CN120738216A - A recombinant Escherichia coli for increasing L-threonine production and a preparation method thereof - Google Patents

Abstract

The invention discloses recombinant escherichia coli for improving the yield of L-threonine and a preparation method thereof, and belongs to the technical field of biology. Firstly, taking an L-threonine production strain such as escherichia coli LMT4 constructed in the early stage of a laboratory as a genetic modification basis, constructing a Calvin cycle by introducing a rubisco-derived ribulose bisphosphate carboxylase mutant and a rhodopseudomonas sphaeroides-derived ribulokinase mutant, reducing carbon loss and improving the production performance of the strain L-threonine. On the other hand, glyceraldehyde-3-phosphate dehydrogenase mutants derived from Thermotoga maritima and Thermus thermophilus were innovatively introduced. Effectively relieves the competition pressure of cofactors of the catalytic steps of aspartate semialdehyde dehydrogenase and homoserine dehydrogenase in the L-threonine synthesis pathway, further improves the production strength of the strain, and has certain significance for promoting the application of escherichia coli in the field of biosynthesis of L-threonine.

Description

Recombinant escherichia coli for improving yield of L-threonine and preparation method thereof

Technical Field

The invention relates to recombinant escherichia coli for improving the yield of L-threonine and a preparation method thereof, belonging to the technical field of biology.

Background

L-threonine is an essential amino acid and has important value in the fields of medicine, food, feed, cosmetics and the like. In the field of medicine, the L-threonine can be used for treating amino acid metabolic diseases such as phenylketonuria and the like, can be used as a key intermediate for synthesizing antibiotic monoamide bacteriocin, can obviously improve the utilization rate of protein by animals, promote the growth performance and improve the meat quality, is especially important for early development of piglets and poultry, and is also favored in the field of cosmetics for repairing skin barrier, moisturizing and whitening. In view of its wide application, the necessity for the biological production of L-threonine is becoming increasingly evident. Compared with chemical synthesis, the microbial fermentation method realizes high-efficiency synthesis by metabolizing engineering strain, reduces the production cost compared with chemical method and accords with the green manufacturing trend (Yang Hao,Hou Ying Jie,Xu Jian Zhong,et al.Metabolic engineering of Escherichia coli for the efficient production of L-threonine.Systems Microbiology and Biomanufacturing,4(2),810-819.).

Although the existing research effectively improves the L-threonine synthesis capability of engineering strains through a systematic metabolic engineering strategy, the current method mainly focuses on metabolic flow enhancement of synthetic pathways, including metabolic node flux optimization (Toan Minh Vo,Joon Young Park,Donghyuk Kim,et al.Use of acetate as substrate for sustainable production of homoserine and threonine by Escherichia coli W3110:a modular metabolic engineering approach.Metabolic engineering,84,13-22.). of key enzyme coding gene overexpression (Zhenqiang Zhao,Jiajia You,Xuanping Shi,et al.Multi-module engineering to guide the development of an efficient L-threonine-producing cell factory.Bioresource Technology,416,131802-131802.)、, however, the natural metabolic network of escherichia coli has carbon flow distribution competition, such as competition of an EMP pathway and a TCA (ternary polymerization) cycle. Enhancement of the L-threonine synthesis pathway may lead to blockage of the upstream pathway, while enhancement of glucose uptake rate may be limited by insufficient efficiency of the EMP pathway. On the other hand, with the improvement of the flux of the synthetic pathway, the steady-state regulation of intracellular cofactors becomes a new limiting bottleneck, and especially, the supply and demand balance of reducing forces such as NADPH/NADH directly influences the distribution of carbon flow and the synthesis efficiency (Zhenqiang Zhao,Rongshuai Zhu,Xuanping Shi,et al.Combining biosensor and metabolic network optimization strategies for enhanced L-threonine production in Escherichia coli.Biotechnology for Biofuels and Bioproducts,18(1),37-37.). of target products, so that the fermentation performance limitation of the current engineering strain is broken through by means of a multidimensional optimization strategy.

Disclosure of Invention

In order to solve the problem of lower production intensity of an engineering strain of escherichia coli L-threonine in the prior art, the invention provides a genetic engineering strain with higher production intensity and high yield of L-threonine, and a construction method and application thereof. In order to obtain L-threonine engineering strains with higher production intensity, the invention firstly constructs a Calvin cycle by introducing a rubisco (Rhodospirillum rubrum) -derived ribulose bisphosphate carboxylase mutant and a rhodopseudomonas sphaeroides (Cereibacter sphaeroides) -derived ribulose phosphate kinase mutant, reduces carbon loss and improves the production performance of L-threonine of the strains. On the other hand, glyceraldehyde-3-phosphate dehydrogenase from Thermomyces maritimus (Thermotoga maritima) and Thermus thermophilus (Thermus thermophilus) is introduced, and key site mutation modification is implemented, so that cofactor competition pressure of an aspartic semialdehyde dehydrogenase and a homoserine dehydrogenase catalytic step in an L-threonine synthesis pathway is effectively relieved, and the production intensity of the strain is further improved. The invention realizes the improvement of the production intensity of the strain by constructing the Calvin cycle and improving the cofactor cycle, and has a certain significance for promoting the application of escherichia coli in the field of biosynthesis of L-threonine.

The invention adopts the technical proposal that

A recombinant escherichia coli for improving the output of L-threonine and a preparation method thereof are provided, wherein the method comprises the steps of introducing a rubisco-derived ribulose bisphosphate carboxylase mutant and a rhodopseudomonas sphaeroides-derived ribulose kinase mutant on a chassis fungus genome by using a CRISPR-Cas9 technology so as to construct a Calvin cycle and reduce carbon loss.

Further, the method further comprises introducing Thermotoga maritima and Thermus thermophilus-derived glyceraldehyde-3-phosphate dehydrogenase on the chassis genome using CRISPR-Cas9 technology, and performing a critical site mutation engineering to enhance the supply of NADPH.

In the natural metabolic network of E.coli, carbon metabolic flux partitioning presents a significant competitive regulatory feature, where carbon source split competition between glycolytic pathway and tricarboxylic acid cycle constitutes a core metabolic contradiction. This competitive partitioning mechanism results in a double challenge in metabolic engineering, namely, firstly, when the expression of key enzymes of the L-threonine biosynthetic pathway is enhanced by a gene editing means, the consumption rate of upstream metabolic intermediates is increased significantly, metabolic flow blocking at the upstream node of the glycolytic pathway may be induced, and precursor accumulation and cell growth inhibition may be caused, and secondly, if glucose uptake efficiency is improved only by heterologously expressing glucose transporter or knocking out metabolic inhibitor, the metabolic pathway is limited by inherent enzyme dynamics limitation of the natural glycolytic pathway, carbon metabolic flow is still difficult to be efficiently directed to the target product synthetic pathway, and on the contrary, the metabolic burden of an acetyl-CoA pool may be aggravated, and carbon overflow phenomenon is induced.

The calvin cycle serves as a core metabolic pathway for the immobilization of autotrophic CO₂, and inorganic carbon is converted into a tricarbose intermediate such as glyceraldehyde-3-phosphate by catalyzing the carboxylation reaction of CO₂ and ribulose-1, 5-biphosphoric acid with ribulose biphosphoric acid carboxylase. The cycle realizes the assimilation of CO₂ and the construction of an organic carbon skeleton by efficiently coupling ATP and NADPH generated by a photosynthetic electron transfer chain. The Carl cycle is introduced into a heterotrophic microorganism metabolic network, and carbon metabolic flow distribution can be reconfigured, so that on one hand, dependence on traditional carbon sources such as glucose and the like is reduced by replacing part of glycolytic pathway functions, and therefore, the cost of raw materials for industrial fermentation is reduced, and on the other hand, triose phosphate generated by the Carl cycle can be converted into oxaloacetic acid through catalysis of phosphoenolpyruvate carboxylase, and the oxaloacetic acid is used as a precursor for L-threonine synthesis, and can directly provide carbon skeleton support for L-threonine biosynthesis.

Specifically, the invention uses the strain E.coli LMT4 (recorded in Chinese invention patent CN) constructed in advance in the laboratory

115011620B) is used as a starting strain, a CRISPR-Cas9 technology is used for introducing a rubisco (Rhodospirillum rubrum) source ribulose bisphosphate carboxylase mutant (rbcL_Rr^T81M、rbcL_Rr^R205K、rbcL_Rr^I240M、rbcL_Rr^L260F、rbcL_Rr^A269P) and a rhodopseudomonas sphaeroides (Cereibacter sphaeroides) source ribulose phosphate kinase mutant (prk_Cs^T108Y、prk_Cs^S140D、prk_Cs^T176S、prk_Cs^W254F、prk_Cs^V283L) to construct a calvin cycle, and carbon flow is redirected from a central metabolism part to an L-threonine synthesis path, so that the production level of the strain L-threonine is improved.

Preferably, the chassis fungus is a strain E.coli LMT4 which is constructed in the earlier stage of a laboratory and is described in the Chinese patent No. 115011620B.

The invention provides a mutant of a rubisco, which is obtained by mutating threonine at the 81 st position of a rubisco parent enzyme with an amino acid sequence shown as SEQ ID NO.1 into methionine, mutating arginine at the 205 st position into lysine, mutating isoleucine at the 240 th position into methionine, mutating leucine at the 260 th position into phenylalanine, mutating alanine at the 269 th position into proline, respectively ：rbcL_Rr^T81M、rbcL_Rr^R205K、rbcL_Rr^I240M、rbcL_Rr^L260F、rbcL_Rr^A269P.

The wild-type amino acid sequence of rbcL_Rr (SEQ ID NO. 1) is as follows:

MDQSSRYVNLALKEEDLIAGGEHVLCAYIMKPKAGYGYVATAAHFAAESSTGTNVEVCTTDDFTRGVDALVYEVDEARELTKIAYPVALFHRNITDGKAMIASFLTLTMGNNQGMGDVEYAKMHDFYVPEAYRALFDGPSVNISALWKVLGRPEVDGGLVVGTIIKPKLGLRPKPFAEACHAFWLGGDFIKNDEPQGNQPFAPLRDTIALVADAMRRAQDETGEAKLFSANITADDPFEIIARGEYVLETFGENASHVALLVDGYVAGAAAITTARRRFPDNFLHYHRAGHGAVTSPQSKRGYTAFVHCKMARLQGASGIHTGTMGFGKMEGESSDRAIAYMLTQDEAQGPFYRQSWGGMKACTPIISGGMNALRMPGFFENLGNANVILTAGGGAFGHIDGPVAGARSLRQAWQAWRDGVPVLDYAREHKELARAFESFPGDADQIYPGWRKALGVEDTRSALPA

the wild-type nucleotide sequence of rbcL_Rr (SEQ ID NO. 2) is as follows:

atggatcagagcagccgctatgtgaacctggcgctgaaagaagaagatctgattgcgggcggcgaacatgtgctgtgcgcgtatattatgaaaccgaaagcgggctatggctatgtggcgaccgcggcgcattttgcggcggaaagcagcaccggcaccaacgtggaagtgtgcaccaccgatgattttacccgcggcgtggatgcgctggtgtatgaagtggatgaagcgcgcgaactgaccaaaattgcgtatccggtggcgctgtttcatcgcaacattaccgatggcaaagcgatgattgcgagctttctgaccctgaccatgggcaacaaccagggcatgggcgatgtggaatatgcgaaaatgcatgatttttatgtgccggaagcgtatcgcgcgctgtttgatggcccgagcgtgaacattagcgcgctgtggaaagtgctgggccgcccggaagtggatggcggcctggtggtgggcaccattattaaaccgaaactgggcctgcgcccgaaaccgtttgcggaagcgtgccatgcgttttggctgggcggcgattttattaaaaacgatgaaccgcagggcaaccagccgtttgcgccgctgcgcgataccattgcgctggtggcggatgcgatgcgccgcgcgcaggatgaaaccggcgaagcgaaactgtttagcgcgaacattaccgcggatgatccgtttgaaattattgcgcgcggcgaatatgtgctggaaacctttggcgaaaacgcgagccatgtggcgctgctggtggatggctatgtggcgggcgcggcggcgattaccaccgcgcgccgccgctttccggataactttctgcattatcatcgcgcgggccatggcgcggtgaccagcccgcagagcaaacgcggctataccgcgtttgtgcattgcaaaatggcgcgcctgcagggcgcgagcggcattcataccggcaccatgggctttggcaaaatggaaggcgaaagcagcgatcgcgcgattgcgtatatgctgacccaggatgaagcgcagggcccgttttatcgccagagctggggcggcatgaaagcgtgcaccccgattattagcggcggcatgaacgcgctgcgcatgccgggcttttttgaaaacctgggcaacgcgaacgtgattctgaccgcgggcggcggcgcgtttggccatattgatggcccggtggcgggcgcgcgcagcctgcgccaggcgtggcaggcgtggcgcgatggcgtgccggtgctggattatgcgcgcgaacataaagaactggcgcgcgcgtttgaaagctttccgggcgatgcggatcagatttatccgggctggcgcaaagcgctgggcgtggaagatacccgcagcgcgctgccggcgtaa

The nucleotide sequence of the ribulose bisphosphate carboxylase mutant rbcL_Rr^T81M is formed by mutating ACC at 241 th to 243 th positions of nucleotide of a parent enzyme of the ribulose bisphosphate carboxylase shown in SEQ ID NO.2 into ATG;

the nucleotide sequence of the ribulose bisphosphate carboxylase mutant rbcL_Rr^R205K is characterized in that CGC at 613-615 th positions of the nucleotide of a parent enzyme of the ribulose bisphosphate carboxylase shown in SEQ ID NO.2 is mutated into AAA;

the nucleotide sequence of the ribulose bisphosphate carboxylase mutant rbcL_Rr^I240M is characterized in that ATT at 718-720 positions of the nucleotide of the parent enzyme of the ribulose bisphosphate carboxylase shown in SEQ ID NO.2 is mutated into ATG;

the nucleotide sequence of the ribulose bisphosphate carboxylase mutant rbcL_Rr^L260F is formed by mutating CTG at 778 th to 780 th positions of nucleotides of a parent enzyme of the ribulose bisphosphate carboxylase shown in SEQ ID NO.2 into TTT;

the nucleotide sequence of the ribulose bisphosphate carboxylase mutant rbcL_Rr^A269P is characterized in that GCG at 805 th to 807 th positions of the nucleotide of a parent enzyme of the ribulose bisphosphate carboxylase shown in SEQ ID NO.2 is mutated into CCG.

The present invention also provides a mutant of phosphoribulokinase prk_Cs, which is obtained by mutating threonine at position 108 of phosphoribulokinase prk_Cs (NCBI NO.P12033.2, SEQ ID NO. 3) from rhodopseudomonas sphaeroides Cereibacter sphaeroides to tyrosine, mutating serine at position 140 to aspartic acid, mutating threonine at position 176 to serine, mutating tryptophan at position 254 to phenylalanine, mutating valine at position 283 to leucine, respectively ：prk_Cs^T108Y、prk_Cs^S140D、prk_Cs^T176S、prk_Cs^W254F、prk_Cs^V283L.

The prk_Cs amino acid sequence (SEQ ID NO. 3) is as follows:

MSKKHPIISVTGSSGAGTSTVKHTFDQIFRREGVKAVSIEGDAFHRFNRADMKAELDRRYAAGDATFSHFSYEANELKELERVFREYGETGQGRTRTYVHDDAEAARTGVAPGNFTDWRDFDSDSHLLFYEGLHGAVVNSEVNIAGLADLKIGVVPVINLEWIQKIHRDRATRGYTTEAVTDVILRRMHAYVHCIVPQFSQTDINFQRVPVVDTSNPFIARWIPTADESVVVIRFRNPRGIDFPYLTSMIHGSWMSRANSIVVPGNKLDLAMQLILTPLIDRVVRESKVA

the prk_Cs nucleotide sequence (SEQ ID NO. 4) is as follows:

atgagcaaaaaacatccgattattagcgtgaccggcagcagcggcgcgggcaccagcaccgtgaaacatacctttgatcagatttttcgccgcgaaggcgtgaaagcggtgagcattgaaggcgatgcgtttcatcgctttaaccgcgcggatatgaaagcggaactggatcgccgctatgcggcgggcgatgcgacctttagccattttagctatgaagcgaacgaactgaaagaactggaacgcgtgtttcgcgaatatggcgaaaccggccagggccgcacccgcacctatgtgcatgatgatgcggaagcggcgcgcaccggcgtggcgccgggcaactttaccgattggcgcgattttgatagcgatagccatctgctgttttatgaaggcctgcatggcgcggtggtgaacagcgaagtgaacattgcgggcctggcggatctgaaaattggcgtggtgccggtgattaacctggaatggattcagaaaattcatcgcgatcgcgcgacccgcggctataccaccgaagcggtgaccgatgtgattctgcgccgcatgcatgcgtatgtgcattgcattgtgccgcagtttagccagaccgatattaactttcagcgcgtgccggtggtggataccagcaacccgtttattgcgcgctggattccgaccgcggatgaaagcgtggtggtgattcgctttcgcaacccgcgcggcattgattttccgtatctgaccagcatgattcatggcagctggatgagccgcgcgaacagcattgtggtgccgggcaacaaactggatctggcgatgcagctgattctgaccccgctgattgatcgcgtggtgcgcgaaagcaaagtggcgtaa

The nucleotide sequence of the mutant prk_Cs^T108Y of the phosphoribulokinase is formed by mutating ACC at the 322 th to 324 th positions of the nucleotide of the parent enzyme of the phosphoribulokinase shown in SEQ ID NO.4 into TAT;

The nucleotide sequence of the phosphoribulokinase prk_Cs^S140D mutant is that AGC at 418 th to 420 th positions of nucleotide of a phosphoribulokinase parent enzyme shown in SEQ ID NO.4 is mutated into GAT;

The nucleotide sequence of the phosphoribulokinase prk_Cs^T176S mutant is characterized in that ACC at 526-528 positions of nucleotide of a phosphoribulokinase parent enzyme shown in SEQ ID NO.4 is mutated into AGC;

The nucleotide sequence of the mutant of the phosphoribulokinase prk_Cs^W254F is formed by mutating TGG at 760 th to 762 th positions of nucleotides of a parent enzyme of the phosphoribulokinase shown in SEQ ID NO.4 into TTT;

The nucleotide sequence of the mutant prk_Cs^V283L of the phosphoribulokinase is that GTG at 847-849 positions of nucleotide of a parent enzyme of the phosphoribulokinase shown in SEQ ID NO.4 is mutated into CTG;

The invention provides a high-yield L-threonine genetic engineering bacterium which is E.coli T06, is constructed by introducing encoding genes of rbcL_Rr^R205K and prk_Cs^T108Y on a genome of E.coli LMT4 by using a CRISPR-CAS9 technology, and achieves the L-threonine yield of 28.6g/L after shaking fermentation.

Second, cofactor recycling is considered a critical process in biochemical production. NADPH is used as a reducing force in the biosynthesis of L-threonine and the synthesis of other valuable chemicals. To solve the ubiquitous cofactor imbalance problem, it is necessary to provide sufficient NADPH. Therefore, it is important to increase the efficiency of E.coli production of target chemicals by increasing the NADPH/NADP+ ratio. Coli produces NADPH as an L-threonine producing strain in two major ways, the pentose phosphate pathway and the citrate cycle. However, both of these approaches have limited ability to produce NADPH in bacteria, and the introduction of exogenous highly efficient NADPH synthase is required to obtain more NADPH supply. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the central carbon metabolic pathway, and is capable of catalyzing NADP⁺ and glyceraldehyde-3-phosphate (G3P) to generate NADPH and 3-phosphate-D-glycerophosphate, and is a main target for NADPH supply and solving the problem of cofactor imbalance.

The invention utilizes a molecular transformation technology to mutate glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima (Thermotoga maritima) and Thermus thermophilus (Thermus thermophilus) to obtain a beneficial mutant gapA_Tm^L175M、gapA_Tm^Y181F、gapA_Tm^V220L、gapA_Tm^R268K、gapA_Tm^Y181F/L175M、gapA_Tm^Y181F/V220L、gapA_Tm^Y181F/R268K、gapA_Tt^T98V、gapA_Tt^L185Y、gapA_Tt^P232A、gapA_Tt^Y272F、gapA_Tt^P232A/T98V、gapA_Tt^P232A/L185Y、gapA_Tt^P232A/Y272F),, and over-expresses the mutant on the genome of an escherichia coli L-threonine production strain after adaptive evolution, so that the supply of NADPH in the L-threonine synthesis process is increased, and the capacity of producing L-threonine by fermentation is improved.

The present invention also provides a mutant in which tyrosine 181 of glyceraldehyde-3-phosphate dehydrogenase gapA_Tm (NCBI No. WP_004081074.1,SEQ ID NO.5) derived from Thermotoga maritima Thermotoga maritima is mutated to phenylalanine, arginine 268 is mutated to lysine, tyrosine 181 is mutated to phenylalanine, arginine 268 is mutated to lysine, and the amino acid sequences of gapA_Tm^Y181F、gapA_Tm^R268K、gapA_Tm^Y181F/R268K.gapA_Tm (SEQ ID NO. 5) are respectively named as follows:

MARVAINGFGRIGRLVYRIIYERKNPDIEVVAINDLTDTKTLAHLLKYDSVHKKFPGKVEYTENSLIVDGKEIKVFAEPDPSKLPWKDLGVDFVIESTGVFRNREKAELHLQAGAKKVIITAPAKGEDITVVIGCNEDQLKPEHTIISCASCTTNSIAPIVKVLHEKFGIVSGMLTTVHSYTNDQRVLDLPHKDLRRARAAAVNIIPTTTGAAKAVALVVPEVKGKLDGMAIRVPTPDGSITDLTVLVEKETTVEEVNAVMKEATEGRLKGIIGYNDEPIVSSDIIGTTFSGIFDATITNVIGGKLVKVASWYDNEYGYSNRVVDTLELLLKM

The gapA_Tm nucleotide sequence (SEQ ID NO. 6) is as follows:

atggcgcgcgtggcgattaacggctttggccgcattggccgcctggtgtatcgcattatttatgaacgcaaaaacccggatattgaagtggtggcgattaacgatctgaccgataccaaaaccctggcgcatctgctgaaatatgatagcgtgcataaaaaatttccgggcaaagtggaatataccgaaaacagcctgattgtggatggcaaagaaattaaagtgtttgcggaaccggatccgagcaaactgccgtggaaagatctgggcgtggattttgtgattgaaagcaccggcgtgtttcgcaaccgcgaaaaagcggaactgcatctgcaggcgggcgcgaaaaaagtgattattaccgcgccggcgaaaggcgaagatattaccgtggtgattggctgcaacgaagatcagctgaaaccggaacataccattattagctgcgcgagctgcaccaccaacagcattgcgccgattgtgaaagtgctgcatgaaaaatttggcattgtgagcggcatgctgaccaccgtgcatagctataccaacgatcagcgcgtgctggatctgccgcataaagatctgcgccgcgcgcgcgcggcggcggtgaacattattccgaccaccaccggcgcggcgaaagcggtggcgctggtggtgccggaagtgaaaggcaaactggatggcatggcgattcgcgtgccgaccccggatggcagcattaccgatctgaccgtgctggtggaaaaagaaaccaccgtggaagaagtgaacgcggtgatgaaagaagcgaccgaaggccgcctgaaaggcattattggctataacgatgaaccgattgtgagcagcgatattattggcaccacctttagcggcatttttgatgcgaccattaccaacgtgattggcggcaaactggtgaaagtggcgagctggtatgataacgaatatggctatagcaaccgcgtggtggataccctggaactgctgctgaaaatgtaa

The nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gapA_Tm^Y181F mutant is that TAT at 541-543 bits of nucleotide of a parent enzyme of the phosphoribulokinase shown in SEQ ID NO.6 is mutated into TTT;

The nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gapA_Tm^R268K mutant is characterized in that CGC at the 802 th to 804 th positions of nucleotides of a parent enzyme of the phosphoribulokinase shown in SEQ ID NO.6 is mutated into AAA;

The nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gapA_Tm^Y181F/R268K mutant is that TAT at 541 th to 543 rd positions of nucleotide of a parent enzyme of the phosphoribulokinase shown in SEQ ID NO.6 is mutated into TTT, and CGC at 802 th to 804 th positions is mutated into AAA.

The present invention also provides a mutant of glyceraldehyde-3-phosphate dehydrogenase gapA_Tt (NCBI No.1VC2_A, SEQ ID No. 7) derived from Thermotoga maritima Thermus thermophilus, or mutant of proline at position 232 into alanine, or mutant of tyrosine at position 272 into phenylalanine, or mutant of proline at position 232 into alanine, respectively named gapA_Tt^P232A、gapA_Tt^Y272F、gapA_Tt^P232A/Y272F.

The gapA_Tt amino acid sequence is as follows (SEQ ID NO. 7):

MKVGINGFGRIGRQVFRILHERGVEVALINDLTDNKTLAHLLKYDSTYGRFPGAVGYDEENLYVDGKAIRATAIKDPREIPWKQAGVGVVVESTGVFTDGEKARAHLEAGAKKVIITAPAKNEDITVVLGVNHEQYDPAKHHILSNASCTTNSLAPVMKVLEKAFGVEKALMTTVHSYTNDQRLLDLPHKDLRRARAAALNIIPTTTGAAKATALVLPSLKGRFDGMALRVPTPTGSISDITALLKREVTAEEVNAALKAAAEGPLKGILAYTEDEIVLRDIVMDPHSSIVDGKLTKAIGNLVKVFAWYDNEWGYANRVADLVELVLKKGV

the gapA_Tt nucleotide sequence is as follows (SEQ ID NO. 8):

atgaaagtgggcattaacggctttggccgcattggccgccaggtgtttcgcattctgcatgaacgcggcgtggaagtggcgctgattaacgatctgaccgataacaaaaccctggcgcatctgctgaaatatgatagcacctatggccgctttccgggcgcggtgggctatgatgaagaaaacctgtatgtggatggcaaagcgattcgcgcgaccgcgattaaagatccgcgcgaaattccgtggaaacaggcgggcgtgggcgtggtggtggaaagcaccggcgtgtttaccgatggcgaaaaagcgcgcgcgcatctggaagcgggcgcgaaaaaagtgattattaccgcgccggcgaaaaacgaagatattaccgtggtgctgggcgtgaaccatgaacagtatgatccggcgaaacatcatattctgagcaacgcgagctgcaccaccaacagcctggcgccggtgatgaaagtgctggaaaaagcgtttggcgtggaaaaagcgctgatgaccaccgtgcatagctataccaacgatcagcgcctgctggatctgccgcataaagatctgcgccgcgcgcgcgcggcggcgctgaacattattccgaccaccaccggcgcggcgaaagcgaccgcgctggtgctgccgagcctgaaaggccgctttgatggcatggcgctgcgcgtgccgaccccgaccggcagcattagcgatattaccgcgctgctgaaacgcgaagtgaccgcggaagaagtgaacgcggcgctgaaagcggcggcggaaggcccgctgaaaggcattctggcgtataccgaagatgaaattgtgctgcgcgatattgtgatggatccgcatagcagcattgtggatggcaaactgaccaaagcgattggcaacctggtgaaagtgtttgcgtggtatgataacgaatggggctatgcgaaccgcgtggcggatctggtggaactggtgctgaaaaaaggcgtgtaa

the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gapA_Tt^P232A mutant is characterized in that CCG at 694 th to 696 th positions of nucleotide of a parent enzyme of the phosphoribulokinase shown in SEQ ID NO.6 is mutated into GCG;

the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gapA_Tt^Y272F mutant is that TAT at 814-816 th positions of nucleotide of a parent enzyme of the phosphoribulokinase shown in SEQ ID NO.6 is mutated into TTT;

The nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gapA_Tt^P232A/Y272F mutant is characterized in that CCG at 694 th to 696 th positions of nucleotide of a parent enzyme of phosphoribulokinase shown in SEQ ID NO.6 is mutated into GCG, and TAT at 814 th to 816 th positions is mutated into TTT

The invention also provides the genetically engineered bacterium which is constructed by the method and is used for high-yield L-threonine.

The invention provides a recombinant escherichia coli which overexpresses the rubidium rubrum (Rhodospirillum rubrum) -derived ribulose bisphosphate carboxylase mutant, the rhodopseudomonas sphaeroides (Cereibacter sphaeroides) -derived ribulose kinase mutant, the Thermotoga maritima (Thermotoga maritima) -derived glyceraldehyde-3-phosphate dehydrogenase mutant and the Thermus thermophilus (Thermus thermophilus) -derived glyceraldehyde-3-phosphate dehydrogenase mutant.

In one embodiment, the mutant ribulose bisphosphate carboxylase, mutant ribulokinase and mutant glyceraldehyde-3-phosphate dehydrogenase are expressed by Trc promoter;

in one embodiment, the nucleotide sequence encoding the Trc promoter is set forth in SEQ ID No. 13;

In one embodiment, the method comprises the steps of knocking out mbhA genes on the genome of the escherichia coli and integrating the ribulose bisphosphate carboxylase mutant at mbhA, knocking out ylbE genes on the genome of the escherichia coli and integrating the ribulose phosphate kinase mutant at ylbE, knocking out arpB genes on the genome of the escherichia coli and integrating glyceraldehyde-3-phosphate dehydrogenase mutant derived from Thermotoga maritima at arpB, knocking out yeeL genes on the genome of the escherichia coli and integrating glyceraldehyde-3-phosphate dehydrogenase mutant derived from Thermus thermophilus at yeeL;

In one embodiment, the mbhA Gene ID at NCBI is 62676021, the ylbE Gene ID at NCBI is 4056025, the arpB Gene ID at NCBI is 948935, and the yeeL Gene ID at NCBI is 2847764.

In one embodiment, the genetically engineered bacterium for high L-threonine production is E.coli T24. The genetically engineered bacterium for high-yield L-threonine is constructed by introducing coding genes of gapA_Tm^R268K and gapA_Tt^P232A/Y272F on a genome of E.coli T06 by using a CRISPR-Cas9 technology, and the yield of L-threonine reaches 164.9g/L after fed-batch fermentation by a 5L fermentation tank.

The invention also provides application of the genetically engineered bacterium for producing L-threonine in producing L-threonine. The method for application preferably comprises inoculating the genetically engineered bacterium with high L-threonine yield into a fermentation medium,

Preferably, the temperature condition of the fermentation is that the fermentation is performed at 30-40 ℃, or at 35-37 ℃, or at 30-31 ℃, or at 32-33 ℃, or at 34-35 ℃, or at 36-37 ℃, or at 38-39 ℃;

Preferably, the fermentation is carried out under the pH condition of 6.8-7.5, or under the pH condition of 6.8-7.0, or under the pH condition of 7.2-7.3, or under the pH condition of 7.0-7.2;

preferably, the fermentation time is at least 24 hours;

preferably, fermentation culture is carried out at 28-37 ℃ and 100-500 rpm for 40-48 hours, and after fermentation, L-threonine is obtained by separation.

Specifically, the genetically engineered bacterium for high-yield of L-threonine produces L-threonine through shake flask fermentation or 5L fermentation tank fermentation. The shake flask fermentation comprises the steps of inoculating the genetically engineered bacteria into a baffle shake flask containing 30mL of fermentation medium, culturing for 48h at 37 ℃ and 220rpm until fermentation is finished, obtaining fermentation liquor containing L-threonine, and separating and purifying the fermentation liquor to obtain the L-threonine. The fermentation in the 5L fermentation tank comprises the steps of inoculating the genetically engineered bacteria to an LB culture medium, activating at 37 ℃ and 220rpm overnight, inoculating the genetically engineered bacteria to a seed culture medium to prepare a seed solution, culturing for 9 hours, and inoculating the seed solution to the fermentation culture medium with an inoculum size of 20% by volume concentration. And inoculating the genetically engineered bacteria into a fermentation medium, performing fermentation culture for 40-48 hours at 28-37 ℃ and 100-500 rpm, and separating after fermentation to obtain L-threonine. The fermentation is carried out in a 5L fermentation tank, and in order to further improve the fermentation efficiency, the invention preferably adopts a feed fermentation mode to carry out fermentation culture on the genetically engineered bacteria. The fed-batch fermentation generally means that when the initial sugar consumption in the fermentation tank is finished and the pH value of a fermentation system is higher than 6.80, automatic feeding is started, and feeding is stopped when the feeding culture medium is fed at a speed of 0-50 mL/h until the pH value is lower than 6.80, so that the residual sugar in the fermentation tank is maintained at a lower level, and the sugar concentration is maintained at 0-5 g/L.

The invention also provides a method for improving threonine yield of escherichia coli, which is characterized in that the mutant of the ribulose bisphosphate carboxylase, the mutant of the ribulose phosphate kinase and the mutant of the glyceraldehyde-3-phosphate dehydrogenase are overexpressed in the escherichia coli.

The invention also provides application of the recombinant escherichia coli in preparing L-threonine or improving the yield of L-threonine and/or the sugar acid conversion rate.

The invention also provides a recombinant nucleic acid, which comprises a coding gene of a ribulose bisphosphate carboxylase mutant rbcL_Rr^R205K, a coding gene of a phosphoribulokinase mutant prk_Cs^T108Y, a coding gene of glyceraldehyde-3-phosphate dehydrogenase mutant gapA_Tm^R268K and a coding gene of gapA_Tt^P232A/Y272F, wherein the coding gene of the ribulose bisphosphate carboxylase mutant has a sequence shown as SEQ ID NO.9, the coding gene of the phosphoribulokinase mutant has a sequence shown as SEQ ID NO.10, and the coding gene of the glyceraldehyde-3-phosphate dehydrogenase mutant has a sequence shown as SEQ ID NO. 11-12.

Advantageous effects

Compared with the prior art, on the one hand, the invention takes the threonine producing strain E.coli LMT4 (recorded in the Chinese patent CN 115011620B) constructed before as an original strain. Using CRISPR-Cas9 gene editing techniques, a ribulose bisphosphate carboxylase mutant (rbcL_Rr^R205K) derived from rhodospirillum rubrum (Rhodospirillum rubrum) and a ribulose phosphate kinase mutant (prk_Cs^T108Y) derived from rhodopseudomonas globosa (Cereibacter sphaeroides) were introduced into the target strain to construct the calvin cycle. The metabolic flow inside the strain can be regulated by introducing the Calvin cycle, so that more carbon flow is effectively redirected to the synthesis path of L-threonine, and the level of L-threonine produced by the strain is improved. On the other hand, the invention improves the production efficiency of L-threonine by improving the ratio of NADPH/NADP+ in Escherichia coli, and in order to increase the intracellular NADPH pool, the beneficial mutants (gapA_Tm^R268K and gapA_Tt^P232A/Y272F) are obtained by mutating glyceraldehyde-3-phosphate dehydrogenase derived from Thermotoga maritima (Thermotoga maritima) and Thermus thermophilus (Thermus thermophilus) by using a molecular engineering technology, and the mutants are heterologously expressed in the Escherichia coli L-threonine producing strain obtained by early engineering, so that the supply of NADPH in the L-threonine synthesis process is increased, and the capacity of the strain for producing L-threonine by fermentation is improved.

The genetic engineering strain obtained by the system metabolic engineering strategy can realize the effective accumulation of L-threonine, the shake flask yield of L-threonine reaches 45.8g/L, the feed fermentation yield of a 5L fermentation tank reaches 168g/L, the sugar acid conversion rate reaches 63%, the production strength reaches 3.36g/L/h, and a foundation is laid for the subsequent industrialized application of the L-threonine engineering strain.

Drawings

FIG. 1 shows the results of 5L tank fermentation of T24 strain.

Detailed Description

The following specific examples are presented to illustrate the present invention, and those skilled in the art will readily appreciate the additional advantages and capabilities of the present invention as disclosed herein. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict. The methods used in the examples of the present invention are conventional methods, and the reagents used are commercially available.

The genotypes of the strains involved in the following examples are shown in Table 1 below:

TABLE 1 list of genetic alterations of strains

Strain name	Genetic manipulation
		LMT4	LMT4 in patent CN115011620B
T01	LMT4,mbhA::Ptrc-rbcLRrT81M
		T02	LMT4,mbhA::Ptrc-rbcLRrR205K
T03	LMT4,mbhA::Ptrc-rbcLRrI240M
		T04	LMT4,mbhA::Ptrc-rbcLRrL260F
T05	LMT4,mbhA::Ptrc-rbcLRrA269P
		T06	T02,ylbE::Ptrc-prkCsT108Y
T07	T02,ylbE::Ptrc-prkCsS140D
		T08	T02,ylbE::Ptrc-prkCsT176S
T09	T02,ylbE::Ptrc-prkCsW254F
		T10	T02,ylbE::Ptrc-prkCsV283L
T11	T06,arpB::Ptrc-gapATmL175M
		T12	T06,arpB::Ptrc-gapATmY181F
T13	T06,arpB::Ptrc-gapATmV220L
		T14	T06,arpB::Ptrc-gapATmR268K
T15	T06,arpB::Ptrc-gapATmY181F/L175M
		T16	T06,arpB::Ptrc-gapATmY181F/V220L
T17	T06,arpB::Ptrc-gapATmY181F/R268K
		T18	T14,yeeL::Ptrc-gapATtT98V
T19	T14,yeeL::Ptrc-gapATtL185Y
		T20	T14,yeeL::Ptrc-gapATtP232A
T21	T14,yeeL::Ptrc-gapATtY272F
		T22	T14,yeeL::Ptrc-gapATtP232A/T98V
T23	T14,yeeL::Ptrc-gapATtP232A/L185Y
		T24	T14,yeeL::Ptrc-gapATtP232A/Y272F

The primer sequences referred to in the following examples are shown in Table 2 below.

TABLE 2 primers according to the present invention

LB medium, 5g/L yeast extract, 10g/L peptone, 10g/L sodium chloride.

30G/L glucose, 3g/L yeast extract, 30mL/L corn steep liquor, 2g/L citric acid ,0.5g/LMgSO₄·7H₂O,10mg/L FeSO₄·7H₂O,10mg/L MnSO₄·H₂O,0.2mg/L vitamin H,0.3mg/L vitamin B₁, and the balance water, wherein the pH is 7.0-7.2.

20G/L glucose, 2g/L yeast extract, 15mL/L corn steep liquor, 2g/L citric acid ,1g/LMgSO₄·7H₂O,10mg/L FeSO₄·7H₂O,5mg/L MnSO₄·H₂O,2g/L potassium dihydrogen phosphate, 0.2mg/L vitamin H,0.3mg/L vitamin B₁, 15g/L ammonium sulfate, and the balance of water, wherein the pH value is 7.0-7.2.

Feed medium, 800g/L glucose.

The detection method involved in the following examples:

Detection of L-threonine production

The yield of the L-threonine is calculated by a high performance liquid chromatography, the concentration of the L-threonine and other amino acids is measured by a pre-column derived phthalic aldehyde (OPA) method, the liquid chromatography is carried out under the conditions that the column temperature is 40 ℃, the wavelength of an ultraviolet detector 338nm, and the flow rate of a mobile phase is 1mL/min.

Calculation of sugar acid conversion

The remaining glucose content, consumption rate, and Sugar acid conversion rate of the resulting fermentation broth were separately measured, wherein the Sugar acid conversion rate (Sugar-to-Acid Conversion Rate, SACR) was defined as the percentage of Sugar material converted to L-threonine per unit mass, and the calculation method was:

SACR=(M_sugar/M_L-Threonine)×100%

wherein, M_L-Threonine is the mass (g) of the L-threonine produced, and M_sugar is the mass (g) of the saccharide consumed, namely the difference between the total mass of the added glucose and the mass of the glucose remained in the system at the end of fermentation.

The detection method of the ratio of NADPH/NADP+ is as follows:

the assay was performed using a commercially available fluorescent detection kit for the ratio of 50140NADP/NADPH from the company Saint. NAD (P) H can reduce the probe in the kit to a highly fluorescent product, and the signal can be quantified by a fluorescence enzyme-labeled instrument (Ex/Em=540/590 nm)

EXAMPLE 1 construction of ribulose bisphosphate carboxylase and mutant thereof

Construction of a mutant of rbcL_Rr of rubisco Rhodospirillum rubrum-derived ribulose bisphosphate, the specific steps are as follows:

(1) Construction of pTrc-99a-rbcL_Rr

The ribulose bisphosphate carboxylase rbcL_Rr (NCBI NO.P04718.1, SEQ ID No. 1) derived from Rhodospirillum rubrum Rhodospirillum rubrum was codon-optimized and ligated to plasmid pTrc-99a (available for synthesis by the company) and designated pTrc-99a-rbcL_Rr.

The rbcL_Rr amino acid sequence (SEQ ID NO. 1) is as follows:

MDQSSRYVNLALKEEDLIAGGEHVLCAYIMKPKAGYGYVATAAHFAAESSTGTNVEVCTTDDFTRGVDALVYEVDEARELTKIAYPVALFHRNITDGKAMIASFLTLTMGNNQGMGDVEYAKMHDFYVPEAYRALFDGPSVNISALWKVLGRPEVDGGLVVGTIIKPKLGLRPKPFAEACHAFWLGGDFIKNDEPQGNQPFAPLRDTIALVADAMRRAQDETGEAKLFSANITADDPFEIIARGEYVLETFGENASHVALLVDGYVAGAAAITTARRRFPDNFLHYHRAGHGAVTSPQSKRGYTAFVHCKMARLQGASGIHTGTMGFGKMEGESSDRAIAYMLTQDEAQGPFYRQSWGGMKACTPIISGGMNALRMPGFFENLGNANVILTAGGGAFGHIDGPVAGARSLRQAWQAWRDGVPVLDYAREHKELARAFESFPGDADQIYPGWRKALGVEDTRSALPA

the rbcL_Rr nucleotide sequence (SEQ ID NO. 2) is as follows:

atggatcagagcagccgctatgtgaacctggcgctgaaagaagaagatctgattgcgggcggcgaacatgtgctgtgcgcgtatattatgaaaccgaaagcgggctatggctatgtggcgaccgcggcgcattttgcggcggaaagcagcaccggcaccaacgtggaagtgtgcaccaccgatgattttacccgcggcgtggatgcgctggtgtatgaagtggatgaagcgcgcgaactgaccaaaattgcgtatccggtggcgctgtttcatcgcaacattaccgatggcaaagcgatgattgcgagctttctgaccctgaccatgggcaacaaccagggcatgggcgatgtggaatatgcgaaaatgcatgatttttatgtgccggaagcgtatcgcgcgctgtttgatggcccgagcgtgaacattagcgcgctgtggaaagtgctgggccgcccggaagtggatggcggcctggtggtgggcaccattattaaaccgaaactgggcctgcgcccgaaaccgtttgcggaagcgtgccatgcgttttggctgggcggcgattttattaaaaacgatgaaccgcagggcaaccagccgtttgcgccgctgcgcgataccattgcgctggtggcggatgcgatgcgccgcgcgcaggatgaaaccggcgaagcgaaactgtttagcgcgaacattaccgcggatgatccgtttgaaattattgcgcgcggcgaatatgtgctggaaacctttggcgaaaacgcgagccatgtggcgctgctggtggatggctatgtggcgggcgcggcggcgattaccaccgcgcgccgccgctttccggataactttctgcattatcatcgcgcgggccatggcgcggtgaccagcccgcagagcaaacgcggctataccgcgtttgtgcattgcaaaatggcgcgcctgcagggcgcgagcggcattcataccggcaccatgggctttggcaaaatggaaggcgaaagcagcgatcgcgcgattgcgtatatgctgacccaggatgaagcgcagggcccgttttatcgccagagctggggcggcatgaaagcgtgcaccccgattattagcggcggcatgaacgcgctgcgcatgccgggcttttttgaaaacctgggcaacgcgaacgtgattctgaccgcgggcggcggcgcgtttggccatattgatggcccggtggcgggcgcgcgcagcctgcgccaggcgtggcaggcgtggcgcgatggcgtgccggtgctggattatgcgcgcgaacataaagaactggcgcgcgcgtttgaaagctttccgggcgatgcggatcagatttatccgggctggcgcaaagcgctgggcgtggaagatacccgcagcgcgctgccggcgtaa

(2) Construction of recombinant plasmids containing mutants

First, pTrc-99a-rbcL_Rr was used as a template and amplified with primers (Table 2) to obtain recombinant vectors containing mutants T81M, R205K, I240M, L260F, A269P ：pTrc-99a-rbcL_Rr^T81M,pTrc-99a-rbcL_Rr^R205K,pTrc-99a-rbcL_Rr^I240M,pTrc-99a-rbcL_Rr^L260F,pTrc-99a-rbcL_Rr^A269P.

(3) Construction of recombinant Strain containing rbcL_Rr mutant

The ribulose bisphosphate carboxylase mutant encoding gene rbcL_Rr^T81M、rbcL_Rr^R205K、rbcL_Rr^I240M、rbcL_Rr^L260F、rbcL_Rr^A269P from rhodospirillum rubrum Rhodospirillum rubrum was integrated into the mbhA (NCBI accession number: 62676021) site of LMT4 strain, respectively.

The E.coli W3110 genome was used as a template, and the upstream homology arm primers (mbhA-1, mbhA-2) and the downstream homology arm primers (mbhA-5, mbhA-6) were designed based on mbhA gene sequences. Then, primers mbhA-3 and mbhA-4 were designed using the pTrc-99a-rbcL_Rr^T81M、pTrc-99a-rbcL_Rr^R205K、pTrc-99a-rbcL_Rr^I240M、pTrc-99a-rbcL_Rr^L260F、pTrc-99a-rbcL_Rr^A269P plasmid constructed in example 2 as a template. Wherein, the P_trc promoter sequence is designed on the primers mbhA-2 and mbhA-3, each fragment is obtained through PCR amplification, and then fusion PCR is carried out by taking the fragments as templates, thus respectively obtaining the integrated fragments of the P_trc-rbcL_Rr^T81M gene, the P_trc-rbcL_Rr^R205K gene, the P_trc-rbcL_Rr^I240M gene, the P_trc-rbcL_Rr^L260F gene and the P_trc-rbcL_Rr^A269P gene. The DNA fragments obtained after annealing the primers gRNA-mbhA-1 and gRNA-mbhA-2 were ligated with plasmid pGRB to construct plasmid pGRB-mbhA. Plasmid pGRB-mbhA and the integrated fragments of P_trc-rbcL_Rr^T81M gene, P_trc-rbcL_Rr^R205K gene, P_trc-rbcL_Rr^I240M gene, P_trc-rbcL_Rr^L260F gene and P_trc-rbcL_Rr^A269P gene are simultaneously and electrically transformed into electrotransformation competent cells of LMT4 strain containing pREDCas9 to obtain positive transformants, and after the plasmids are eliminated, E.coli T01(E.coli LMT4,ΔmbhA::P_trc-rbcL_Rr^T81M)、E.coli T02(E.coli LMT4,ΔmbhA::P_trc-rbcL_Rr^R205K)、E.coli T03(E.coli LMT4,ΔmbhA::P_trc-rbcL_Rr^I240M)、E.coli T04(E.coli LMT4,ΔmbhA::P_trc-rbcL_Rr^L260F)、E.coli T05(E.coli LMT4,ΔmbhA::P_trc-rbcL_Rr^A269P) strain is obtained respectively.

(4) Shake flask fermentation of recombinant strains

Inoculating the prepared recombinant strains E.coli T01, E.coli T02, E.coli T03, E.coli T04 and E.coli T05 into LB culture medium, culturing at 37 ℃ for 12h for full activation, then inoculating into a 500mL round bottom triangular flask filled with 30mL seed culture medium according to 20% (v/v) inoculum size, sealing with nine layers of gauze, culturing at 37 ℃ and 220rpm for 8-10h, and preparing seed liquid.

Inoculating the seed solution into a 500mL baffle triangular flask containing 30mL of fermentation medium according to the inoculation amount of 15% (v/v), sealing nine layers of gauze, culturing at 37 ℃ and 240r/min, controlling the pH value to be 7.0-7.2 by adding 25% ammonia water in the fermentation process, and maintaining the fermentation by adding 60% (m/v) glucose solution to a final concentration of 5g/L when the glucose in the medium is exhausted, wherein the fermentation period is 36h, and preparing the fermentation liquid. After the fermentation, the production of L-threonine in the fermentation broth was separately examined. According to the above method, a recombinant strain E.coli WT (E.coli LMT4, delta mbhA:: P_trc-rbcL_Rr) was prepared in which the recombinant strain expressed wild-type ribulose bisphosphate carboxylase. After fermentation according to the above method, the yield of L-threonine was examined. The results are shown in Table 3 below.

TABLE 3:L production of threonine

Strains containing different mutants	Yield (g/L)	Sugar acid conversion (%)
			LMT4	31.5	40.2
E.coli WT	32.3	40.6
			T01(T81M)	30.1	38.4
T02(R205K)	35.2	42.4
			T03(I240M)	33.5	41.1
T04(L260F)	34.9	42
			T05(A269P)	32.8	40.9

The results show that the T02 strain has higher L-threonine yield and conversion rate, so that the T02 strain (E.coli LMT4, delta mbhA: P_trc-rbcL_Rr^R205K) is selected to be continuously transformed, and the Kalman cycle is supplemented.

EXAMPLE 2 construction of ribulokinase phosphate and mutants thereof

Construction of a mutant of ribulokinase prk_Cs derived from rhodopseudomonas sphaeroides Cereibacter sphaeroides, comprising the following specific steps:

(1) Construction of pTrc-99a-prk_Cs

The ribulose phosphate kinase prk_Cs (NCBI NO.P12033.2, SEQ ID No. 3) from P.globosus Cereibacter sphaeroides was codon-optimized and ligated to the plasmid pTrc-99a (available for synthesis by the company) and designated pTrc-99a-prk_Cs.

The prk_Cs amino acid sequence (SEQ ID NO. 3) is as follows:

MSKKHPIISVTGSSGAGTSTVKHTFDQIFRREGVKAVSIEGDAFHRFNRADMKAELDRRYAAGDATFSHFSYEANELKELERVFREYGETGQGRTRTYVHDDAEAARTGVAPGNFTDWRDFDSDSHLLFYEGLHGAVVNSEVNIAGLADLKIGVVPVINLEWIQKIHRDRATRGYTTEAVTDVILRRMHAYVHCIVPQFSQTDINFQRVPVVDTSNPFIARWIPTADESVVVIRFRNPRGIDFPYLTSMIHGSWMSRANSIVVPGNKLDLAMQLILTPLIDRVVRESKVA

the prk_Cs nucleotide sequence (SEQ ID NO. 4) is as follows:

atgagcaaaaaacatccgattattagcgtgaccggcagcagcggcgcgggcaccagcaccgtgaaacatacctttgatcagatttttcgccgcgaaggcgtgaaagcggtgagcattgaaggcgatgcgtttcatcgctttaaccgcgcggatatgaaagcggaactggatcgccgctatgcggcgggcgatgcgacctttagccattttagctatgaagcgaacgaactgaaagaactggaacgcgtgtttcgcgaatatggcgaaaccggccagggccgcacccgcacctatgtgcatgatgatgcggaagcggcgcgcaccggcgtggcgccgggcaactttaccgattggcgcgattttgatagcgatagccatctgctgttttatgaaggcctgcatggcgcggtggtgaacagcgaagtgaacattgcgggcctggcggatctgaaaattggcgtggtgccggtgattaacctggaatggattcagaaaattcatcgcgatcgcgcgacccgcggctataccaccgaagcggtgaccgatgtgattctgcgccgcatgcatgcgtatgtgcattgcattgtgccgcagtttagccagaccgatattaactttcagcgcgtgccggtggtggataccagcaacccgtttattgcgcgctggattccgaccgcggatgaaagcgtggtggtgattcgctttcgcaacccgcgcggcattgattttccgtatctgaccagcatgattcatggcagctggatgagccgcgcgaacagcattgtggtgccgggcaacaaactggatctggcgatgcagctgattctgaccccgctgattgatcgcgtggtgcgcgaaagcaaagtggcgtaa

(2) Construction of recombinant plasmids containing mutants

First, pTrc-99a-prk_Cs was used as a template and amplified with primers (Table 2) to obtain recombinant vectors containing mutants T108Y, S140D, T176S, W254F, V283L ：pTrc-99a-prk_Cs^T108Y,pTrc-99a-prk_Cs^S140D,pTrc-99a-prk_Cs^T176S,pTrc-99a-prk_Cs^W254F,pTrc-99a-prk_Cs^V283L.

(3) Construction of recombinant strains containing prk_Cs mutants

The ribulokinase phosphate mutant-encoding gene prk_Cs^T108Y、prk_Cs^S140D、prk_Cs^T176S、prk_Cs^W254F、prk_Cs^V283L from P.globosus Cereibacter sphaeroides was integrated into the ylbE (NCBI accession number: 4056025) site of the T02 strain, respectively.

The E.coli W3110 genome was used as a template, and the upstream homology arm primers (ylbE-1, ylbE-2) and the downstream homology arm primers (ylbE-5, ylbE-6) were designed based on ylbE gene sequences. Then, primers ylbE-3 and ylbE-4 were designed using the pTrc-99a-prk_Cs^T108Y、pTrc-99a-prk_Cs^S140D、pTrc-99a-prk_Cs^T176S、pTrc-99a-prk_Cs^W254F、pTrc-99a-prk_Cs^V283L plasmid thus constructed as a template. Wherein, the P_trc promoter sequence is designed on the primers ylbE-2 and ylbE-3, each fragment is obtained through PCR amplification, and then fusion PCR is carried out by taking the fragments as templates, thus respectively obtaining the integrated fragments of the P_trc-prk_Cs^T108Y gene, the P_trc-prk_Cs^S140D gene, the P_trc-prk_Cs^T176S gene, the P_trc-prk_Cs^W254F gene and the P_trc-prk_Cs^V283L gene. The DNA fragments obtained after annealing the primers gRNA-ylbE-1 and gRNA-ylbE-2 were ligated with plasmid pGRB to construct plasmid pGRB-ylbE. The plasmids pGRB to ylbE and the integrated fragments of the P_trc-prk_Cs^T108Y gene, the P_trc-prk_Cs^S140D gene, the P_trc-prk_Cs^T176S gene, the P_trc-prk_Cs^W254F gene and the P_trc-prk_Cs^V283L gene are simultaneously and electrically transformed into electrotransformation competent cells of the T02 strain containing pREDCas to obtain positive transformants, and after the plasmids are eliminated, E.coli T06(E.coli T02,ΔylbE::P_trc-prk_Cs^T108Y)、E.coli T07(E.coli T02,ΔylbE::P_trc-prk_Cs^S140D)、E.coli T08(E.coli T02,ΔylbE::P_trc-prk_Cs^T176S)、E.coli T09(E.coli T02,ΔylbE::P_trc-prk_Cs^W254F)、E.coli T10(E.coli T02,ΔylbE::P_trc-prk_Cs^V283L) strains are respectively obtained.

(4) Shake flask fermentation of recombinant strains

Shake flask fermentation of the recombinant strain, determination of the L-threonine content, and calculation of the sugar acid conversion rate of the strain were performed as in example 1. According to the above method, a recombinant strain T02 WT (E.coli T02, delta ylbE:: P_trc-prk_Cs) was prepared in which the recombinant strain expressed wild-type ribulose phosphate kinase. After fermentation according to the above method, the yield of L-threonine and the sugar acid conversion rate of the strain were examined. The results are shown in Table 4 below:

TABLE 4:L production of threonine

Strains containing different mutants	Yield (g/L)	Sugar acid conversion (%)
			T02	35.2	42.4
T02WT	35.6	42.7
			T06(T02T108Y)	38.7	44.2
T07(T02S140D)	36.2	43.6
			T08(T02T176S)	35.8	42.8
T09(T02W254F)	33.5	42
			T10(T02V283L)	37.5	43.9

The results show that the T06 strain (E.coli LMT4,. DELTA. mbhA: P_trc-rbcL_Rr^R205KΔylbE::P_trc-prk_Cs^T108Y) has a higher L-threonine production and conversion, so that the T06 strain was selected for further engineering.

EXAMPLE 3 construction of glyceraldehyde-3-phosphate dehydrogenase and mutants thereof

The method comprises the following specific steps:

1. Construction of glyceraldehyde-3-phosphate dehydrogenase gapA_Tm mutant derived from Thermotoga maritima Thermotoga maritima (1) construction of pTrc-99a-gapA_Tm

Glyceraldehyde-3-phosphate dehydrogenase gapA_Tm (NCBI No. WP_004081074.1,SEQ ID NO.5) derived from Thermotoga maritima Thermotoga maritima was codon-optimized and ligated to plasmid pTrc-99a (available for synthesis by the company) and designated pTrc-99a-gapA_Tm.

The gapA_Tm amino acid sequence (SEQ ID NO. 5) is as follows:

MARVAINGFGRIGRLVYRIIYERKNPDIEVVAINDLTDTKTLAHLLKYDSVHKKFPGKVEYTENSLIVDGKEIKVFAEPDPSKLPWKDLGVDFVIESTGVFRNREKAELHLQAGAKKVIITAPAKGEDITVVIGCNEDQLKPEHTIISCASCTTNSIAPIVKVLHEKFGIVSGMLTTVHSYTNDQRVLDLPHKDLRRARAAAVNIIPTTTGAAKAVALVVPEVKGKLDGMAIRVPTPDGSITDLTVLVEKETTVEEVNAVMKEATEGRLKGIIGYNDEPIVSSDIIGTTFSGIFDATITNVIGGKLVKVASWYDNEYGYSNRVVDTLELLLKM

The gapA_Tm nucleotide sequence (SEQ ID NO. 6) is as follows:

atggcgcgcgtggcgattaacggctttggccgcattggccgcctggtgtatcgcattatttatgaacgcaaaaacccggatattgaagtggtggcgattaacgatctgaccgataccaaaaccctggcgcatctgctgaaatatgatagcgtgcataaaaaatttccgggcaaagtggaatataccgaaaacagcctgattgtggatggcaaagaaattaaagtgtttgcggaaccggatccgagcaaactgccgtggaaagatctgggcgtggattttgtgattgaaagcaccggcgtgtttcgcaaccgcgaaaaagcggaactgcatctgcaggcgggcgcgaaaaaagtgattattaccgcgccggcgaaaggcgaagatattaccgtggtgattggctgcaacgaagatcagctgaaaccggaacataccattattagctgcgcgagctgcaccaccaacagcattgcgccgattgtgaaagtgctgcatgaaaaatttggcattgtgagcggcatgctgaccaccgtgcatagctataccaacgatcagcgcgtgctggatctgccgcataaagatctgcgccgcgcgcgcgcggcggcggtgaacattattccgaccaccaccggcgcggcgaaagcggtggcgctggtggtgccggaagtgaaaggcaaactggatggcatggcgattcgcgtgccgaccccggatggcagcattaccgatctgaccgtgctggtggaaaaagaaaccaccgtggaagaagtgaacgcggtgatgaaagaagcgaccgaaggccgcctgaaaggcattattggctataacgatgaaccgattgtgagcagcgatattattggcaccacctttagcggcatttttgatgcgaccattaccaacgtgattggcggcaaactggtgaaagtggcgagctggtatgataacgaatatggctatagcaaccgcgtggtggataccctggaactgctgctgaaaatgtaa

(2) Construction of recombinant plasmids containing mutants

First, pTrc-99a-gapA_Tm was used as a template and amplified with primers (Table 2) to obtain recombinant vectors containing mutants L175M, Y181F, V220L, R268K, Y181F/L175M, Y181F/V220L, Y181F/R268K ：pTrc-99a-gapA_Tm^L175M,pTrc-99a-gapA_Tm^Y181F,pTrc-99a-gapA_Tm^V220L,pTrc-99a-gapA_Tm^R268K,pTrc-99a-gapA_Tm^Y181F/L175M,pTrc-99a-gapA_Tm^Y181F/V220L,pTrc-99a-gapA_Tm^Y181F/R268K.

(3) Construction of recombinant Strain containing gapA_Tm

The glyceraldehyde-3-phosphate dehydrogenase encoding genes gapA_Tm^L175M mutant, gapA_Tm^Y181F mutant, gapA_Tm^V220L mutant, gapA_Tm^R268K mutant, gapA_Tm^Y181F/L175M mutant, gapA_Tm^Y181F/V220L mutant and gapA_Tm^Y181F/R268K mutant from Thermotoga maritima (Thermotoga maritima) were integrated into the arpB (NCBI number: 948935) site of the T06 strain, respectively.

The E.coli W3110 genome is used as a template, an upstream homology arm primer (arpB-1, arpB-2) and a downstream homology arm primer (arpB-5, arpB-6) are designed according to arpB gene sequences, then the constructed pTrc-99a-gapA_Tm^L175M,pTrc-99a-gapA_Tm^Y181F,pTrc-99a-gapA_Tm^V220L,pTrc-99a-gapA_Tm^R268K,pTrc-99a-gapA_Tm^Y181F/L175M,pTrc-99a-gapA_Tm^Y181F/V220L,pTrc-99a-gapA_Tm^Y181F/R268K plasmid is used as a template, primers arpB-3 and arpB-4 are designed, wherein the P_trc promoter sequences are designed on the primers arpB-2 and arpB-3, each fragment is obtained through PCR amplification, and fusion PCR is carried out by using the fragments as templates to respectively obtain integrated fragments of the P_trc-gapA_Tm^L175M gene, the P_trc-gapA_Tm^Y181F gene, the P_trc-gapA_Tm^V220L gene, the P_trc-gapA_Tm^R268K gene, the P_trc-gapA_Tm^Y181F/L175M gene and the P_trc-gapA_Tm^Y181F/V220L、P_trc-gapA_Tm^Y181F/R268K gene. The DNA fragments obtained after annealing the primers gRNA-arpB-1 and gRNA-arpB-2 were ligated with plasmid pGRB to construct plasmid pGRB-arpB. The integrated fragments of plasmids pGRB-arpB and P_trc-gapA_Tm^L175M、P_trc-gapA_Tm^Y181F、P_trc-gapA_Tm^V220L、P_trc-gapA_Tm^R268K、P_trc-gapA_Tm^Y181F/L175M、P_trc-gapA_Tm^Y181F/V220L、P_trc-gapA_Tm^Y181F/R268K, respectively, were simultaneously electrotransformed into electrotransformed competent cells of strain T06 containing pREDCas9 to obtain positive transformants, and after removal of the plasmids, strain E.coli T11(E.coli T06,ΔarpB::P_trc-gapA_Tm^L175M)、E.coli T12(E.coli T06,ΔarpB::P_trc-gapA_Tm^Y181F)、E.coli T13(E.coli T06,ΔarpB::P_trc-gapA_Tm^V220L)、E.coli T14(E.coli T06,ΔarpB::P_trc-gapA_Tm^R268K)、E.coli T15(E.coli T06,ΔarpB::P_trc-gapA_Tm^Y181F/L175M)、E.coli T16(E.coli T06,ΔarpB::P_trc-gapA_Tm^Y181F/V220L)、E.coli T17(E.coli T06,ΔarpB::P_trc-gapA_Tm^Y181F/R268K) was obtained, respectively.

(4) Shake flask fermentation of recombinant strains

Shake flask fermentation of the recombinant strain, determination of L-threonine content, and calculation of the sugar acid conversion rate of the strain were performed as in example 1, and the ratio of NADPH/NADP+ in the recombinant strain after the end of the shake flask fermentation was detected. According to the above method, a recombinant strain T06 WT (E.coli T06, delta arpB:: P_trc-gapA_Tm) was prepared in which the recombinant strain expressed wild-type glyceraldehyde-3-phosphate dehydrogenase. After fermentation according to the above method, the yield of L-threonine and the sugar acid conversion rate of the strain were examined. And the ratio of NADPH/NADP+ in the recombinant strain after the completion of shake flask fermentation was examined, and the results are shown in Table 5.

TABLE 5 Effect of strains containing different mutants

The results show that T14 strain (E.coli LMT4,ΔmbhA::P_trc-rbcL_Rr^R205KΔylbE::P_trc-prk_Cs^T108YΔarpB::P_trc-gapA_Tm^R268K) has higher L-threonine production and conversion, and thus T14 strain selection continues to be engineered.

2. Construction of glyceraldehyde-3-phosphate dehydrogenase gapA_Tt mutant derived from Thermus thermophilus Thermus thermophilus (1) construction of pTrc-99a-gapA_Tt

Glyceraldehyde-3-phosphate dehydrogenase gapA_Tt (NCBI No.1VC2_A, SEQ ID No. 7) derived from Thermotoga maritima Thermus thermophilus was codon-optimized and ligated to plasmid pTrc-99a (available for synthesis by the company) and designated pTrc-99a-gapA_Tt.

The gapA_Tt amino acid sequence is as follows (SEQ ID NO. 7):

MKVGINGFGRIGRQVFRILHERGVEVALINDLTDNKTLAHLLKYDSTYGRFPGAVGYDEENLYVDGKAIRATAIKDPREIPWKQAGVGVVVESTGVFTDGEKARAHLEAGAKKVIITAPAKNEDITVVLGVNHEQYDPAKHHILSNASCTTNSLAPVMKVLEKAFGVEKALMTTVHSYTNDQRLLDLPHKDLRRARAAALNIIPTTTGAAKATALVLPSLKGRFDGMALRVPTPTGSISDITALLKREVTAEEVNAALKAAAEGPLKGILAYTEDEIVLRDIVMDPHSSIVDGKLTKAIGNLVKVFAWYDNEWGYANRVADLVELVLKKGV

the gapA_Tt nucleotide sequence is as follows (SEQ ID NO. 8):

atgaaagtgggcattaacggctttggccgcattggccgccaggtgtttcgcattctgcatgaacgcggcgtggaagtggcgctgattaacgatctgaccgataacaaaaccctggcgcatctgctgaaatatgatagcacctatggccgctttccgggcgcggtgggctatgatgaagaaaacctgtatgtggatggcaaagcgattcgcgcgaccgcgattaaagatccgcgcgaaattccgtggaaacaggcgggcgtgggcgtggtggtggaaagcaccggcgtgtttaccgatggcgaaaaagcgcgcgcgcatctggaagcgggcgcgaaaaaagtgattattaccgcgccggcgaaaaacgaagatattaccgtggtgctgggcgtgaaccatgaacagtatgatccggcgaaacatcatattctgagcaacgcgagctgcaccaccaacagcctggcgccggtgatgaaagtgctggaaaaagcgtttggcgtggaaaaagcgctgatgaccaccgtgcatagctataccaacgatcagcgcctgctggatctgccgcataaagatctgcgccgcgcgcgcgcggcggcgctgaacattattccgaccaccaccggcgcggcgaaagcgaccgcgctggtgctgccgagcctgaaaggccgctttgatggcatggcgctgcgcgtgccgaccccgaccggcagcattagcgatattaccgcgctgctgaaacgcgaagtgaccgcggaagaagtgaacgcggcgctgaaagcggcggcggaaggcccgctgaaaggcattctggcgtataccgaagatgaaattgtgctgcgcgatattgtgatggatccgcatagcagcattgtggatggcaaactgaccaaagcgattggcaacctggtgaaagtgtttgcgtggtatgataacgaatggggctatgcgaaccgcgtggcggatctggtggaactggtgctgaaaaaaggcgtgtaa

(2) Construction of recombinant plasmids containing mutants

First, pTrc-99a-gapA_Tt was used as a template and amplified with primers (Table 2) to obtain recombinant vectors containing mutants T98V, L185Y, P232A, Y272F, P232A/T98V, P232A/L185Y, P232A/Y272F ：pTrc-99a-gapA_Tt^T98VpTrc-99a-gapA_Tt^L185Y、pTrc-99a-gapA_Tt^P232A、pTrc-99a-gapA_Tt^Y272F,pTrc-99a-gapA_Tt^P232A/T98V,pTrc-99a-gapA_Tt^P232A/L185Y,pTrc-99a-gapA_Tt^P232A/Y272F.

(3) Construction of recombinant Strain containing gapA_Tt

Glyceraldehyde-3-phosphate dehydrogenase encoding genes gapA_Tt^T98V mutant, gapA_Tt^L185Y mutant, gapA_Tt^P232A mutant, gapA_Tt^Y272F mutant, gapA_Tt^P232A/T98V mutant, gapA_Tt^P232A/L185Y mutant and gapA_Tt^P232A/Y272F mutant from Thermus thermophilus (Thermus thermophilus) were integrated into the yeeL locus of the T14 strain (NCBI accession number: 2847764), respectively.

The E.coli W3110 genome was used as a template, and the upstream homology arm primers (yeeL-1, yeeL-2) and the downstream homology arm primers (yeeL-5) were designed based on yeeL gene sequences, yeeL-6), then, designing primers yeeL-3 and yeeL-4 by taking the constructed pTrc-99a-gapA_Tt^T98VpTrc-99a-gapA_Tt^L185Y、pTrc-99a-gapA_Tt^P232A、pTrc-99a-gapA_Tt^Y272F,pTrc-99a-gapA_Tt^P232A/T98V,pTrc-99a-gapA_Tt^P232A/L185Y,pTrc-99a-gapA_Tt^P232A/Y272F plasmid as a template, wherein the P_trc promoter sequence is designed on the primers yeeL-2 and yeeL-3, obtaining each fragment through PCR amplification, and then carrying out fusion PCR by taking the fragment as the template to respectively obtain the P_trc-gapA_Tt^T98V gene, P_trc-gapA_Tt^L185Y gene, P_trc-gapA_Tt^P232A gene, P_trc-gapA_Tt^Y272F gene, P_trc-gapA_Tt^P232A/T98V gene, P_trc-gapA_Tt^P232A/L185Y gene, an integrated fragment of the P_trc-gapA_Tt^P232A/Y272F gene. the DNA fragments obtained after annealing the primers gRNA-yeeL-1 and gRNA-yeeL-2 were ligated with plasmid pGRB to construct plasmid pGRB-yeeL. The plasmid pGRB-yeeL was isolated from the gene P_trc-gapA_Tt^T98V, the gene P_trc-gapA_Tt^L185Y, the gene P_trc-gapA_Tt^P232A, the gene P_trc-gapA_Tt^Y272F, The integrated fragments of the P_trc-gapA_Tt^P232A/T98V gene, the P_trc-gapA_Tt^P232A/L185Y gene and the P_trc-gapA_Tt^P232A/Y272F gene are simultaneously and electrically transformed into electrically transformed competent cells containing pREDCas E.coli T14 to obtain positive transformants, and after plasmid elimination, E.coli T18(E.coli T14,ΔyeeL::P_trc-gapA_Tt^T98V)、E.coli T19(E.coli T14,ΔyeeL::P_trc-gapA_Tt^L185Y)、E.coli T20(E.coli T14,ΔyeeL::P_trc-gapA_Tt^P232A) strains, namely, E.coli T21 (E.coli T14,. DELTA. yeeL: P_trc-gapA_Tt^Y272F) strain, E.coli T22 (E.coli T14,. DELTA. yeeL: P_trc-gapA_Tt^P232A/T98V) strain, E.coli T23 (E.coli T14,. DELTA. yeeL: P_trc-gapA_Tt^P232A/L185Y) strain, E.coli T24 (E.coli T14,. DELTA. yeeL: P_trc-gapA_Tt^P232A/Y272F) strain.

(4) Detection of the ratio of NADPH/NADP+

Shake flask fermentation of the recombinant strain, determination of L-threonine content and calculation of the sugar acid conversion rate of the strain were performed as described above (example 1), and the ratio of NADPH/NADP+ in the recombinant strain after the end of shake flask fermentation was detected. According to the above method, a recombinant strain T14 WT (E.coli T14, delta yeeL:: P_trc-gapA_Tt) was prepared in which the recombinant strain expressed wild-type glyceraldehyde-3-phosphate dehydrogenase. After fermentation according to the above method, the yield of L-threonine and the sugar acid conversion rate of the strain were examined. And the ratio of NADPH/NADP+ in the recombinant strain after the completion of shake flask fermentation was examined, and the results are shown in Table 6.

TABLE 6 Effect of strains containing different mutants

Strains containing different mutants	Yield (g/L)	Sugar acid conversion (%)	NADPH/NADP+
				T14	42.3	46.6	0.73
T14WT	43.2	47.8	1
				T18(T14T98V)	42.8	47	0.81
T19(T14L185Y)	43	47.5	0.92
				T20(T14P232A)	44.9	48.9	1.13
T21(T14Y272F)	43.8	48.3	1.08
				T22(T14P232A/T98V)	40.2	43.2	0.64
T23(T14P232A/L185Y)	41.9	44.6	0.69
				T24(T14P232A/Y272F)	45.8	49.7	1.48

The results showed that T24 strain (E.coli LMT4,ΔmbhA::P_trc-rbcL_Rr^R205KΔylbE::P_trc-prk_Cs^T108YΔarpB::P_trc-gapA_Tm^R268KΔyeeL::P_trc-gapA_Tt^P232A/Y272F) had higher L-threonine production and conversion, so that the selection of T24 strain continued to perform the 5L fermenter fed-batch fermentation.

Example 6 feed fermentation of strains T14, T24 5L fermentors

Preparation of L-threonine Using E.coli T14 and E.coli T24, respectively

(1) And (3) seed culture, namely pouring a proper amount of sterile water into the inclined plane, suspending the thalli by using an inoculating loop, and then inoculating the bacterial suspension into a seed culture medium for culture. The culture temperature is 37 ℃, the initial ventilation is 2L/min, the initial stirring rotation speed is 200r/min, the pH of the culture medium is controlled to be 7.0-7.2 by automatically feeding 25% ammonia water, the dissolved oxygen is controlled to be 25-30% by stirring and ventilation, and when the OD₆₀₀ reaches 15-20, the culture medium is ready to be accessed.

The seed culture medium comprises 30g/L glucose, 3g/L yeast extract, 30mL/L corn steep liquor, 2g/L citric acid ,0.5g/L MgSO₄·7H₂O,10mg/L FeSO₄·7H₂O,10mg/L MnSO₄·H₂O,0.2mg/L vitamin H,0.3mg/L vitamin B₁, and water for the rest, and has pH of 7.0-7.2.

(2) Fermenting culture, inoculating the seed solution into 2L fermentation culture medium according to 15% (v/v) inoculum size, wherein the culture temperature is 37 ℃, controlling pH of the culture medium to 7.0-7.2 by automatically feeding 25% ammonia water, controlling dissolved oxygen to 25-30% by stirring and ventilation, automatically feeding 80% glucose solution when glucose in the culture medium is exhausted, and controlling glucose residual sugar concentration in the fermentation liquid to 1-10g/L.

The fermentation medium comprises 20g/L glucose, 2g/L yeast extract, 15mL/L corn steep liquor, 2g/L citric acid ,1g/L MgSO₄·7H₂O,10mg/L FeSO₄·7H₂O,5mg/L MnSO₄·H₂O,2g/L potassium dihydrogen phosphate, 0.2mg/L vitamin H,0.3mg/L vitamin B₁, 15g/L ammonium sulfate, and the balance of water, wherein the pH value is 7.0-7.2.

The E.coli T24 is fermented for 58 hours in a 5L fermentation tank, and the yield is improved by 15.2% compared with the E.coli T14 (the yield is 129.6 g/L) and reaches 149.3g/L.

Example 7 optimization of fermentation conditions in Strain T24 5L fermentors

(1) Seed culture, pouring proper amount of sterile water into the inclined plane, and suspending the thallus with inoculating loop. Then, the bacterial suspension is inoculated into a seed culture medium for culture. The culture temperature is 37 ℃, the initial ventilation is 2L/min, the initial stirring rotation speed is 200r/min, the pH of the culture medium is controlled to be 7.0-7.2 by automatically feeding 25% ammonia water, the dissolved oxygen is controlled to be 25-30% by stirring and ventilation, and when the OD₆₀₀ reaches 15-20, the culture medium is ready to be accessed.

The seed culture medium comprises 35g/L glucose, 5g/L yeast powder, 3g/L peptone, 1.5g/L KH₂PO₄,0.5g/LMgSO₄·7H₂ O,1g/L citric acid, 1g/L L-threonine, 10mg/L FeSO₄·7H₂O,10mg/L MnSO₄·H₂O,1mg/L V_H and 0.5mg/L V_B1, and the balance of water, wherein the pH is 7.0-7.2.

(2) Inoculating the seed solution into 2L fermentation culture medium according to 20% (v/v) inoculum size, culturing at 37 deg.C, controlling pH of the culture medium to 7.0-7.2 by automatically feeding 25% ammonia water, controlling dissolved oxygen to 25-30% by stirring and ventilation, automatically feeding 80% glucose solution and 1g/L betaine when glucose in the culture medium is exhausted, controlling glucose residual sugar concentration in the fermentation liquid to 1-10g/L, and feeding feed culture medium at flow rate of 14mL/h between 14-20h of fermentation.

The fermentation medium consists of 10g/L glucose, 12g/L corn steep liquor, 4g/L yeast powder, 3g/L peptone, 4g/L KH₂PO₄,1g/L MgSO₄·7H₂ O,2g/L citric acid, 1g/L L-threonine, 10mg/L FeSO₄·7H₂O,10mg/LMnSO₄·H₂O,0.2mg/L V_H and 0.3mg/L V_B1, and the balance being water, and the pH is 7.0-7.2. The fed-batch culture medium consists of 4g/L yeast powder, 3g/L peptone, 4g/L KH₂PO₄,1g/L MgSO₄·7H₂ O,2g/L citric acid, 10mg/L FeSO₄·7H₂O,10mg/L MnSO₄·H₂O,0.5mg/L V_H and 0.5mg/L V_B1.

E, fermenting the coll T24 in a 5L fermentation tank for 50h, wherein the fermentation process curve is shown in figure 1, the yield of L-threonine is up to 168g/L, the sugar acid conversion rate is up to 63%, and the production intensity is up to 3.36g/L/h.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. The recombinant nucleic acid comprises a ribulose bisphosphate carboxylase mutant encoding gene rbcL, a phosphoribulokinase mutant encoding gene prk and a glyceraldehyde-3-phosphate dehydrogenase mutant encoding gene gapA, wherein the sequence of the coding gene of the ribulose bisphosphate carboxylase mutant is shown as SEQ ID NO.9, the sequence of the coding gene of the phosphoribulokinase mutant is shown as SEQ ID NO.10, and the sequence of the coding gene of the glyceraldehyde-3-phosphate dehydrogenase mutant is shown as SEQ ID NO. 11-12.

2. A recombinant escherichia coli, characterized in that the recombinant escherichia coli overexpresses a mutant of rubisco (Rhodospirillum rubrum) -derived rubisco-derived ribulose bisphosphate carboxylase, a mutant of rubisco globosa (Cereibacter sphaeroides) -derived ribulose phosphate kinase, a mutant of thermomyces maritimus (Thermotoga maritima) -derived glyceraldehyde-3-phosphate dehydrogenase, and a mutant of thermomyces thermophilus (Thermus thermophilus) -derived glyceraldehyde-3-phosphate dehydrogenase;

The mutant of the rubisco is obtained by mutating threonine at the 81 st position of a parent enzyme of the rubisco with an amino acid sequence shown as SEQ ID NO.1 into methionine, mutating arginine at the 205 st position into lysine, mutating isoleucine at the 240 th position into methionine, mutating leucine at the 260 th position into phenylalanine, mutating alanine at the 269 th position into proline;

The mutant of the phosphoribulokinase is obtained by mutating threonine at position 108 of the phosphoribulokinase with an amino acid sequence shown as SEQ ID NO.3 into tyrosine, mutating serine at position 140 into aspartic acid, mutating threonine at position 176 into serine, mutating tryptophan at position 254 into phenylalanine, mutating valine at position 283 into leucine;

the mutant of the glyceraldehyde-3-phosphate dehydrogenase derived from the Thermotoga maritima is obtained by mutating the 181 th tyrosine of the glyceraldehyde-3-phosphate dehydrogenase with an amino acid sequence shown as SEQ ID NO.5 into phenylalanine, mutating the 268 th arginine into lysine, mutating the 181 th tyrosine into phenylalanine and mutating the 268 th arginine into lysine;

the mutant of the glyceraldehyde-3-phosphate dehydrogenase derived from the thermophilic thermus is obtained by mutating the 232 rd proline of the glyceraldehyde-3-phosphate dehydrogenase with the amino acid sequence shown as SEQ ID NO.7 into alanine, or mutating the 272 rd tyrosine into phenylalanine, or mutating the 232 rd proline into alanine and mutating the 272 rd tyrosine into phenylalanine.

3. The recombinant escherichia coli according to claim 2, wherein the mutant ribulose bisphosphate carboxylase, the mutant ribulokinase and the mutant glyceraldehyde-3-phosphate dehydrogenase are expressed by Trc promoters;

Preferably, the nucleotide sequence encoding the Trc promoter is set forth in SEQ ID No. 13;

Preferably, the method comprises the steps of knocking out mbhA genes on the genome of the escherichia coli and integrating the ribulose bisphosphate carboxylase mutant at mbhA, knocking out ylbE genes on the genome of the escherichia coli and integrating the ribulose phosphate kinase mutant at ylbE, knocking out arpB genes on the genome of the escherichia coli and integrating glyceraldehyde-3-phosphate dehydrogenase mutant from Thermotoga maritima at arpB, knocking out yeeL genes on the genome of the escherichia coli and integrating glyceraldehyde-3-phosphate dehydrogenase mutant from Thermus thermophilus at yeeL;

Preferably, the mbhA Gene ID at NCBI is 62676021, the ylbE Gene ID at NCBI is 4056025, the arpB Gene ID at NCBI is 948935, and the yeeL Gene ID at NCBI is 2847764.

4. A mutant of the rubisco is characterized in that the mutant is obtained by mutating threonine at the 81 st position of a rubisco parent enzyme with an amino acid sequence shown as SEQ ID NO.1 into methionine, mutating arginine at the 205 st position into lysine, mutating isoleucine at the 240 th position into methionine, mutating leucine at the 260 th position into phenylalanine, mutating alanine at the 269 th position into proline.

5. A mutant of phosphoribulokinase, characterized in that the mutant is obtained by mutating threonine at position 108 of phosphoribulokinase with an amino acid sequence shown as SEQ ID NO.3 into tyrosine, mutating serine at position 140 into aspartic acid, mutating threonine at position 176 into serine, mutating tryptophan at position 254 into phenylalanine, and mutating valine at position 283 into leucine.

6. A glyceraldehyde-3-phosphate dehydrogenase mutant is characterized in that the mutant is obtained by mutating the 181 th tyrosine of glyceraldehyde-3-phosphate dehydrogenase with an amino acid sequence shown in SEQ ID NO.5 from Thermotoga maritima into phenylalanine, mutating the 268 th arginine into lysine, mutating the 181 th tyrosine into phenylalanine and mutating the 268 th arginine into lysine;

or the mutant is obtained by mutating proline at position 232 of glyceraldehyde-3-phosphate dehydrogenase with an amino acid sequence shown in SEQ ID NO.7 from Thermus thermophilus into alanine, or mutating tyrosine at position 272 into phenylalanine, or mutating proline at position 232 into alanine and tyrosine at position 272 into phenylalanine.

7. A gene encoding the mutant according to any one of claims 4 to 6 or a recombinant vector carrying the gene or a recombinant strain expressing the mutant according to any one of claims 4 to 6.

8. A method for improving threonine production in escherichia coli, wherein the mutant of ribulose bisphosphate carboxylase according to claim 4, the mutant of phosphoribulokinase according to claim 5 and the mutant of glyceraldehyde-3-phosphate dehydrogenase according to claim 6 are overexpressed in escherichia coli;

Preferably, the mutant ribulose bisphosphate carboxylase, mutant phosphoribulokinase and mutant glyceraldehyde-3-phosphate dehydrogenase are all expressed by the promotion of Trc promoter;

Preferably, the nucleotide sequence encoding the Trc promoter is set forth in SEQ ID No. 13;

Preferably, the method comprises the steps of knocking out mbhA genes on the genome of the escherichia coli and integrating the mutant of the rubisco at mbhA, knocking out ylbE genes on the genome of the escherichia coli and integrating the mutant of the rubisco at ylbE, knocking out arpB genes on the genome of the escherichia coli and integrating the mutant of the glyceraldehyde-3-phosphate dehydrogenase of the thermomyces maritima at arpB, knocking out yeeL genes on the genome of the escherichia coli and integrating the mutant of the glyceraldehyde-3-phosphate dehydrogenase of the thermomyces thermophilus at yeeL;

Preferably, the mbhA Gene ID at NCBI is 62676021, the ylbE Gene ID at NCBI is 4056025, the arpB Gene ID at NCBI is 948935, and the yeeL Gene ID at NCBI is 2847764.

9. A method for preparing threonine, which is characterized in that the method is prepared by adopting the recombinant escherichia coli according to claim 2 or 3 for fermentation;

Preferably, the temperature condition of the fermentation is that the fermentation is performed at 30-40 ℃, or at 35-37 ℃, or at 30-31 ℃, or at 32-33 ℃, or at 34-35 ℃, or at 36-37 ℃, or at 38-39 ℃;

Preferably, the fermentation is carried out under the pH condition of 6.8-7.5, or under the pH condition of 6.8-7.0, or under the pH condition of 7.2-7.3, or under the pH condition of 7.0-7.2;

preferably, the fermentation time is at least 24 hours.

10. Use of the recombinant escherichia coli of claim 2 or 3 for the preparation of L-threonine or for increasing the yield of L-threonine and/or the conversion of sugar acids.