命名为ST13。
    (7)将测序验证正确的含有pCas质粒的大肠杆菌突变体ST11ΔfadR,ΔP fadD::P CPA1(ST13)接种在LB液体培养基中，37℃培养过夜以消除pCas质粒，将过夜培养后的菌株在LB固体平板上划线，37℃培养过夜培养，得到不含质粒的大肠杆菌突变体ST11ΔfadR,ΔP fadD::P CPA1，简称ST13。
  6. 根据权利要求5所述的L-高丝氨酸高产菌株的构建方法，其特征在于，还包括制备电转感受态细胞的步骤：将pCas质粒利用化学转化法转化大肠杆菌ST11，在含卡那霉素的LB平板上30℃培养筛选阳性克隆，阳性克隆接种在含2g/L阿拉伯糖的LB液体培养基中30℃培养至OD 600约为0.6后，制备电转感受态细胞。
  7. 根据权利要求5所述的L-高丝氨酸高产菌株的构建方法，其特征在于，步骤(1)中所述正向引物pTarget-fadR-F的核苷酸序列如SEQ ID NO.9所示，所述反向引物pTarget-fadR-R的核苷酸序列如SEQ ID NO.10所示；所述正向引物pTarget-fadDp-F的核苷酸序列如SEQ ID NO.11所示，所述反向引物pTarget-fadDp-R的核苷酸序列如SEQ ID NO.12所示；
    所述PCR扩增体系为：5X SF Buffer 10ul、dNTP Mix(10mM each)1ul、模板pTargetF 20ng、引物(10uM)各2ul、Phanta Super-Fidelity DNA Polymerase 1ul、蒸馏水34ul，总体积为50ul；
    所述PCR扩增条件为：95℃预变性2分钟(1个循环)；95℃变性10秒、55℃退火20秒、72℃延伸1.5分钟(30个循环)；72℃延伸10分钟(1个循环)。
  8. 根据权利要求5所述的L-高丝氨酸高产菌株的构建方法，其特征在于，步骤(2)中所述正向引物fadR-up500-F的核苷酸序列如SEQ ID NO.19所示，所述反向引物fadR-up500-R的核苷酸序列如SEQ ID NO.20所示；所述正向引物fadR-down500-F的核苷酸序 列如SEQ ID NO.21所示，所述反向引物fadR-down500-R的核苷酸序列如SEQ ID NO.22所示；所述正向引物fadD-up500-F的核苷酸序列如SEQ ID NO.13所示，所述反向引物fadD-up500-R的核苷酸序列如SEQ ID NO.14所示；所述正向引物CPA1-fadD-F的核苷酸序列如SEQ ID NO.15所示，所述反向引物CPA1-fadD-R的核苷酸序列如SEQ ID NO.16所示；所述正向引物fadD-down500-F的核苷酸序列如SEQ ID NO.17所示，所述反向引物fadD-down500-R的核苷酸序列如SEQ ID NO.18所示；
    所述PCR扩增体系为：5X SF Buffer 10ul、dNTP Mix(10mM each)1ul、模板5-20ng、引物(10uM)各2ul、Phanta Super-Fidelity DNA Polymerase 1ul、蒸馏水34ul，总体积为50ul；
    所述PCR扩增条件为：95℃预变性2分钟(1个循环)；95℃变性10秒、55℃退火20秒、72℃延伸0.5-2分钟(30秒/kb)(30个循环)；72℃延伸10分钟(1个循环)。
  9. 根据权利要求5所述的L-高丝氨酸高产菌株的构建方法，其特征在于，步骤(3)中所述正向引物fadR-up700-F的核苷酸序列如SEQ ID NO23所示，所述反向引物fadR-down700-R的核苷酸序列如SEQ ID NO.24所示；步骤(5)所述正向引物fadD-up700-F的核苷酸序列如SEQ ID NO.25所示，所述反向引物fadD-down700-R的核苷酸序列如SEQ ID NO.26所示。
  10. 一种如权利要求1所述的L-高丝氨酸高产菌株的应用，其特征在于，用于制备L-高丝氨酸。
  11. 根据权利要求10所述的L-高丝氨酸高产菌株的应用，其特征在于，该应用是将活化后的高效发酵生产L-高丝氨酸菌株接种于发酵培养基中并采用生物发酵法制备L-高丝氨酸，所述方法包括：
    温度为37℃，初始空气通量为2vvm，搅拌转速300rpm，此时的溶解氧浓度设定为100％，菌体生长过程中调节空气通量直至3vvm，同时将搅拌转速与DO值关联来控制溶解氧浓度始终大于30％，当初始葡萄糖消耗完时，开启补料培养基，发酵过程采用氨水控制pH在7.0，当菌体密度达到600nm的吸光度(OD 600)为30时，加入终浓度2g/L的L-阿拉伯糖诱导蛋白表达，诱导4h后加入终浓度2g/L的棕榈酸，之后每隔4h补加2g/L棕榈酸，直至发酵结束，当补料培养基耗尽即结束发酵。
  12. 根据权利要求10所述的L-高丝氨酸高产菌株的应用，其特征在于，所述发酵培养基组成为：柠檬酸1-5g/L，磷酸二氢钾1-20g/L，氮源1-5g/L，聚醚类消泡剂150uL/L，葡萄糖5-30g/L，MgSO 4·7H 2O 0.3-1g/L，VB1 5-10mg/L，赖氨酸0.1-1g/L，甲硫氨酸0.1-1g/L，异亮氨酸0.1-1g/L，苏氨酸0.1-1g/L，微量无机盐I 1-10mL/L，pH7.0±0.5；
    所述补料培养基组成为：葡萄糖100-800g/L，MgSO 4·7H 2O 1-5g/L，赖氨酸1-10g/L，甲硫氨酸1-10g/L，异亮氨酸1-10g/L，苏氨酸1-10g/L，棕榈酸2-5g/L，微量无机盐II 1-10mL/L。
  13. 根据权利要求12所述的L-高丝氨酸高产菌株的应用，其特征在于，所述发酵培养基中所述微量无机盐I组成为：EDTA 840mg/L，CoCl 2·6H 2O 250mg/L，MnCl 2·4H 2O 1500mg/L，CuCl 2·2H 2O 150mg/L，H 3BO 3300mg/L，Na 2MoO 4·2H 2O 250mg/L，Zn(CH 3COO) 2·2H 2O 1300mg/L，柠檬酸铁10g/L；所述氮源选自氯化铵、乙酸铵、硫酸铵和磷酸铵中的一种或多种；
    所述补料培养基中所述微量无机盐II组成为：EDTA 1300mg/L，CoCl 2·6H 2O 400mg/L，MnCl 2·4H 2O 2350mg/L，CuCl 2·2H 2O 250mg/L，H 3BO 3500mg/L，Na 2MoO 4·2H 2O 400mg/L，Zn(CH 3COO) 2·2H 2O 1600mg/L，柠檬酸铁4g/L。
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