Recombinant klebsiella michiganensis for producing 1,3-propanediol and its construction method and application

Abstract

The invention provides a genetically engineered recombinant Klebsiella michiganensis having reduced or inactivated glucose-specific PTS transport system, glycerol kinase, glycerol dehydrogenase, alcohol dehydrogenase, acetolactate synthase, acetolactate decarboxylase, fumarate reductase and 3-phosphoglyceraldehyde dehydrogenase, having, preferably enhanced, NADPH-dependent alcohol dehydrogenase, diol dehydratase, 3-phosphoglycerate dehydrogenase and 3-phosphoglycerate phosphatase activities, methods of making said recombinant Klebsiella michiganensis, and uses and methods of producing 1,3-propanediol using said recombinant Klebsiella michiganensis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a novel recombinant Klebsiella michiganensis for producing 1,3-propanediol and a construction method and application thereof. BACKGROUND

[0002] 1,3-propanediol is an important diol, which can be used to synthesize polyester polymer materials by polycondensation with dicarboxylic acid, and can also be used to produce plasticizers, surfactants, emulsifiers, demulsifiers, lubricating oils, dyes, inks, antifreezes, cosmetics, etc.

[0003] Propylene aldehyde method and ethylene oxide method are currently the main chemical synthesis methods for 1,3-propanediol. These two technologies are mature and reliable, but the production cost is high, and waste that pollutes the environment is generated, which limits large-scale application.

[0004] The biosynthesis method is more environmentally friendly than the chemical synthesis method, and there are many natural high-yield 1,3-propanediol strains in nature. For example, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, etc. These strains synthesize 1,3-propanediol by metabolizing glycerol under anaerobic or microaerobic conditions and simultaneously obtain substances and energy required for growth. However, the conversion rate of glycerol to 1,3-propanediol is only 60-70%, and the price of glycerol is relatively high, resulting in high fermentation cost. In order to further reduce the production cost of 1,3-propanediol, fermentation synthesis of 1,3-propanediol from glucose has become a research target.

[0005] DuPont developed an Escherichia coli engineering strain (CN100471946C) for one-step fermentation synthesis of 1,3-propanediol from glucose, which greatly reduced the production cost of 1,3-propanediol, and the yield of 1,3-propanediol reached 135 g / L, and the mass conversion rate reached 51%. However, this technology has many defects. For example, E. coli lacks key proteins for 1,3-propanediol synthesis, and the use of plasmid expression of related proteins not only requires the addition of multiple antibiotics during cultivation, but also has expression defects caused by plasmid loss, thereby causing unstable fermentation effect; E. coli itself does not have the synthesis and repair ability of key coenzyme B12, so the addition amount of vitamin B12 is large, but the price of vitamin B12 is extremely high.

[0006] To reduce the dosage of vitamin B12 to reduce costs, Suman Lama et al. transformed Klebsiella pneumoniae J2B, which naturally produces 1,3-propanediol at a high yield, into an engineered strain capable of synthesizing 1,3-propanediol using glucose as a carbon source (https: / / doi.org / 10.1016 / j.ymben.2020.09.001). Since Klebsiella pneumoniae J2B has a complete vitamin B12 synthesis and repair system, only 5.4 mg / L of vitamin B12 needs to be added during fermentation, which is one-tenth of the amount required by the DuPont technology. However, this technology has some drawbacks. In terms of fermentation indicators, the mass conversion rate reached 53.6%, but the 1,3-propanediol yield was only 62 g / L, which is much lower than the technical indicators of DuPont. The expression of key module proteins in the processes of glycerol synthesis, dehydration, and 3-hydroxypropanal reduction is still carried out using plasmids as vectors, and there are still problems such as antibiotic addition and plasmid loss.

[0007] Currently available 1,3-propanediol fermentation synthesis technologies have not achieved comprehensive improvements in the dosage of vitamin B12, mass conversion rate, and 1,3-propanediol yield, and all require the use of plasmids to express key proteins, thus requiring the addition of antibiotics during fermentation and having problems such as unstable fermentation. Therefore, the biosynthesis technology of 1,3-propanediol still needs further improvement. SUMMARY

[0008] To optimize the biosynthesis technology of 1,3-propanediol, the present inventors first isolated and screened a Klebsiella michiganensis strain from soil, which has a vitamin B12 synthesis and repair system. During metabolic engineering modification, the strain was modified using genome integration regulation throughout, without using plasmids, avoiding the use of antibiotics and greatly improving the stability of fermentation effect. Under the conditions of using glucose as raw material and without adding vitamin B12, the 1,3-propanediol yield of the modified strain YY-P023 (deposited at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on March 23, 2022, with the accession number CGMCC No. 24576) can reach 92 g / L, and the mass conversion rate can reach 54%. The fermentation technology using the modified strain YY-P023 has achieved great comprehensive advantages in 1,3-propanediol yield, mass conversion rate, dosage of vitamin B12, and use of antibiotics, significantly reducing the comprehensive cost of 1,3-propanediol biosynthesis.

[0009] In one aspect, the present application provides a genetically engineered recombinant Klebsiella michiganensis having reduced or inactivated activities of glucose-specific PTS transport system, glycerol kinase, glycerol dehydrogenase, alcohol dehydrogenase, acetolactate synthase, acetolactate decarboxylase, 3-phosphoglyceraldehyde dehydrogenase and fumarate reductase, having, preferably enhanced, activities of NADPH-dependent alcohol dehydrogenase, diol dehydratase, 3-phosphoglycerate dehydrogenase and 3-phosphoglycerate phosphatase. In particular, the recombinant Klebsiella michiganensis can produce 1,3-propanediol without the need of adding extra vitamin B12 and / or antibiotics.

[0010] In one embodiment, the genetically engineered recombinant Klebsiella michiganensis has attenuated or inactivated ptsG, glpK, gldAl, gldA2, adhE, GapA, budB, budA genes and frdABCD gene cluster, has expressed, preferably overexpressed, exogenous yqhd gene and has expressed, preferably overexpressed, pdu gene cluster, GPD1 and GPP2.

[0011] In one embodiment, the genetically engineered recombinant Klebsiella michiganensis is the strain having the accession number of CGMCC No. 24576 deposited with CGMCC.

[0012] In one aspect, the present application provides a method for preparing a Klebsiella michiganensis producing 1,3-propanediol, in particular without the need of adding extra vitamin B12 and / or antibiotics, comprising: attenuating or inactivating activities of glucose-specific PTS transport system, glycerol kinase, glycerol dehydrogenase, alcohol dehydrogenase, acetolactate synthase, acetolactate decarboxylase, 3-phosphoglyceraldehyde dehydrogenase and fumarate reductase in an initial Klebsiella michiganensis strain, and conferring or enhancing activities of NADPH-dependent alcohol dehydrogenase, diol dehydratase, 3-phosphoglycerate dehydrogenase and 3-phosphoglycerate phosphatase, to obtain a genetically engineered recombinant Klebsiella michiganensis.

[0013] In one embodiment, the method comprises: attenuating or inactivating ptsG, glpK, gldAl, gldA2, adhE, budB, budA, GapA genes and frdABCD gene cluster and expressing, preferably overexpressing, yqhd, pdu gene cluster, GPD1 and GPP2 genes in an initial Klebsiella michiganensis strain to obtain a genetically engineered recombinant Klebsiella michiganensis.

[0014] In one embodiment, the genetically engineered recombinant Klebsiella michiganensis is the strain having the accession number of CGMCC No. 24576 deposited with CGMCC.

[0015] In one aspect, the present application provides the use of the genetically engineered recombinant K. michiganensis according to the present application or obtained by the method of producing a genetically engineered K. michiganensis for producing 1,3-propanediol, in particular without the need of adding additional vitamin B12 and / or antibiotics.

[0016] In one aspect, the present application provides a method of producing 1,3-propanediol, comprising cultivating a genetically engineered recombinant K. michiganensis according to the present application or obtained by the method of producing a genetically engineered K. michiganensis for producing 1,3-propanediol. Preferably, the method of producing 1,3-propanediol does not require the addition of additional vitamin B12 and / or antibiotics. DETAILED DESCRIPTION

[0017] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

[0018] In one aspect, the present application provides a genetically engineered recombinant K. michiganensis having reduced or inactivated activity of a glucose-specific PTS transport system, e.g. components IIBC, a glycerol kinase, a glycerol dehydrogenase, an alcohol dehydrogenase, an acetolactate synthase, an acetolactate decarboxylase, a 3-phosphoglyceraldehyde dehydrogenase, and a fumarate reductase, having, preferably enhanced, activity of an NADPH-dependent alcohol dehydrogenase, a diol dehydratase, a 3-phosphoglycerol dehydrogenase, and a 3-phosphoglycerol phosphatase.

[0019] As used herein, "genetically engineered" refers to a strain that has been artificially altered by biological means, which has one or more alterations, e.g. gene deletions, amplifications, or mutations, compared to the original strain prior to the engineering, thereby having altered biological properties, e.g. improved production performance. As used herein, the original strain can be a natural strain to which the genetic engineering is to be performed or a strain having other genetic engineering.

[0020] As used herein, "reduced or inactive" means at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more, or even 100% reduction in activity compared to a reference activity (e.g., the corresponding activity in an initial or wild-type strain).

[0021] The activity of a protein (e.g., enzyme) can be reduced or inactivated by any appropriate means known in the art, for example including but not limited to use of a weak promoter, a weakened or inactivated corresponding gene encoding the protein, introduction of a mutation that results in a reduction or inactivation of the activity of the protein, use of an antagonist or inhibitor of the protein (e.g., an antibody, a ligand, etc.).

[0022] As used herein, "has an activity" means has a detectable activity compared to a reference that does not have the activity (e.g., an initial or wild-type strain).

[0023] As used herein, "has an enhanced activity" means at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or more increase in activity compared to a reference that has the activity (e.g., an initial or wild-type strain).

[0024] The activity of a protein (e.g., enzyme) can be produced or enhanced by any appropriate means known in the art, for example including but not limited to expression or overexpression (e.g., by a vector such as a plasmid) of a corresponding gene encoding the protein in the strain, introduction of a mutation that results in an increase in the activity of the protein, etc.

[0025] In some embodiments, in the genetically engineered 1,3-propanediol-producing strains described herein, one or more copies of a gene of interest or a homolog thereof can be integrated into the genome (e.g., by homologous recombination), optionally at any site in the genome (as long as such integration does not significantly negatively affect the growth and production of the strain), for example, one copy of any gene in the genome is replaced by one or more copies of a gene of interest or a homolog thereof. Those skilled in the art know how to integrate a transgene and select for strains that have integrated the transgene.

[0026] As used herein, a glucose-specific PTS transport system mediates the transport of glucose across the plasma membrane and combines translocation with substrate phosphorylation (https: / / doi.org / 10.1016 / S0021-9258(19)50244-7). It consists of two subunits, IIBC GLc and IIA Glc . IIBCGlc (51 kDa) consists of two domains, a 36 kDa N-terminal domain (IIC Glc ) predicted to span the membrane eight times and a 12 kDa C-terminal cytoplasmic domain (IIB Glc ) (Buhr A and Erni B, J. Biol. Chem. 268, 11599-1160 (1993)); IIA Glc is an 18.1-kDa cytoplasmic protein (Meadow et al., Annu. Rev. Biochem. 59, 497-542, (1990)). In the present context, a glucose-specific PTS transport system with reduced activity or an inactivated glucose-specific PTS transport system means a glucose-specific PTS transport system with reduced or lost PTS transport function.

[0027] As used herein, glucose-specific PTS transport system component IIBC is encoded by the gene ptsG and functions to constitute glucose-specific PTS transport system key component IIBC.

[0028] In one embodiment, the ptsG gene comprises the sequence set forth in SEQ ID NO: 102 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence with glucose-specific PTS transport system component IIBC activity.

[0029] As used herein, glycerol kinase (EC 2.7.1.30) is encoded by the glpK gene and catalyzes the conversion of glycerol to 3-phosphoglycerol and coenzyme ADP with ATP as coenzyme. In the present context, a glycerol kinase with reduced activity or an inactivated glycerol kinase means a glycerol kinase with reduced or lost activity to catalyze the conversion of glycerol to 3-phosphoglycerol and coenzyme ADP.

[0030] In one embodiment, the glpK gene comprises the sequence set forth in SEQ ID NO: 103 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence with glycerol kinase activity.

[0031] As used herein, glycerol dehydrogenase (EC 1.1.1.6) is encoded by the gldA gene and catalyzes the conversion of glycerol to 1,3-propanediol with NAD +Glycerol dehydrogenase (EC 1.1.1.6) is encoded by gldA1 and gldA2 genes. Glycerol dehydrogenase catalyzes the conversion of glycerol to dihydroxyacetone and coenzyme NADH. In this context, a reduced activity or inactivated glycerol dehydrogenase refers to a reduction or loss of the activity of the enzyme to catalyze the conversion of glycerol to dihydroxyacetone and coenzyme NADH. Both copies of glycerol dehydrogenase, gldA1 and gldA2, have 75% amino acid sequence similarity and both copies can achieve the catalytic activity of glycerol dehydrogenase.

[0032] In one embodiment, the gldA1 gene comprises the sequence set forth in SEQ ID NO: 104 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence with glycerol dehydrogenase activity.

[0033] In one embodiment, the gldA2 gene comprises the sequence set forth in SEQ ID NO: 105 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence with glycerol dehydrogenase activity.

[0034] As used herein, alcohol dehydrogenase (EC 1.2.1.10, EC 1.1.1.1) is encoded by the adhE gene, which is a multifunctional enzyme that can catalyze the synthesis of acetaldehyde, NAD + and coenzyme A from acetyl-coenzyme A and NADH; the synthesis of ethanol and NAD + from acetaldehyde and NADH. In this context, a reduced activity or inactivated alcohol dehydrogenase refers to a reduction or loss of one or more, in particular all, of the catalytic activities of the enzyme.

[0035] In one embodiment, the adhE gene comprises the sequence set forth in SEQ ID NO: 106 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence with alcohol dehydrogenase activity.

[0036] As used herein, acetolactate synthase (EC 2.2.1.6) is encoded by the budB gene, which catalyzes the conversion of 2 molecules of pyruvate to 1 molecule of acetolactate and the release of 1 molecule of CO2. In this context, a reduced activity or inactivated acetolactate synthase refers to a reduction or loss of the activity of the enzyme to catalyze the conversion of pyruvate to acetolactate.

[0037] In one embodiment, the budB gene comprises the sequence set forth in SEQ ID NO: 107 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence having acetolactate synthase activity.

[0038] As used herein, acetolactate decarboxylase (EC 4.1.1.5) is encoded by the budA gene, which catalyzes the decarboxylation of acetolactate to produce acetoin and carbon dioxide. Herein, a reduced activity or inactivated acetolactate decarboxylase refers to a reduction or loss of activity of the enzyme catalyzing the decarboxylation of acetolactate to produce acetoin and carbon dioxide.

[0039] In one embodiment, the budA gene comprises the sequence set forth in SEQ ID NO: 108 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence having acetolactate decarboxylase activity.

[0040] As used herein, NADPH-dependent alcohol dehydrogenase (EC 1.1.1.2) is encoded by the yqhd gene, which catalyzes the formation of 1,3-propanediol and NADP+ from 3- hydroxypropanal and coenzyme NADPH. + Herein, having or having enhanced NADPH-dependent alcohol dehydrogenase activity refers to a strain having or having increased activity of catalyzing the formation of 1,3-propanediol and NADP+ from 3-hydroxypropanal and coenzyme NADPH. +

[0041] In one embodiment, the yqhd gene is from E. coli, for example the yqhd gene comprises the sequence set forth in SEQ ID NO: 100 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence having NADPH-dependent alcohol dehydrogenase activity.

[0042] As used herein, diol dehydratase (EC 4.2.1.28) is encoded by the pdu gene cluster, which can catalyze the dehydration of glycerol to produce 3-hydroxypropanal. Herein, having or having enhanced diol dehydratase activity refers to a strain having or having increased activity of catalyzing the dehydration of glycerol to produce 3-hydroxypropanal.

[0043] ​In one embodiment, the pdu gene cluster comprises the sequence shown in SEQ ID NO: 109 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having diol dehydratase activity.

[0044] As used in this article, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is encoded by the GPD1 gene. This enzyme catalyzes the conversion of dihydroxyacetone phosphate and coenzyme NADH to glycerol-3-phosphate and coenzyme NAD. + In this article, having or having enhanced glycerol-3-phosphate dehydrogenase activity refers to the strain having or having increased catalytic activity in the production of glycerol-3-phosphate and NAD from dihydroxyacetone phosphate and coenzyme NADH. + The activity.

[0045] In one embodiment, the GPD1 gene comprises the sequence shown in SEQ ID NO: 110 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having glycerol-3-phosphate dehydrogenase activity.

[0046] As used herein, glycerol-3-phosphate phosphatase (EC 3.1.3.21) is encoded by the GPP2 gene, which catalyzes the hydrolysis of glycerol-3-phosphate to glycerol and phosphate ions. In this text, having or having enhanced glycerol-3-phosphate phosphatase activity means that the strain has or has increased activity in catalyzing the hydrolysis of glycerol-3-phosphate to glycerol and phosphate ions.

[0047] In one embodiment, the GPP2 gene comprises the sequence shown in SEQ ID NO: 111 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having glycerol-3-phosphate phosphatase activity.

[0048] As used in this article, glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) is encoded by the GapA gene, which catalyzes the reaction of glyceraldehyde-3-phosphate, phosphate ions, and the coenzyme NAD. + It is converted into 1,3-diphosphoglycerate and the coenzyme NADH. In this article, having or having enhanced 3-phosphoglyceraldehyde dehydrogenase activity refers to the strain having or having increased catalytic activity for 3-phosphoglyceraldehyde, phosphate ions, and the coenzyme NAD. +activity of converting 1,3-bisphosphoglycerate and coenzyme NADH.

[0049] In one embodiment, the GapA gene comprises the sequence set forth in SEQ ID NO: 112 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence having 3-phosphoglycerate dehydrogenase activity.

[0050] As used herein, fumarate reductase (EC 1.3.5.1) is encoded by the frdABCD gene cluster, which catalyzes the conversion of fumarate and reduced coenzyme Q to succinate and oxidized coenzyme Q. In the present context, a reduced activity or inactivated fumarate reductase refers to a reduced or lost activity of the enzyme catalyzing the conversion of fumarate and reduced coenzyme Q to succinate and oxidized coenzyme Q.

[0051] In one embodiment, the frdABCD gene cluster comprises the sequence set forth in SEQ ID NO: 113 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence having fumarate reductase activity.

[0052] In one embodiment, the recombinant K. michiganensis further has a reduced activity or inactivated phosphogluconate dehydratase and / or an acetolactate synthase, and / or has, preferably enhanced, levansucrase activity and / or has chloramphenicol acetyltransferase activity.

[0053] As used herein, phosphogluconate dehydratase (EC 4.2.1.12) is encoded by the edd gene, which catalyzes the dehydration of 6-phosphogluconate to 2-keto-3-deoxy-6- phosphogluconate. In the present context, a reduced activity or inactivated phosphogluconate dehydratase refers to a reduced or lost activity of the enzyme catalyzing the dehydration of 6- phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate.

[0054] In one embodiment, the edd gene comprises the sequence set forth in SEQ ID NO: 114 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence having phosphogluconate dehydratase activity.

[0055] As used herein, a methylglyoxal synthase (EC 4.2.3.3) is encoded by the mgsA gene, which catalyzes the conversion of dihydroxyphosphonate to methylglyoxal and a phosphate ion. As used herein, a methylglyoxal synthase with reduced activity or inactivated refers to a reduction or loss of the enzyme's activity to catalyze the conversion of dihydroxyphosphonate to methylglyoxal and a phosphate ion.

[0056] In one embodiment, the mgsA gene comprises the sequence set forth in SEQ ID NO: 115 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence having methylglyoxal synthase activity.

[0057] As used herein, a levansucrose fructosyltransferase (EC 2.4.1.10) is encoded by the sacB gene, which catalyzes the synthesis of levansucrose from sucrose. As used herein, having or having enhanced levansucrose fructosyltransferase activity refers to a strain having or having increased activity to catalyze the synthesis of levansucrose from sucrose.

[0058] In one embodiment, the sacB gene comprises the sequence set forth in SEQ ID NO: 116 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence having levansucrose fructosyltransferase activity.

[0059] In one embodiment, the recombinant K. michiganensis further has a chloramphenicol resistance gene cat.

[0060] As used herein, a chloramphenicol resistance gene cat encodes a chloramphenicol acetyltransferase (EC 2.3.1.28), which catalyzes the formation of chloramphenicol-3-acetate from chloramphenicol and acetyl-CoA.

[0061] In one embodiment, the cat gene comprises the sequence set forth in SEQ ID NO: 117 or a degenerate sequence thereof, or a nucleotide sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and encoding an amino acid sequence conferring chloramphenicol resistance.

[0062] In one embodiment, the genetically engineered recombinant K. michiganensis has a reduced or inactivated glucose-specific PTS transport system, e.g. components IIBC, glycerol kinase, glycerol dehydrogenase, alcohol dehydrogenase, acetolactate synthase, acetolactate decarboxylase, 3-phosphoglyceraldehyde dehydrogenase, and fumarate reductase, has NADPH-dependent alcohol dehydrogenase activity, and has enhanced diol dehydratase, 3-phosphoglycerate dehydrogenase, and 3-phosphoglycerate phosphatase activities.

[0063] In one embodiment, the genetically engineered recombinant K. michiganensis has an inactivated glucose-specific PTS transport system, e.g. components IIBC, glycerol kinase, glycerol dehydrogenase, alcohol dehydrogenase, acetolactate synthase, acetolactate decarboxylase, phosphogluconate dehydratase, acetone aldehyde synthase, and fumarate reductase, has a reduced 3-phosphoglyceraldehyde dehydrogenase activity, has a levansucrose transferase, chloramphenicol acetyltransferase, NADPH-dependent alcohol dehydrogenase activity, and has enhanced diol dehydratase, 3-phosphoglycerate dehydrogenase, and 3-phosphoglycerate phosphatase.

[0064] In one embodiment, the genetically engineered recombinant K. michiganensis has attenuated or inactivated ptsG, glpK, gldAl, gldA2, adhE, budB, budA, GapA genes and frdABCD gene cluster, has an expressed, preferably overexpressed, exogenous yqhd gene, and an expressed, preferably overexpressed, pdu gene cluster, GPD1 and GPP2 genes. In a further embodiment, the genetically engineered recombinant K. michiganensis also has attenuated or inactivated edd and / or mgsA genes, and / or has a sacB gene, and / or has a cat gene.

[0065] As used herein, "attenuated or inactivated gene" means that the activity, e.g. expression level (as a protein-encoding gene) or regulatory performance (as a regulatory element), of the gene is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even is not detectable, compared to a reference (e.g. the corresponding gene in the initial or wild-type strain). In case the gene encodes a protein, e.g. an enzyme, "attenuated or inactivated gene" also encompasses that the activity level of the protein expressed by the gene is reduced, e.g. by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even 100%, compared to the activity level of the corresponding protein in the initial or wild-type strain.

[0066] In the present context, the reference can be a wild type microorganism or a microorganism prior to the desired genetic manipulation (e.g. the initial microorganism used for performing the genetic manipulation to increase the activity of a gene). In the present context, the terms parent microorganism and initial microorganism are used interchangeably to refer to the microorganism on which the desired genetic manipulation (e.g. to enhance or attenuate the activity of a gene or protein) is performed.

[0067] As used herein, the terms "polypeptide", "amino acid sequence", "peptide" and "protein" are used interchangeably herein to refer to a chain of amino acids of any length, which can include modified amino acids and / or can be interrupted by non-amino acids. The term also encompasses an amino acid chain that has been modified by natural processes, such as post- translational modifications, or by human intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.

[0068] As used herein, the expressions "gene", "nucleic acid sequence", "polynucleotide" and "nucleotide sequence" are used interchangeably to refer to a chain of nucleotides, including DNA and RNA. "Expression of a gene" refers to the transcription of a DNA region operably linked to appropriate regulatory regions, in particular a promoter, into a biologically active RNA and the ability of the RNA to be translated into a biologically active protein or peptide.

[0069] As used herein, a degenerate sequence refers to a nucleotide sequence that, as a result of the degeneracy of the genetic code, encodes the same amino acid sequence as a specified sequence but differs in nucleotide sequence.

[0070] As used herein, the terms "homology", "sequence identity" and the like are used interchangeably herein. Sequence identity can be detected by the number of identical nucleotide bases between a polynucleotide and a reference polynucleotide, for example, as determined by a standard alignment algorithm program using the default gap penalties set by each vendor. Whether two nucleic acid molecules have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical" nucleotide sequences can be determined using known computer algorithms, such as BLASTN, FASTA, DNAStar, and Gap (University of Wisconsin Genetics Computer Group (UWG), Madison WI, USA). For example, the percent identity of two nucleic acid molecules can be determined by comparing sequence information, for example, using the GAP computer program (e.g., Needleman et al. J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981)). Briefly, the GAP program defines similarity as the number of identical aligned symbols (i.e., nucleotides) divided by the total number of symbols in the shorter of the two sequences.

[0071] In one embodiment, the genetically engineered recombinant K. michiganensis has deleted ptsG, glpK, gldAl, gldA2, adhE, budB, budA and frdABCD gene clusters, has attenuated GapA gene, has expressed, preferably overexpressed, yqhd gene and expressed, preferably overexpressed, pdu gene cluster, GPD1 and GPP2 genes, optionally further has deleted edd and / or mgsA genes, and / or has sacB gene, and / or has cat gene.

[0072] In one embodiment, the yqhd, pdu, GPD1 and / or GPP2 genes are under control of strong promoters (e.g. Yqhd-2 (SEQ ID NO: 94), Pdu-31 (SEQ ID NO: 95), GP-28 (SEQ ID NO: 96)) and / or the GapA gene is under control of a weak promoter (e.g. GapA-42 (SEQ ID NO: 97) promoter).

[0073] In one embodiment, the yqhd gene is under control of the promoter Yqhd-2 (SEQ ID NO: 94).

[0074] In one embodiment, the yqhd gene is located at the edd gene site and / or the GPD1 and GPP2 are located at the mgsA gene site. In one embodiment, the yqhd gene is exogenous, e.g. from E. coli.

[0075] In one embodiment, the pdu gene cluster is under control of the promoter Pdu-31 (SEQ ID NO: 95).

[0076] In one embodiment, the GPD1 and / or GPP2 genes are under control of the promoter GP-28 (SEQ ID NO: 96). In one embodiment, the genetically engineered recombinant K. michiganensis expresses, preferably overexpresses, GPD1 and GPP2 fusion protein encoding genes (e.g. as shown in SEQ ID NO: 98), e.g. under control of the promoter GP-28 (SEQ ID NO: 96).

[0077] In one embodiment, the GapA gene is under control of the promoter GapA-42 (SEQ ID NO: 97).

[0078] In one embodiment, the genetically engineered recombinant K. michiganensis has a deleted ptsG, glpK, gldAl, gldA2, adhE, budB, budA, edd, mgsA genes and frdABCD gene cluster, attenuated GapA gene, has a sacB gene, has a cat gene, and an exogenous yqhd gene and overexpressed pdu gene cluster, GPD1 and GPP2 genes.

[0079] In one embodiment, the genetically engineered recombinant K. michiganensis is the strain deposited at CGMCC with the accession number CGMCC No. 24576.

[0080] In one aspect, the present application provides a method of preparing a genetically engineered K. michiganensis producing 1,3-propanediol, comprising: reducing or inactivating the activity of glucose-specific PTS transport system, e.g., components IIBC, glycerol kinase, glycerol dehydrogenase, alcohol dehydrogenase, acetolactate synthase, acetolactate decarboxylase, 3-phosphoglyceraldehyde dehydrogenase, and fumarate reductase in an initial K. michiganensis strain, conferring or enhancing the activity of NADPH-dependent alcohol dehydrogenase, diol dehydratase, 3-phosphoglycerate dehydrogenase, and 3-phosphoglycerate phosphatase.

[0081] In one embodiment, the method further comprises reducing or inactivating phosphogluconate dehydratase and / or methylglyoxal synthase, and / or conferring or enhancing levansucrose fructosyltransferase activity and / or chloramphenicol acetyltransferase activity.

[0082] As used herein, an initial K. michiganensis strain refers to a microorganism in which the genetic manipulations described in the present application (e.g., conferring, enhancing, or attenuating the activity of a gene or protein) are to be performed. In one embodiment, the initial K. michiganensis strain is a wild-type K. michiganensis (e.g., K. michiganensis strains Biosolid 27 (CP054159.1), KM41 (CP090078.1), AR375 (CP029141.1), and CZ598 (CP073305.1)) or a K. michiganensis isolated from a natural environment (e.g., the PDO-046 strain described in the present application).

[0083] As used herein, reducing or inactivating the activity of a protein, such as an enzyme, means making the activity of the protein at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more, or even not detectable. A variety of means for reducing or inactivating are known in the art, including, for example, inhibiting gene expression such as knockdown (e.g., using small interfering RNA), using a weak promoter (when the gene is a polypeptide-encoding gene), etc.; gene knockout, deleting part or all of the gene or polypeptide sequence; mutating certain sites in the gene or polypeptide, such as the coding sequence or active domain, to reduce gene expression or the activity of the expression product or regulatory activity; and using antagonists or inhibitors (e.g., including but not limited to antibodies, interfering RNAs, etc.).

[0084] As used herein, "conferring...an activity" means producing an activity in a genetically engineered 1,3-propanediol-producing strain that does not exist in the original strain prior to the genetic engineering.

[0085] As used herein, "enhancing...an activity" means increasing the activity, such as by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or more.

[0086] A variety of methods for conferring or enhancing the activity of a desired protein are known in the art, including, for example, but not limited to, expressing or overexpressing a protein-encoding gene and mutations or other modifications that increase the activity of the protein.

[0087] As used herein, "overexpressing" means that the expression level of a gene is elevated, such as by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or more, relative to the level prior to the genetic manipulation. Methods of overexpressing a gene are well known in the art, including, for example, but not limited to, using a strong promoter, increasing the copy number of the gene, enhancers, etc. Increasing the copy number of a gene can be achieved, for example, but not limited to, by introducing one or more copies of an exogenous gene or an endogenous gene, such as by an expression vector or integrated into the genome.

[0088] As used herein, "exogenous gene" means a gene from another cell or organism, such as from the same species or a different species.

[0089] As used herein, "endogenous gene" means a gene of the cell or organism itself.

[0090] In one embodiment, the method comprises: attenuating or inactivating the ptsG, glpK, gldAl, gldA2, adhE, budB, budA, GapA genes and frdABCD gene cluster in an initial Klebsiella michiganensis strain, and expressing, preferably overexpressing the yqhd, pdu gene cluster, GPD1 and GPP2 genes to obtain a genetically engineered recombinant Klebsiella michiganensis.

[0091] As used herein, attenuating or inactivating a gene means reducing the expression level (when the gene is a protein-encoding gene) or regulatory activity (when the gene is a regulatory element) of the gene by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable. Various means of attenuating or inactivating a gene are known in the art, including, for example, inhibiting gene expression such as knockdown (e.g., using small interfering RNA), using a weak promoter (when the gene is a polypeptide-encoding gene), etc.; gene knockout, deleting part or all of the gene sequence; mutating certain sites in the gene, e.g., the coding sequence, to reduce gene expression or regulatory activity or activity of the expression product, etc. In one embodiment, the attenuation or inactivation is achieved by deleting or knocking out the gene encoding the corresponding protein.

[0092] In one embodiment, the method further comprises attenuating or inactivating the edd, mgsA genes and / or expressing, preferably overexpressing the sacB gene. In one embodiment, the method further comprises expressing the cat gene.

[0093] In one embodiment, the method comprises:

[0094] (1) placing the yqhd, pdu, GPD1 and / or GPP2 genes under the control of a promoter, preferably a strong promoter, such as Yqhd-2 (SEQ ID NO: 94), Pdu-31 (SEQ ID NO: 95), GP-28 (SEQ ID NO: 96).

[0095] (2) placing the GapA gene under the control of a weak promoter, such as GapA-42 promoter (SEQ ID NO: 97)

[0096] (3) placing the yqhd gene at the site of the edd gene, and / or

[0097] (4) placing the GPD1 and GPP2 genes (e.g., the GPD1 and GPP2 fusion protein-encoding gene shown in SEQ ID NO: 98) at the site of the mgsA gene.

[0098] In one embodiment, the method comprises placing the yqhd gene under the control of the promoter Yqhd-2 (SEQ ID NO: 94).

[0099] In one embodiment, the method comprises placing the pdu gene cluster under the control of the promoter Pdu-31 (SEQ ID NO: 95).

[0100] In one embodiment, the method comprises placing the GPD1 and / or GPP2 genes under the control of the promoter GP-28 (SEQ ID NO: 96). In one embodiment, the method comprises placing a GPD1 and GPP2 fusion protein encoding gene (e.g. as set forth in SEQ ID NO: 98) under the control of the promoter GP-28 (SEQ ID NO: 96).

[0101] In one embodiment, the method comprises placing the GapA gene under the control of the promoter GapA-42 (SEQ ID NO: 97).

[0102] In one embodiment, the genetically engineered recombinant K. michiganensis is the strain deposited at CGMCC with the accession number CGMCC No. 24576.

[0103] In one aspect, the present application provides the use of the genetically engineered recombinant K. michiganensis of the present application or obtained by the method of producing genetically engineered K. michiganensis for producing 1,3-propanediol, e.g. without the need of adding additional vitamin B12 and / or antibiotics.

[0104] In one aspect, the present application provides a method of producing 1,3-propanediol, comprising culturing the genetically engineered recombinant K. michiganensis of the present application or obtained by the method of producing genetically engineered K. michiganensis for producing 1,3-propanediol according to the present application under conditions suitable for fermentation to produce 1,3-propanediol, optionally comprising isolating and purifying the produced 1,3-propanediol. The method can produce 1,3-propanediol without the need of adding additional vitamin B12 and / or antibiotics.

[0105] Conditions for fermentation culture of K. michiganensis for fermentation to produce 1,3-propanediol are known in the art, including for example but not limited to pH, temperature, medium composition, fermentation time, etc.

[0106] In one embodiment, the method of producing 1,3-propanediol comprises performing a seed culture in a seed culture medium and then performing a fermentation in a fermentation medium.

[0107] In one embodiment, the seed culture medium comprises: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract. In one embodiment, the fermentation medium comprises: glucose (170 g / L), yeast extract (1 g / L-3 g / L), NH4H2PO4(0.5 g / L-5 g / L), (NH4)2HPO4(1 g / L-10 g / L), MgSO4-7H2O (0.1 g / L-5 g / L), and KCl (10 mg / L-100 mg / L), FeCl3-6H2O (0.2 mg / L-5 mg / L), CoCl2-6H2O (0.05 mg / L-5 mg / L), CuCl2-2H2O (0.05 mg / L-5 mg / L), ZnCl2(0.05 mg / L-5 mg / L), Na2MoO4-2H2O (0.05 mg / L-5 mg / L), H3BO3(0.05 mg / L-5 mg / L), and MnCl2-4H2O (0.05 mg / L-5 mg / L)

[0108] The temperature for fermentation of Klebsiella michiganensis to produce 1,3-propanediol is known in the art, for example, about 25-37°C. In one embodiment, the 1,3-propanediol production strain of the present application is cultured at 37°C to produce 1,3-propanediol.

[0109] The Klebsiella michiganensis of the present application can be fermented at a suitable pH known in the art, for example, about 6.5-7.5. In one embodiment, the 1,3-propanediol production strain of the present application is cultured at about pH 7.0 to produce 1,3-propanediol.

[0110] The Klebsiella michiganensis of the present application can be fermented using a suitable aeration rate known in the art, for example, about 0.1-2vvm. In one embodiment, the 1,3-propanediol production strain of the present application is cultured at about 0.5vvm to produce 1,3-propanediol.

[0111] In one embodiment, the 1,3-propanediol production strain of the present application is fermented under microaerobic conditions. The microaerobic conditions refer to a dissolved oxygen concentration of less than 1 mg / L.

[0112] The Klebsiella michiganensis of the present application can be cultured for a suitable time to produce 1,3-propanediol, for example, about 12-96 hours. In one embodiment, the 1,3-propanediol production strain of the present application is fermented for about 24-72 hours, for example, about 72 hours.

[0113] The Klebsiella michiganensis described herein can be fermented under shaking conditions (e.g., about 100-400 rpm, such as about 150, about 200, about 250, about 300, about 350 rpm) to produce L-1,3-propanediol.

[0114] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, an optionally included step refers to the existence or nonexistence of that step.

[0115] As used herein, the term "about" refers to a range of values including the particular value, which a person of skill in the art would reasonably consider analogous to the particular value. In some embodiments, the term "about" refers to within standard error of the measurements using accepted measurement techniques in the art. In some embodiments, about refers to + / - 10% of a particular value.

[0116] The ranges disclosed herein are to be understood to include all possible subranges and individual values within the range. For example, a description of a range from 1 to 6 should be considered to have explicitly disclosed subranges and individual values from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual values of 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0117] The application will be further illustrated by the following non-limiting examples, to which however, the application is not limited, and many modifications can be made by those skilled in the art without departing from the spirit of the application, and such modifications are to be considered within the scope of the application.

[0118] The following experimental methods are routine methods unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.

[0119] Examples

[0120] Example 1: Isolation and screening of chassis

[0121] 1.1 Enrichment of strains

[0122] A portion of river beach mud was excavated from the beach of the North Pond Drainage River at Chigan Bridge in Dongli District, Tianjin, 25.0 g of which was mixed with 250 mL of phosphate buffer (pH 7.0), and allowed to stand for 40 min. The supernatant was collected, centrifuged at 5000 rpm for 10 min, and the precipitate was resuspended with 1 ml of phosphate buffer (pH 7.0).

[0123] The suspension was inoculated into a 250 mL flask containing 50 mL of enrichment medium, and incubated at 37°C for 72 h. The fermentation broth was diluted and spread on LB solid plates, which were incubated at 37°C for 24-36 h. The enrichment medium contained the following components: 1,2-propanediol (50 g / L), NH4H2PO4(1 g / L), (NH4)2HPO4(3 g / L), MgSO4·7H2O (1 g / L), KCl (74 mg / L), FeCl3·6H2O (2.4 mg / L), CoCl2·6H2O (0.3 mg / L), CuCl2·2H2O (0.15 mg / L), ZnCl2(0.3 mg / L), Na2MoO4·2H2O (0.3 mg / L), H3BO3(0.1 mg / L), and MnCl2·4H2O (0.5 mg / L).

[0124] 1.2 Strain screening

[0125] Forty-eight single colonies were randomly picked from the LB plates and inoculated into 350 mL fermenters containing 250 mL of screening medium. The culture conditions were as follows: 37°C, 250 rpm, anaerobic, and pH 7.0. After 72 h of cultivation, the fermentation broth was collected and subjected to liquid chromatography to determine the 1,3-propanediol yield. The strain PDO-046 with the highest 1,3-propanediol yield was selected. The screening medium contained the following components: glycerol (50 g / L), NH4H2PO4(1 g / L), (NH4)2HPO4(3 g / L), MgSO4·7H2O (1 g / L), KCl (74 mg / L), FeCl3·6H2O (2.4 mg / L), CoCl2·6H2O (0.3 mg / L), CuCl2·2H2O (0.15 mg / L), ZnCl2(0.3 mg / L), Na2MoO4·2H2O (0.3 mg / L), H3BO3(0.1 mg / L), and MnCl2·4H2O (0.5 mg / L).

[0126] Liquid chromatography detection conditions: mobile phase: 5 mM sulfuric acid, flow rate: 0.5 ml / min, differential refractive index detector, Bore HPX-87H chromatographic column, column temperature: 35°C, sample injection volume: 20 μL.

[0127] 1.3 Strain identification

[0128] Strain PDO-046 was identified by 16S rRNA sequence determination and comparison.

[0129]

[0130] The 16S rRNA gene sequence obtained was compared with the GenBank database using BLAST software. The results showed that the 16S rRNA gene sequence of PDO-046 was most similar to the 16S rRNA sequences of the sequenced and published Klebsiella Michigani strains Biosolid 27 (CP054159.1), KM41 (CP090078.1), AR375 (CP029141.1), CZ598 (CP073305.1), and several other unlisted Klebsiella Michigani strains, with a similarity of 100%. Based on the 16S rRNA sequencing and analysis results, strain PDO-046 was identified as Klebsiella Michigani.

[0131] 1.4 Drawing the complete genome map

[0132] Using the Promega Genome Extraction Kit ( The PDO-046 genome was extracted using the Genomic DNA Purification Kit and sent to a gene sequencing company. A combined third-generation Nanopore sequencing and second-generation Illumina sequencing were used to create a complete genome map. Gene function annotation was performed using eight major databases: UniProt, KEGG and KEGGPathway, GO, Pfam, COG, TIGERFAMS, RefSeq, and NR. The predicted gene sequences were BLAST+ (2.9.0+) aligned with functional databases such as COG, KEGG, Swiss-Prot, and RefSeq to obtain gene function annotation results. Functional annotation was performed using hmmer (3.3.1) software based on the Pfam and TIGERFAMS databases, thus completing the genome map and gene function annotation.

[0133] Example 2: Metabolic Engineering of PDO-046

[0134] 2.1 Inactivation of ptsG, the gene encoding a component of the glucose-specific PTS transport system in PDO-046

[0135] Starting with Klebsiella Michigani PDO-046, a two-step homologous recombination method was used to inactivate the ptsG gene, which encodes a component of the glucose-specific PTS transport system. The specific steps are as follows:

[0136] The first step was to amplify the basic gene fragment of ptsG, the gene encoding the inactivated glucose-specific PTS transport system component in PDO-046.

[0137] The genomic DNA of PDO-046 was used as a template, and primers ΔptsG up arm up (SEQ ID NO: 4) and ΔptsG up arm down (SEQ ID NO: 5) were used to perform PCR amplification to obtain a DNA fragment ΔptsG-1. The DNA fragment has a size of 613 bp and is an upstream homologous arm of the ptsG gene.

[0138] The genomic DNA of PDO-046 was used as a template, and primers ΔptsG down arm up (SEQ ID NO: 6) and ΔptsG down arm down (SEQ ID NO: 7) were used to perform PCR amplification to obtain a DNA fragment ΔptsG-2. The DNA fragment has a size of 423 bp and is a downstream homologous arm of the ptsG gene.

[0139] The genomic DNA of PDO-046 was used as a template, and primers ΔptsG down arm up (SEQ ID NO: 6) and ΔptsG down arm down (SEQ ID NO: 7) were used to perform PCR amplification to obtain a DNA fragment ΔptsG-2. The DNA fragment has a size of 423 bp and is a downstream homologous arm of the ptsG gene.

[0140] The genomic DNA of PDO-046 was used as a template, and primers ΔptsG down arm up (SEQ ID NO: 6) and ΔptsG down arm down (SEQ ID NO: 7) were used to perform PCR amplification to obtain a DNA fragment ΔptsG-2. The DNA fragment has a size of 423 bp and is a downstream homologous arm of the ptsG gene.

[0141] In the second step, a two-step homologous recombination fragment for inactivating the ptsG gene encoding a component of a glucose-specific PTS transport system in PDO-046 was constructed.

[0142] The genomic DNA of PDO-046 was used as a template, and primers ΔptsG down arm up (SEQ ID NO: 6) and ΔptsG down arm down (SEQ ID NO: 7) were used to perform PCR amplification to obtain a DNA fragment ΔptsG-2. The DNA fragment has a size of 423 bp and is a downstream homologous arm of the ptsG gene.

[0143] The DNA fragment ΔptsG-II was obtained by PCR amplification using the primer ΔptsG up arm up (SEQ ID NO: 4) and ΔptsG down arm down (SEQ ID NO: 7) with the equimolar mixture of the fragments ΔptsG-1, ΔptsG-4 as template. The size of the DNA fragment ΔptsG-II is 1036 bp, which is the splicing product of the two fragments ΔptsG-1 and ΔptsG-4, and is a DNA fragment containing the homologous arms of the upstream and downstream of ptsG.

[0144] Step 3, construction of the recombinant strain YY-P001

[0145] The fragment ΔptsG-I was used for the first homologous recombination: first, the pKD46-km plasmid was transformed into the strain PDO-046 by calcium chloride transformation method, and then the DNA fragment ΔptsG-I was electroporated into the PDO-046 with the pKD46-km. The specific transformation steps are as follows:

[0146] First, prepare the electrocompetent cells of the Michigan Klebsiella strain PDO-046 with the pKD46-km plasmid (the specific preparation steps of the competent cells are described in the literature "Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation [J]. Nucleic Acids Res, 1998, 16(13): 6127-6145"); 50 μl of competent cells were placed on ice, 50 ng of DNA fragment ΔptsG-I was added, and the mixture was placed on ice for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electroporation cup. The MicroPulser (Bio-Rad) electroporation instrument was used, and the voltage was 2 kv. After the electric shock, 1 ml of LB medium was quickly transferred to the electric shock cup, and then the mixture was blown 5 times and transferred to a test tube, and then incubated at 75 rpm and 30°C for 2 h. 200 μl of bacterial solution was spread on LB plates containing chloramphenicol (final concentration of 34 μg / ml) and kanamycin (final concentration of 50 μg / ml), and then incubated at 37°C overnight. After incubation, 8 single colonies were selected and used for PCR verification using the primers ΔptsG up arm up (SEQ ID NO: 4) and ΔptsG down arm down (SEQ ID NO: 7). The size of the correct colony amplification product was 3654 bp, and one correct single colony was selected and named YY-P001.

[0147] Step 4, construction of the recombinant strain YY-P002

[0148] Second homologous recombination with fragment ΔptsG-II: DNA fragment ΔptsG-II was electroporated into strain YY-P001. The specific transformation steps are as follows:

[0149] First, prepare the electroporation competent cells of Michigan Klebsiella YY-P001 with pKD46-km plasmid; place 50 μl of competent cells on ice, add 50 ng of DNA fragment ΔptsG-II, and place on ice for 2 minutes, then transfer to a 0.2 cm Bio-Rad electroporation cup. Use a MicroPulser (Bio-Rad) electroporation instrument, and the electric shock parameters are voltage 2 kv. After electric shock, quickly transfer 1 ml of LB medium into the electric shock cup, shake 5 times, then transfer to a test tube, incubate at 75 rpm and 30°C for 4 h. Transfer the bacterial solution to LB liquid medium containing 10% sucrose without sodium chloride (50 ml medium in a 250 ml flask), and incubate for 24 h, then streak on LB solid medium containing 6% sucrose without sodium chloride. Select a single colony and perform PCR verification using primers ΔptsG up arm up (SEQ ID NO: 4) and ΔptsG down arm down (SEQ ID NO: 7), and the correct colony has an amplified product size of 1036 bp. Select one correct single colony and name it YY-P002.

[0150] 2.2 Integration and expression of NADPH-dependent alcohol dehydrogenase encoding gene yqhd from Escherichia coli at the edd site of phosphogluconate dehydratase encoding gene in YY-P002.

[0151] Starting from recombinant bacteria YY-P002, the yqhd gene was inserted at the edd site by a two-step homologous recombination method, and the specific steps are as follows:

[0152] First, amplify the basic DNA fragment of the yqhd gene inserted at the edd gene site of recombinant bacteria YY-P002.

[0153] Using the PDO-046 genome as a template, primers Edd:yqhd up arm up (SEQ ID NO: 11) and Edd:yqhd up arm down (SEQ ID NO: 12) were used for PCR amplification to obtain DNA fragment Edd:yqhd-1. The size of the DNA fragment is 588 bp, which is the homologous arm upstream of the edd gene.

[0154] The DNA fragment Edd:yqhd-2 was obtained by PCR amplification using primers Edd:yqhd down arm up (SEQ ID NO: 13) and Edd:yqhd down arm down (SEQ ID NO: 14) with the genome of PDO-046 as template. The DNA fragment Edd:yqhd-2 was 401 bp in size, which was the downstream homologous arm of the edd gene.

[0155] The DNA fragment Edd:yqhd-3 was obtained by PCR amplification using primers Edd:yqhd cat up (SEQ ID NO: 15) and Edd:yqhd sacB down (SEQ ID NO: 16) with pXZ-CS as template. The DNA fragment Edd:yqhd-3 was 2661 bp in size, which contained the chloramphenicol resistance gene (cat), levan sucrose transferase gene (sacB), and a 20 bp downstream of the fragment Edd:yqhd-1 and a 23 bp upstream of the promoter Yqhd-2 overlapping region.

[0156] The gene fragment Edd:yqhd-4 was obtained by PCR amplification using primers Edd:yqhd yq2 up (SEQ ID NO: 17) and Edd:yqhd yq2 down (SEQ ID NO: 18) with the plasmid Yqhd-2 as template. The DNA fragment Edd:yqhd-4 was 89 bp in size, which contained the coding sequence of the promoter Yqhd-2.

[0157] The gene fragment Edd:yqhd-5 was obtained by PCR amplification using primers Edd:yqhd yqhd up (SEQ ID NO: 19) and Edd:yqhd yqhd down (SEQ ID NO: 20) with the plasmid Yqhd as template. The DNA fragment Edd:yqhd-5 was 1211 bp in size, which contained 27 bp downstream of Edd:yqhd-4 and 20 bp upstream of Edd:yqhd-2.

[0158] The DNA fragment Edd:yqhd-6 was obtained by PCR amplification using primers Edd:yqhd two-step up (SEQ ID NO: 21) and Edd:yqhd yq2 down (SEQ ID NO: 18) with the plasmid Yqhd-2 as template. The DNA fragment Edd:yqhd-6 was 109 bp in size, which contained 20 bp downstream of Edd:yqhd-1 and the coding sequence of the promoter Yqhd-2.

[0159] The DNA fragment Edd:yqhd-7 is obtained by PCR amplification using the plasmid Yqhd as a template and the primers Edd:yqhd yqhd up (SEQ ID NO: 19) and Edd:yqhd two-step down (SEQ ID NO: 22). The DNA fragment Edd:yqhd-7 is 511 bp in size and contains the 511 bp upstream of Edd:yqhd-5.

[0160] The second step is to construct the two-step homologous recombination fragment for integrating yqhd at the edd site in YY-P002.

[0161] The DNA fragment Edd:yqhd-I is obtained by PCR amplification using the molar mixture of the fragments Edd:yqhd-1, Edd:yqhd-2, Edd:yqhd-3, Edd:yqhd-4, and Edd:yqhd-5 as a template and the primers Edd:yqhd up arm up (SEQ ID NO: 11) and Edd:yqhd down arm down (SEQ ID NO: 14). The DNA fragment Edd:yqhd-I is 4860 bp in size and is a splicing product of the five fragments Edd:yqhd-1, Edd:yqhd-2, Edd:yqhd-3, Edd:yqhd-4, and Edd:yqhd-5. It is a DNA fragment containing the upstream homologous arm of edd, the chloramphenicol resistance gene (cat), the levansucrose transferase gene (sacB), the Yqhd-2 promoter, the yqhd coding sequence, and the downstream homologous arm of edd.

[0162] The DNA fragment Edd:yqhd-II is obtained by PCR amplification using the molar mixture of the fragments Edd:yqhd-1, Edd:yqhd-6, and Edd:yqhd-7 as a template and the primers Edd:yqhd up arm up (SEQ ID NO: 11) and Edd:yqhd two-step down (SEQ ID NO: 22). The DNA fragment Edd:yqhd-II is 1161 bp in size and is a splicing product of the three fragments Edd:yqhd-1, Edd:yqhd-6, and Edd:yqhd-7. It is a DNA fragment containing the upstream homologous arm of edd, the Yqhd-2 promoter, and the 484 bp upstream of the yqhd gene coding region.

[0163] The third step is the construction of the recombinant bacteria YY-P003

[0164] The fragment Edd:yqhd-I is used for the first homologous recombination: first, the pKD46-km plasmid is transformed into the strain YY-P002 by the calcium chloride transformation method, and then the DNA fragment is electroporated into YY-P002 with pKD46-km. The specific transformation steps are as follows:

[0165] Firstly, prepare the electrotransformation competent cells of the recombinant bacteria YY-P002 with pKD46-km plasmid; 50 μl of competent cells were placed on ice, 50 ng of DNA fragment Edd:yqhd-I was added, and the mixture was placed on ice for 2 min, and then transferred to a 0.2 cm Bio-Rad electroporation cup. The MicroPulser (Bio-Rad) electroporation instrument was used, and the voltage was 2 kv. After electroporation, 1 ml of LB medium was quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 2 h. 200 μl of bacterial solution was spread on an LB plate containing chloramphenicol (final concentration 34 μg / ml) and kanamycin (final concentration 50 μg / ml), and then incubated at 37°C overnight. Eight single colonies were selected and verified by PCR using primers Edd:yqhd up arm up (SEQ ID NO: 11) and Edd:yqhd down arm down (SEQ ID NO: 14). The correct colony had an amplified product of 4860 bp, and one correct single colony was selected and named YY-P003.

[0166] Fourthly, construction of recombinant bacteria YY-P004

[0167] Secondly, use of fragment Edd:yqhd-II for the second homologous recombination: the DNA fragment Edd:yqhd-II was electroporated into strain YY-P001. The specific transformation steps are as follows:

[0168] Firstly, prepare the electrotransformation competent cells of the recombinant bacteria YY-P002 with pKD46-km plasmid; 50 μl of competent cells were placed on ice, 50 ng of DNA fragment Edd:yqhd-I was added, and the mixture was placed on ice for 2 min, and then transferred to a 0.2 cm Bio-Rad electroporation cup. The MicroPulser (Bio-Rad) electroporation instrument was used, and the voltage was 2 kv. After electroporation, 1 ml of LB medium was quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 2 h. 200 μl of bacterial solution was spread on an LB plate containing chloramphenicol (final concentration 34 μg / ml) and kanamycin (final concentration 50 μg / ml), and then incubated at 37°C overnight. Eight single colonies were selected and verified by PCR using primers Edd:yqhd up arm up (SEQ ID NO: 11) and Edd:yqhd down arm down (SEQ ID NO: 14). The correct colony had an amplified product of 4860 bp, and one correct single colony was selected and named YY-P003.

[0169] 2.3 Inactivation of glycerol kinase encoding gene glpK in YY-P004

[0170] The glycerol kinase encoding gene glpK was inactivated by two-step homologous recombination from recombinant K. michiganii YY-P004. The specific steps were as follows:

[0171] First, the basic gene fragment of inactivated glycerol kinase encoding gene glpK in YY-P004 was amplified.

[0172] The DNA fragment ΔglpK-1 was obtained by PCR amplification using primer ΔglpK up arm up (SEQ ID NO: 23) and ΔglpK up arm down (SEQ ID NO: 24) with PDO-046 genome as template. The size of the DNA fragment was 500 bp, which was the upstream homologous arm of glpK gene.

[0173] The DNA fragment ΔglpK-2 was obtained by PCR amplification using primer ΔglpK down arm up (SEQ ID NO: 25) and ΔglpK down arm down (SEQ ID NO: 26) with PDO-046 genome as template. The size of the DNA fragment was 463 bp, which was the downstream homologous arm of glpK gene.

[0174] The DNA fragment ΔglpK-3 was obtained by PCR amplification using primer ΔglpK cat up (SEQ ID NO: 27) and ΔglpK sacB down (SEQ ID NO: 28) with pXZ-CS as template. The size of the DNA fragment was 2666 bp, which contained chloramphenicol resistance gene (cat), levansucrase gene (sacB) and the fragment of 27 bp downstream of ΔglpK-1 and 21 bp upstream of ΔglpK-2 overlapping region.

[0175] The gene fragment ΔglpK-4 was obtained by PCR amplification using primer ΔglpK two-step up (SEQ ID NO: 29) and ΔglpK down arm down (SEQ ID NO: 26) with PDO-046 genome as template. The size of the DNA fragment was 499 bp, which contained 27 bp downstream of ΔglpK-1 and all of ΔglpK-2 overlapping region.

[0176] Second, the two-step homologous recombination fragment of inactivated glycerol kinase encoding gene glpK in YY-P004 was constructed.

[0177] The DNA fragment ΔglpK-I is obtained by PCR amplification using primer ΔglpK up arm up (SEQ ID NO: 23) and ΔglpK down arm down (SEQ ID NO: 26) with the equimolar mixture of fragments ΔglpK-1, ΔglpK-2, ΔglpK-3 as template. The DNA fragment is 3581 bp in size, which is the splicing product of the three fragments ΔglpK-1, ΔglpK-2, ΔglpK-3, and is a DNA fragment containing chloramphenicol resistance gene (cat) and levansucrase gene (sacB) and the upstream and downstream homologous arms of glpK.

[0178] The DNA fragment ΔglpK-II is obtained by PCR amplification using primer ΔglpK up arm up (SEQ ID NO: 23) and ΔglpK down arm down (SEQ ID NO: 26) with the equimolar mixture of fragments ΔglpK-1, ΔglpK-4 as template. The DNA fragment is 963 bp in size, which is the splicing product of the two fragments ΔglpK-1, ΔglpK-4, and is a DNA fragment containing the upstream and downstream homologous arms of glpK.

[0179] Thirdly, the construction of recombinant bacteria YY-P005

[0180] The fragment ΔglpK-I is used for the first homologous recombination: first, the pKD46-km plasmid is transformed into strain YY-P004 by calcium chloride transformation method, and then the DNA fragment is electroporated into YY-P004 with pKD46-km.

[0181] The specific transformation steps are as follows:

[0182] Firstly, prepare the electroporation competent cells of the Michigan Klebsiella YY-P004 with the pKD46-km plasmid; 50 μl of competent cells are placed on ice, 50 ng of DNA fragment ΔglpK-I is added, and the mixture is placed on ice for 2 min, and then transferred to a 0.2 cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad) electroporation instrument is used, and the voltage is 2 kv. After electroporation, 1 ml of LB medium is quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 2 h. 200 μl of bacterial solution is spread on an LB plate containing chloramphenicol (a final concentration of 34 μg / ml) and kanamycin (a final concentration of 50 μg / ml), and then incubated at 37°C overnight. Eight single colonies are selected and used for PCR verification with primers ΔglpK up arm up (SEQ ID NO: 23) and ΔglpK down arm down (SEQ ID NO: 26). The correct colony has an amplified product of 3581 bp, and one correct single colony is selected and named YY-P005.

[0183] Fourthly, construction of the recombinant strain YY-P006

[0184] The fragment ΔglpK-II is used for the second homologous recombination: the DNA fragment ΔglpK-II is electroporated into the strain YY-P005. The specific transformation steps are as follows:

[0185] Firstly, prepare the electroporation competent cells of the Michigan Klebsiella YY-P005 with the pKD46-km plasmid; 50 μl of competent cells are placed on ice, 50 ng of DNA fragment ΔglpK-II is added, and the mixture is placed on ice for 2 min, and then transferred to a 0.2 cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad) electroporation instrument is used, and the voltage is 2 kv. After electroporation, 1 ml of LB medium is quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 4 h. The bacterial solution is transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml of medium is added to a 250 ml flask), and then incubated at 24 h. A single colony is selected and used for PCR verification with primers ΔglpK up arm up (SEQ ID NO: 23) and ΔglpK down arm down (SEQ ID NO: 26). The correct colony has an amplified product of 963 bp, and one correct single colony is selected and named YY-P006.

[0186] 2.4 Inactivation of the first copy of glycerol dehydrogenase gldA1 in YY-P006

[0187] From recombinant Klebsiella michigan YY-P006, the first copy of glycerol dehydrogenase encoding gene gldA1 was inactivated by two-step homologous recombination method, and the specific steps were as follows:

[0188] First, the basic gene fragment of gldA1 inactivated in YY-P006 was amplified.

[0189] Using primer ΔgldA1 up arm up (SEQ ID NO: 30) and ΔgldA1 up arm down (SEQ ID NO: 31), the DNA fragment ΔgldA1-1 was obtained by PCR amplification using PDO-046 genome as template. The size of the DNA fragment is 580bp, which is the upstream homologous arm of gldA1 gene.

[0190] Using primer ΔgldA1 down arm up (SEQ ID NO: 32) and ΔgldA1 down arm down (SEQ ID NO: 33), the DNA fragment ΔgldA1-2 was obtained by PCR amplification using PDO-046 genome as template. The size of the DNA fragment is 569bp, which is the downstream homologous arm of gldA1 gene.

[0191] Using primer ΔgldA1 cat up (SEQ ID NO: 34) and ΔgldA1 sacB down (SEQ ID NO: 35), the DNA fragment ΔgldA1-3 was obtained by PCR amplification using pXZ-CS as template. The size of the DNA fragment is 2659bp, which contains chloramphenicol resistance gene (cat), levansucrase gene (sacB) and 19bp downstream of ΔgldA1-1 and 22bp upstream of ΔgldA1-2 overlapping region.

[0192] Using primer ΔgldA1 two-step up (SEQ ID NO: 36) and ΔgldA1 down arm down (SEQ ID NO: 33), the gene fragment ΔgldA1-4 was obtained by PCR amplification using PDO-046 genome as template. The size of the DNA fragment is 588 containing 19bp downstream of ΔgldA1-1 and all of ΔgldA1-2.

[0193] Second, the two-step homologous recombination fragment of gldA1 inactivated in YY-P006 was constructed.

[0194] The DNA fragment ΔgldA1-I is obtained by using the primer ΔgldA1 up arm up (SEQ ID NO: 30) and ΔgldA1 down arm down (SEQ ID NO: 33) to perform PCR amplification with the equimolar mixture of fragments ΔgldA1-1, ΔgldA1-2, ΔgldA1-3 as a template. The DNA fragment ΔgldA1-I is 3767 bp in size, which is the splicing product of the three fragments ΔgldA1-1, ΔgldA1-2, ΔgldA1-3, and is a DNA fragment containing chloramphenicol resistance gene (cat) and levansucrose transferase gene (sacB) and the upstream and downstream homologous arms of gldA1.

[0195] The DNA fragment ΔgldA1-II is obtained by using the primer ΔgldA1 up arm up (SEQ ID NO: 30) and ΔgldA1 down arm down (SEQ ID NO: 33) to perform PCR amplification with the equimolar mixture of fragments ΔgldA1-1, ΔgldA1-4 as a template. The DNA fragment ΔgldA1-II is 1149 bp in size, which is the splicing product of the two fragments ΔgldA1-1, ΔgldA1-4, and is a DNA fragment containing the upstream and downstream homologous arms of gldA1.

[0196] Step 3, construction of recombinant bacteria YY-P007

[0197] The fragment ΔgldA1-I is used for the first homologous recombination: first, the pKD46-km plasmid is transformed into strain YY-P006 by calcium chloride transformation method, and then the DNA fragment is electroporated into YY-P006 with pKD46-km. The specific transformation steps are as follows:

[0198] Firstly, prepare the electroporation competent cells of the Michigan Klebsiella YY-P006 with the pKD46-km plasmid; 50 μl of competent cells are placed on ice, 50 ng of DNA fragment ΔgldA1-I is added, and placed on ice for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad) electroporation instrument is used, and the voltage is 2 kv. After electroporation, 1 ml of LB medium is quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 2 hours. 200 μl of bacterial solution is spread on an LB plate containing chloramphenicol (a final concentration of 34 μg / ml) and kanamycin (a final concentration of 50 μg / ml), and then incubated at 37°C overnight. Eight single colonies are selected and used for PCR verification using primers ΔgldA1 up arm up (SEQ ID NO: 30) and ΔgldA1 down arm down (SEQ ID NO: 33). The correct colony has an amplified product of 3767 bp, and one correct single colony is selected and named YY-P007.

[0199] Fourthly, construction of the recombinant strain YY-P008

[0200] Secondly, the fragment ΔgldA1-II is used for the second homologous recombination: the DNA fragment ΔgldA1-II is electroporated into the strain YY-P007. The specific transformation steps are as follows:

[0201] Firstly, prepare the electroporation competent cells of the Michigan Klebsiella YY-P007 with the pKD46-km plasmid; 50 μl of competent cells are placed on ice, 50 ng of DNA fragment ΔgldA1-II is added, and placed on ice for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad) electroporation instrument is used, and the voltage is 2 kv. After electroporation, 1 ml of LB medium is quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 4 hours. The bacterial solution is transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml of medium is added to a 250 ml flask), and then incubated for 24 hours. A single colony is selected and used for PCR verification using primers ΔgldA1 up arm up (SEQ ID NO: 30) and ΔgldA1 down arm down (SEQ ID NO: 33). The correct colony has an amplified product of 1149 bp, and one correct single colony is selected and named YY-P008.

[0202] 2.5 Inactivation of the second copy of glycerol dehydrogenase gldA2 in YY-P008

[0203] From recombinant Klebsiella michigan YY-P008, the first copy of glycerol dehydrogenase encoding gene gldA2 was inactivated by two-step homologous recombination method, and the specific steps were as follows:

[0204] First, the basic gene fragment of gldA2 in YY-P008 was amplified.

[0205] Using primer ΔgldA2 up arm up (SEQ ID NO: 37) and ΔgldA2 up arm down (SEQ ID NO: 38), the DNA fragment ΔgldA2-1 was obtained by PCR amplification using PDO-046 genome as template. The size of the DNA fragment is 691 bp, which is the upstream homologous arm of gldA2 gene.

[0206] Using primer ΔgldA2 down arm up (SEQ ID NO: 39) and ΔgldA2 down arm down (SEQ ID NO: 40), the DNA fragment ΔgldA2-2 was obtained by PCR amplification using PDO-046 genome as template. The size of the DNA fragment is 674 bp, which is the downstream homologous arm of gldA2 gene.

[0207] Using primer ΔgldA2 cat up (SEQ ID NO: 41) and ΔgldA2 sacB down (SEQ ID NO: 42), the DNA fragment ΔgldA2-3 was obtained by PCR amplification using pXZ-CS as template. The size of the DNA fragment is 2663 bp, which contains chloramphenicol resistance gene (cat), levansucrase gene (sacB) and 23 bp downstream of ΔgldA2-1 and 22 bp upstream of ΔgldA2-2 overlapping region.

[0208] Using primer ΔgldA2 two-step up (SEQ ID NO: 43) and ΔgldA2 down arm down (SEQ ID NO: 40), the gene fragment ΔgldA2-4 was obtained by PCR amplification using PDO-046 genome as template. The size of the DNA fragment is 697 bp, which contains 23 bp downstream of ΔgldA2-1 and all of ΔgldA2-2.

[0209] Second, the two-step homologous recombination fragment of gldA2 inactivation in YY-P008 was constructed.

[0210] ΔgldA2-1, ΔgldA2-2, ΔgldA2-3, etc. as a template, using primer ΔgldA2 up arm up (SEQ ID NO: 37) and ΔgldA2 down arm down (SEQ ID NO: 40) for PCR amplification, to obtain DNA fragment ΔgldA2-I, the size of which is 3983 bp, which is the splicing product of three fragments ΔgldA2-1, ΔgldA2-2, ΔgldA2-3, and is a DNA fragment containing chloramphenicol resistance gene (cat) and levansucrose transferase gene (sacB) and gldA2 upstream and downstream homologous arms.

[0211] ΔgldA2-1, ΔgldA2-4, etc. as a template, using primer ΔgldA2 up arm up (SEQ ID NO: 37) and ΔgldA2 down arm down (SEQ ID NO: 40) for PCR amplification, to obtain DNA fragment ΔgldA2-II. The size of this DNA fragment is 1365 bp, which is the splicing product of two fragments ΔgldA2-1 and ΔgldA2-4, and is a DNA fragment containing gldA2 upstream and downstream homologous arms.

[0212] Thirdly, the construction of recombinant bacteria YY-P009

[0213] Fragment ΔgldA2-I was used for the first homologous recombination: first, pKD46-km plasmid was transformed into strain YY-P008 by calcium chloride transformation method, and then the DNA fragment was electroporated into YY-P008 with pKD46-km. The specific transformation steps are as follows:

[0214] Firstly, prepare the electroporation competent cells of the Michigan Klebsiella YY-P008 with the pKD46-km plasmid; 50 μl of the competent cells are placed on ice, 50 ng of the DNA fragment ΔgldA2-I is added, and the mixture is placed on ice for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad) electroporation instrument is used, and the voltage is 2 kv. After the electroporation, 1 ml of LB medium is quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 2 hours. 200 μl of the bacterial solution is spread on an LB plate containing chloramphenicol (a final concentration of 34 μg / ml) and kanamycin (a final concentration of 50 μg / ml), and then incubated at 37°C overnight. Eight single colonies are selected and used for PCR verification by using primers ΔgldA2 up arm up (SEQ ID NO: 37) and ΔgldA2 down arm down (SEQ ID NO: 40). The correct colony has an amplified product with a size of 3983 bp, and one correct single colony is selected and named YY-P009.

[0215] Fourthly, construction of the recombinant strain YY-P010

[0216] The fragment ΔgldA2-II is used for the second homologous recombination: the DNA fragment ΔgldA2-II is electroporated into the strain YY-P009. The specific transformation steps are as follows:

[0217] Firstly, prepare the electroporation competent cells of the Michigan Klebsiella YY-P009 with the pKD46-km plasmid; 50 μl of the competent cells are placed on ice, 50 ng of the DNA fragment ΔgldA2-II is added, and the mixture is placed on ice for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad) electroporation instrument is used, and the voltage is 2 kv. After the electroporation, 1 ml of LB medium is quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 4 hours. The bacterial solution is transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml of the medium is placed in a 250 ml flask), and then incubated for 24 hours. A single colony is selected and used for PCR verification by using primers ΔgldA2 up arm up (SEQ ID NO: 37) and ΔgldA2 down arm down (SEQ ID NO: 40). The correct colony has an amplified product with a size of 1365 bp, and one correct single colony is selected and named YY-P010.

[0218] 2.6 Inactivation of the fumarate reductase gene cluster frdABCD in YY-P010

[0219] From recombinant Klebsiella michigan YY-P010, the fumarate reductase encoding gene cluster frdABCD was inactivated by two-step homologous recombination, and the specific steps were as follows:

[0220] First, the basic gene fragment of frdABCD in YY-P010 was amplified.

[0221] Using the primer ΔfrdABCD up arm up (SEQ ID NO: 44) and ΔfrdABCD up arm down (SEQ ID NO: 45), the DNA fragment ΔfrdABCD-1 was obtained by PCR amplification using PDO-046 genome as a template. The size of the DNA fragment is 623 bp, which is the upper homologous arm for frdABCD gene cluster inactivation.

[0222] Using the primer ΔfrdABCD down arm up (SEQ ID NO: 46) and ΔfrdABCD down arm down (SEQ ID NO: 47), the DNA fragment ΔfrdABCD-2 was obtained by PCR amplification using PDO-046 genome as a template. The size of the DNA fragment is 582 bp, which is the lower homologous arm for frdABCD gene cluster inactivation.

[0223] Using the primer ΔfrdABCD cat up (SEQ ID NO: 48) and ΔfrdABCD sacB down (SEQ ID NO: 49), the DNA fragment ΔfrdABCD-3 was obtained by PCR amplification using pXZ-CS as a template. The size of the DNA fragment is 2658 bp, which contains chloramphenicol resistance gene (cat), levansucrase gene (sacB) and the 20 bp overlapping region downstream of ΔfrdABCD-1 and upstream of ΔfrdABCD-2.

[0224] Using the primer ΔfrdABCD two-step up (SEQ ID NO: 50) and ΔfrdABCD down arm down (SEQ ID NO: 47), the gene fragment ΔfrdABCD-4 was obtained by PCR amplification using PDO-046 genome as a template. The size of the DNA fragment is 602 bp, which contains the 20 bp overlapping region downstream of ΔfrdABCD-1 and the entire ΔfrdABCD-2.

[0225] Second, the two-step homologous recombination fragment for inactivating frdABCD in YY-P010 was constructed.

[0226] PCR amplification was performed using the primer ΔfrdABCD up arm up (SEQ ID NO: 44) and ΔfrdABCD down arm down (SEQ ID NO: 47) with the equimolar mixture of the fragments ΔfrdABCD-1, ΔfrdABCD-2, ΔfrdABCD-3 as templates to obtain the DNA fragment ΔfrdABCD-I. The size of the DNA fragment is 3823 bp, which is the splicing product of the three fragments ΔfrdABCD-1, ΔfrdABCD-2, ΔfrdABCD-3, and is a DNA fragment containing chloramphenicol resistance gene (cat) and levansucrase gene (sacB) and the upper and lower homologous arms for inactivation of gene cluster frdABCD.

[0227] PCR amplification was performed using the primer ΔfrdABCD up arm up (SEQ ID NO: 44) and ΔfrdABCD down arm down (SEQ ID NO: 47) with the equimolar mixture of the fragments ΔfrdABCD-1, ΔfrdABCD-4 as templates to obtain the DNA fragment ΔfrdABCD-II. The size of the DNA fragment is 1205 bp, which is the splicing product of the two fragments ΔfrdABCD-1, ΔfrdABCD-4, and is a DNA fragment containing the upper and lower homologous arms for inactivation of gene cluster frdABCD.

[0228] Step 3, construction of recombinant bacteria YY-P011

[0229] The fragment ΔfrdABCD-I was used for the first homologous recombination: first, the pKD46-km plasmid was transformed into strain YY-P010 by calcium chloride transformation method, and then the DNA fragment was electroporated into YY-P010 with pKD46-km. The specific transformation steps are as follows:

[0230] Firstly, prepare the electrotransformation competent cells of the Michigan Klebsiella YY-P010 with the pKD46-km plasmid; put 50 μl of the competent cells on ice, add 50 ng of the DNA fragment ΔfrdABCD-I, and keep on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cup. Use a MicroPulser (Bio-Rad) electroporation instrument, and the voltage is 2 kv. After the electric shock, quickly transfer 1 ml of LB medium into the electroporation cup, and then transfer into a test tube after 5 times of blowing, and incubate at 75 rpm and 30°C for 2 hours. Take 200 μl of the bacterial solution and spread on an LB plate containing chloramphenicol (the final concentration is 34 μg / ml) and kanamycin (the final concentration is 50 μg / ml), and incubate at 37°C overnight. After the incubation, select 8 single colonies, and use primers ΔfrdABCD up arm up (SEQ ID NO: 44) and ΔfrdABCD down arm down (SEQ ID NO: 47) to perform PCR verification. The correct colony has an amplified product with a size of 3823 bp. Select one correct single colony, and name it YY-P011.

[0231] Fourthly, construction of the recombinant strain YY-P012

[0232] Use the fragment ΔfrdABCD-II for the second homologous recombination: electrically transfer the DNA fragment ΔfrdABCD-II to the strain YY-P011. The specific transformation steps are as follows:

[0233] Firstly, prepare the electrotransformation competent cells of the Michigan Klebsiella YY-P011 with the pKD46-km plasmid; put 50 μl of the competent cells on ice, add 50 ng of the DNA fragment ΔfrdABCD-II, and keep on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cup. Use a MicroPulser (Bio-Rad) electroporation instrument, and the voltage is 2 kv. After the electric shock, quickly transfer 1 ml of LB medium into the electroporation cup, and then transfer into a test tube after 5 times of blowing, and incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial solution to LB liquid medium (50 ml of medium in a 250 ml flask) containing 10% sucrose and no sodium chloride, and incubate for 24 hours. Then, perform streak culture on LB solid medium containing 6% sucrose and no sodium chloride. Select single colonies, and use primers ΔfrdABCD up arm up (SEQ ID NO: 44) and ΔfrdABCD down arm down (SEQ ID NO: 47) to perform PCR verification. The correct colony has an amplified product with a size of 1205 bp. Select one correct single colony, and name it YY-P012.

[0234] 2.7 Inactivation of alcohol dehydrogenase encoding gene adhE in YY-P012

[0235] The alcohol dehydrogenase encoding gene adhE was inactivated by two-step homologous recombination from recombinant K. michiganii YY-P012, and the specific steps were as follows:

[0236] First, the basic gene fragment of inactivated alcohol dehydrogenase encoding gene adhE in YY-P012 was amplified.

[0237] Using the primer ΔadhE up arm up (SEQ ID NO: 51) and ΔadhE up arm down (SEQ ID NO: 52), the DNA fragment ΔadhE-1 was obtained by PCR amplification using PDO-046 genome as a template. The size of the DNA fragment was 496 bp, which was the upstream homologous arm for adhE inactivation.

[0238] Using the primer ΔadhE down arm up (SEQ ID NO: 53) and ΔadhE down arm down (SEQ ID NO: 54), the DNA fragment ΔadhE-2 was obtained by PCR amplification using PDO-046 genome as a template. The size of the DNA fragment was 638 bp, which was the downstream homologous arm for adhE inactivation.

[0239] Using the primer ΔadhE cat up (SEQ ID NO: 55) and ΔadhE sacB down (SEQ ID NO: 56), the DNA fragment ΔadhE-3 was obtained by PCR amplification using pXZ-CS as a template. The size of the DNA fragment was 2658 bp, which contained chloramphenicol resistance gene (cat), levansucrase gene (sacB), and the fragment of 20 bp downstream of ΔadhE-1 and 20 bp upstream of ΔadhE-2 overlapping region.

[0240] Using the primer ΔadhE two-step up (SEQ ID NO: 57) and ΔadhE down arm down (SEQ ID NO: 54), the gene fragment ΔadhE-4 was obtained by PCR amplification using PDO-046 genome as a template. The size of the DNA fragment was 658 bp, which contained 20 bp downstream of ΔadhE-1 overlapping region and all ΔadhE-2.

[0241] Second, the two-step homologous recombination fragment for inactivating alcohol dehydrogenase encoding gene adhE in YY-P010 was constructed.

[0242] The DNA fragment ΔadhE-I is obtained by using the equimolar mixture of fragments ΔadhE-1, ΔadhE-2, ΔadhE-3 as a template, and using primers ΔadhE up arm up (SEQ ID NO: 51) and ΔadhE down arm down (SEQ ID NO: 54) for PCR amplification. The DNA fragment ΔadhE-I is 3752 bp in size, which is the splicing product of three fragments ΔadhE-1, ΔadhE-2, ΔadhE-3, and is a DNA fragment containing chloramphenicol resistance gene (cat) and levansucrose transferase gene (sacB) and the upstream and downstream homologous arms for inactivating gene adhE.

[0243] The DNA fragment ΔadhE-II is obtained by using the equimolar mixture of fragments ΔadhE-1, ΔadhE-4 as a template, and using primers ΔadhE up arm up (SEQ ID NO: 51) and ΔadhE down arm down (SEQ ID NO: 54) for PCR amplification. The DNA fragment ΔadhE-II is 1134 bp in size, which is the splicing product of two fragments ΔadhE-1, ΔadhE-4, and is a DNA fragment containing the upstream and downstream homologous arms for inactivating gene adhE.

[0244] Step 3, construction of recombinant bacteria YY-P013

[0245] The fragment ΔadhE-I is used for the first homologous recombination: first, the pKD46-km plasmid is transformed into strain YY-P012 by calcium chloride transformation method, and then the DNA fragment is electroporated into YY-P012 with pKD46-km.

[0246] The specific transformation steps are as follows:

[0247] Firstly, prepare the electrotransformation competent cells of the recombinant Klebsiella michiganii YY-P013 with pKD46-km plasmid; put 50 μl of the competent cells on ice, add 50 ng of DNA fragment ΔadhE-I, and keep on ice for 2 min, then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporation instrument, and the voltage is 2 kv. After the electric shock, quickly transfer 1 ml of LB medium into the electroporation cuvette, then transfer into a test tube after 5 times of blowing, and incubate at 75 rpm and 30°C for 2 h. Take 200 μl of the bacterial solution and spread on an LB plate containing chloramphenicol (the final concentration is 34 μg / ml) and kanamycin (the final concentration is 50 μg / ml), and incubate at 37°C overnight. After the incubation, select 8 single colonies, and use primers ΔadhE up arm up (SEQ ID NO: 51) and ΔadhE down arm down (SEQ ID NO: 54) to perform PCR verification. The correct colony has an amplified product with a size of 3752 bp. Select one correct single colony, and name it as YY-P013.

[0248] Fourth step, construction of the recombinant strain YY-P014

[0249] Use the fragment ΔadhE-II for the second homologous recombination: electrically transfer the DNA fragment ΔadhE-II to the strain YY-P013. The specific transformation steps are as follows:

[0250] Firstly, prepare the electrotransformation competent cells of the recombinant Klebsiella michiganii YY-P013 with pKD46-km plasmid; put 50 μl of the competent cells on ice, add 50 ng of DNA fragment ΔadhE-I, and keep on ice for 2 min, then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporation instrument, and the voltage is 2 kv. After the electric shock, quickly transfer 1 ml of LB medium into the electroporation cuvette, then transfer into a test tube after 5 times of blowing, and incubate at 75 rpm and 30°C for 2 h. Take 200 μl of the bacterial solution and spread on an LB plate containing chloramphenicol (the final concentration is 34 μg / ml) and kanamycin (the final concentration is 50 μg / ml), and incubate at 37°C overnight. After the incubation, select 8 single colonies, and use primers ΔadhE up arm up (SEQ ID NO: 51) and ΔadhE down arm down (SEQ ID NO: 54) to perform PCR verification. The correct colony has an amplified product with a size of 3752 bp. Select one correct single colony, and name it as YY-P013.

[0251] 2.8 Regulating the expression of diol dehydratase-encoding gene cluster pdu using artificial promoter Pdu-31 in YY-P0014.

[0252] Starting from recombinant bacteria YY-P0014, the expression of diol dehydratase-encoding gene cluster pdu was regulated using Pdu-31 promoter by two-step homologous recombination. The specific steps are as follows:

[0253] First, the DNA fragment for regulating the expression of pdu gene cluster using Pdu-31 promoter in recombinant bacteria YY-P014 was amplified.

[0254] Using PDO-046 genome as template, primers Pdu-31up arm up (SEQ ID NO: 58) and Pdu-31up arm down (SEQ ID NO: 59) were used for PCR amplification to obtain DNA fragment Pdu-31-1. The size of the DNA fragment is 541 bp, which is the upstream homologous arm for regulating the expression of pdu gene cluster.

[0255] Using PDO-046 genome as template, primers Pdu-31down arm up (SEQ ID NO: 60) and Pdu-31down arm down (SEQ ID NO: 61) were used for PCR amplification to obtain DNA fragment Pdu-31-2. The size of the DNA fragment is 535 bp, which is the downstream homologous arm for regulating the expression of pdu gene cluster.

[0256] Using pXZ-CS as template, primers Pdu-31cat up (SEQ ID NO: 62) and Pdu-31sacB down (SEQ ID NO: 63) were used for PCR amplification to obtain DNA fragment Pdu-31-3. The size of the DNA fragment is 2669 bp, which contains chloramphenicol resistance gene (cat), levan sucrose transferase gene (sacB), and the fragment Pdu-31-1 downstream 28 bp and the promoter Pdu-31 upstream 23 bp overlapping region.

[0257] Using plasmid Pdu-31 as template, primers Pdu-31up (SEQ ID NO: 64) and Pdu-31down (SEQ ID NO: 65) were used for PCR amplification to obtain gene fragment Pdu-31-4. The size of the DNA fragment is 113, which contains the coding sequence of promoter Yqhd-2 and the upstream 24 bp of DNA fragment Pdu-31-2.

[0258] The DNA fragment Pdu-31-5 is obtained by PCR amplification using the primer Pdu-31 two-step up (SEQ ID NO: 66) and Pdu-31 down (SEQ ID NO: 65) with the plasmid Pdu-31 genome as the template. The DNA fragment has a size of 141 bp, including 28 bp downstream of Pdu-31-1, the coding sequence of the promoter Yqhd-2, and 24 bp upstream of Pdu-31-2.

[0259] The second step is to construct the two-step homologous recombination fragment for regulating the expression of the pdu gene cluster using the Pdu-31 promoter in the recombinant bacteria YY-P014.

[0260] The DNA fragment Pdu-31-I is obtained by PCR amplification using the primer Pdu-31 up arm up (SEQ ID NO: 58) and Pdu-31 down arm down (SEQ ID NO: 61) with the equimolar mixture of the fragments Pdu-31-1, Pdu-31-2, Pdu-31-3, and Pdu-31-4 as the template. The DNA fragment has a size of 3783 bp, which is the splicing product of the four fragments Pdu-31-1, Pdu-31-2, Pdu-31-3, and Pdu-31-4, and is a DNA fragment containing the upstream homologous arm for regulating the expression of the pdu gene cluster, the chloramphenicol resistance gene (cat), the levansucrose transferase gene (sacB), the Pdu-31 promoter, and the downstream homologous arm for regulating the expression of the pdu gene cluster.

[0261] The DNA fragment Pdu-31-II is obtained by PCR amplification using the primer Pdu-31 up arm up (SEQ ID NO: 58) and Pdu-31 down arm down (SEQ ID NO: 61) with the equimolar mixture of the fragments Pdu-31-1, Pdu-31-2, and Pdu-31-5 as the template. The DNA fragment has a size of 1165 bp, which is the splicing product of the three fragments Pdu-31-1, Pdu-31-2, and Pdu-31-5, and is a DNA fragment containing the upstream homologous arm for regulating the expression of the pdu gene cluster, the Pdu-31 promoter, and the downstream homologous arm for regulating the expression of the pdu gene cluster.

[0262] The third step is the construction of the recombinant bacteria YY-P017

[0263] The fragment Pdu-31-I is used for the first homologous recombination: first, the pKD46-km plasmid is transformed into the strain YY-P014 by the calcium chloride transformation method, and then the DNA fragment is electroporated into the YY-P014 with the pKD46-km. The specific transformation steps are as follows:

[0264] Firstly, prepare the electrotransformation competent cells of recombinant bacteria YY-P014 with pKD46-km plasmid; 50 μl of competent cells are placed on ice, 50 ng of DNA fragment Pdu-31-I is added, and the mixture is placed on ice for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad) electroporation instrument is used, and the voltage is 2 kv. After electroporation, 1 ml of LB medium is quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 2 hours. 200 μl of bacterial solution is spread on an LB plate containing chloramphenicol (a final concentration of 34 μg / ml) and kanamycin (a final concentration of 50 μg / ml), and then incubated at 37°C overnight. Eight single colonies are selected and used for PCR verification with primers Pdu-31up arm up (SEQ ID NO: 58) and Pdu-31down arm down (SEQ ID NO: 61). The correct colony has an amplified product with a size of 3783 bp, and a correct single colony is selected and named YY-P017.

[0265] Fourthly, construction of recombinant bacteria YY-P018

[0266] Secondly, use of fragment Pdu-31-II for the second homologous recombination: DNA fragment Pdu-31-II is electroporated into strain YY-P017. The specific transformation steps are as follows:

[0267] Firstly, prepare the electrotransformation competent cells of recombinant bacteria YY-P014 with pKD46-km plasmid; 50 μl of competent cells are placed on ice, 50 ng of DNA fragment Pdu-31-I is added, and the mixture is placed on ice for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electroporation cup. A MicroPulser (Bio-Rad) electroporation instrument is used, and the voltage is 2 kv. After electroporation, 1 ml of LB medium is quickly transferred to the electroporation cup, and then transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 2 hours. 200 μl of bacterial solution is spread on an LB plate containing chloramphenicol (a final concentration of 34 μg / ml) and kanamycin (a final concentration of 50 μg / ml), and then incubated at 37°C overnight. Eight single colonies are selected and used for PCR verification with primers Pdu-31up arm up (SEQ ID NO: 58) and Pdu-31down arm down (SEQ ID NO: 61). The correct colony has an amplified product with a size of 3783 bp, and a correct single colony is selected and named YY-P017.

[0268] 2.9 The GPD1+GPP2 fusion gene encoding 3-phosphoglycerate dehydrogenase GPD1 and 3-phosphoglycerate phosphatase GPP2 is integrated and expressed at the mgsA site of the gene encoding methylglyoxal synthase in YY-P018.

[0269] The GPD1+GPP2 fusion gene is inserted and expressed at the mgsA site of the recombinant strain YY-P018 by two-step homologous recombination, and the specific steps are as follows:

[0270] First, the basic DNA fragment of the GPD1+GPP2 fusion gene is amplified and inserted and expressed at the mgsA gene site of the recombinant strain YY-P018.

[0271] The DNA fragment mgsA:GP-1 is obtained by PCR amplification using the primer mgsA:GP up arm up (SEQ ID NO: 67) and mgsA:GP up arm down (SEQ ID NO: 68) with the PDO-046 genome as the template. The size of the DNA fragment is 481 bp, which is the upstream homologous arm of the mgsA gene.

[0272] The DNA fragment mgsA:GP-2 is obtained by PCR amplification using the primer mgsA:GP down arm up (SEQ ID NO: 69) and mgsA:GP down arm down (SEQ ID NO: 70) with the PDO-046 genome as the template. The size of the DNA fragment is 488 bp, which is the downstream homologous arm of the mgsA gene.

[0273] The DNA fragment mgsA:GP-3 is obtained by PCR amplification using the primer mgsA:GP cat up (SEQ ID NO: 71) and mgsA:GP sacB down (SEQ ID NO: 72) with the pXZ-CS as the template. The size of the DNA fragment is 2663 bp, which contains the chloramphenicol resistance gene (cat), the levansucrose transferase gene (sacB), and the fragment mgsA:GP-1 downstream 22 bp and the promoter GP-28 upstream 23 bp overlapping region.

[0274] The gene fragment mgsA:GP-4 is obtained by PCR amplification using the primer mgsA:GP GP28 up (SEQ ID NO: 73) and mgsA:GP GP28 down (SEQ ID NO: 74) with the plasmid GP-28 as the template. The size of the DNA fragment is 89, which contains the coding sequence of the promoter GP-28.

[0275] The plasmid GPD1+GPP2 was used as a template, and primers mgsA:GP GP up (SEQ ID NO: 75) and mgsA:GP GP down (SEQ ID NO: 76) were used for PCR amplification to obtain the gene fragment mgsA:GP-5. The size of the DNA fragment was 1989, which contained 25 bp downstream of mgsA:GP-4, 1941 bp of GPD1+GPP2 coding sequence (SEQ ID NO: 98), and 23 bp upstream of mgsA:GP-2.

[0276] The plasmid GP-28 genome was used as a template, and primers mgsA:GP two-step up (SEQ ID NO: 77) and mgsA:GP GP28 down (SEQ ID NO: 74) were used for PCR amplification to obtain the DNA fragment mgsA:GP-6. The size of the DNA fragment was 111 bp, including 20 bp downstream of mgsA:GP-1 and the promoter GP-28 coding sequence.

[0277] The plasmid GPD1+GPP2 was used as a template, and primers mgsA:GP GP up (SEQ ID NO: 75) and mgsA:GP two-step down (SEQ ID NO: 78) were used for PCR amplification to obtain the DNA fragment mgsA:GP-7. The size of the DNA fragment was 589 bp, containing 511 bp upstream of GPD1+GPP2.

[0278] In the second step, the two-step homologous recombination fragment of GPD1+GPP2 fusion gene was inserted and expressed at the mgsA gene site of the recombinant bacteria YY-P018.

[0279] An equimolar mixture of fragments mgsA:GP-1, mgsA:GP-2, mgsA:GP-3, mgsA:GP-4, mgsA:GP-5 was used as a template, and primers mgsA:GP up arm up (SEQ ID NO: 67) and mgsA:GP down arm down (SEQ ID NO: 70) were used for PCR amplification to obtain the DNA fragment mgsA:GP-I. The size of the DNA fragment was 5617 bp, which was the splicing product of the five fragments mgsA:GP-1, mgsA:GP-2, mgsA:GP-3, mgsA:GP-4, and mgsA:GP-5, and was a DNA fragment containing the mgsA upstream homologous arm, the chloramphenicol resistance gene (cat), the levansucrose transferase gene (sacB), the GP-28 promoter, the GPD1+GPP2 coding sequence, and the mgsA downstream homologous arm.

[0280] The DNA fragment mgsA:GP-II was obtained by PCR amplification using the primer mgsA:GP up arm up (SEQ ID NO: 67) and mgsA:GP two-step down (SEQ ID NO: 78) with the molar mixture of the fragments mgsA:GP-1, mgsA:GP-6, mgsA:GP-7 as templates. The DNA fragment mgsA:GP-II is 1159 bp in size, which is the splicing product of the three fragments mgsA:GP-1, mgsA:GP-6, mgsA:GP-7, and is a DNA fragment containing the upstream homologous arm of mgsA, the GP-28 promoter, and the upstream 589 bp of the GPD1+GPP2 gene coding region.

[0281] Step 3, construction of the recombinant strain YY-P019

[0282] The fragment mgsA:GP-I was used for the first homologous recombination: first, the pKD46-km plasmid was transformed into the strain YY-P018 by the calcium chloride transformation method, and then the DNA fragment was electroporated into the YY-P018 with the pKD46-km. The specific transformation steps are as follows:

[0283] First, the electrocompetent cells of the recombinant strain YY-P018 with the pKD46-km plasmid were prepared; 50 μl of competent cells were placed on ice, 50 ng of DNA fragment mgsA:GP-I was added, and the mixture was placed on ice for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electroporation cup. The MicroPulser (Bio-Rad) electroporation instrument was used, and the voltage was 2 kv. After electroporation, 1 ml of LB medium was quickly transferred to the electroporation cup, and then the mixture was transferred to a test tube after being blown 5 times, and then incubated at 75 rpm and 30°C for 2 h. 200 μl of bacterial solution was spread on an LB plate containing chloramphenicol (final concentration of 34 μg / ml) and kanamycin (final concentration of 50 μg / ml), and then incubated at 37°C overnight. After incubation, 8 single colonies were selected and verified by PCR using the primers mgsA:GP up arm up (SEQ ID NO: 67) and mgsA:GP down arm down (SEQ ID NO: 70). The correct colony amplified a product of 5617 bp in size, and one correct single colony was selected and named YY-P019.

[0284] Step 4, construction of the recombinant strain YY-P020

[0285] The fragment mgsA:GP-II was used for the second homologous recombination: the DNA fragment mgsA:GP-II was electroporated into the strain YY-P019. The specific transformation steps are as follows:

[0286] Firstly, prepare the electroporation competent cells of Michigan Klebsiella YY-P019 with pKD46-km plasmid; place 50 μl of the competent cells on ice, add 50 ng of DNA fragment mgsA:GP-II, and place on ice for 2 min, and then transfer to a 0.2 cm Bio-Rad electroporation cup. Use a MicroPulser (Bio-Rad) electroporation instrument, and the voltage is 2 kv. After the electric shock, quickly transfer 1 ml of LB medium into the electric shock cup, blow 5 times, and then transfer to a test tube, incubate at 75 rpm and 30°C for 4 h. Transfer the bacterial solution to LB liquid medium containing 10% sucrose without sodium chloride (50 ml medium in a 250 ml flask), and incubate for 24 h. Then, streak culture on LB solid medium containing 6% sucrose without sodium chloride. Select a single colony, and use primers mgsA:GP up arm up (SEQ ID NO: 67) and mgsA:GP two-step down (SEQ ID NO: 78) to perform PCR verification. The correct colony has an amplified product of 1159 bp, and a correct single colony is selected and named YY-P020.

[0287] 2.10 Inactivation of acetyl-CoA synthase encoding gene budB and acetolactate decarboxylase encoding gene budA in YY-P020

[0288] From the recombinant Michigan Klebsiella YY-P020, inactivate the acetyl-CoA synthase encoding gene budB and the acetolactate decarboxylase encoding gene budA by using a two-step homologous recombination method, and the specific steps are as follows:

[0289] Firstly, amplify the basic gene fragment for inactivating the acetyl-CoA synthase encoding gene budB and the acetolactate decarboxylase encoding gene budA in YY-P020.

[0290] Use the PDO-046 genome as a template, and use primers ΔbudBA up arm up (SEQ ID NO: 79) and ΔbudBA up arm down (SEQ ID NO: 80) to perform PCR amplification, to obtain a DNA fragment ΔbudBA-1. The DNA fragment has a size of 586 bp, and is an upstream homologous arm for inactivating budB and budA.

[0291] Use the PDO-046 genome as a template, and use primers ΔbudBA down arm up (SEQ ID NO: 81) and ΔbudBA down arm down (SEQ ID NO: 82) to perform PCR amplification, to obtain a DNA fragment ΔbudBA-2. The DNA fragment has a size of 685 bp, and is a downstream homologous arm for inactivating budB and budA.

[0292] The DNA fragment ΔbudBA-3 was obtained by PCR amplification using primers ΔbudBA cat up (SEQ ID NO: 83) and ΔbudBA sacb down (SEQ ID NO: 84) with pXZ-CS as template. The DNA fragment was 2658 bp in size, which contained chloramphenicol resistance gene (cat), levan sucrose transferase gene (sacB) and 20 bp overlapping region downstream of fragment ΔbudBA-1 and 20 bp overlapping region upstream of ΔbudBA-2.

[0293] The gene fragment ΔbudBA-4 was obtained by PCR amplification using primers ΔbudBA two step up (SEQ ID NO: 85) and ΔbudBA down arm down (SEQ ID NO: 82) with PDO-046 genome as template. The DNA fragment was 705 bp in size, which contained 20 bp overlapping region downstream of ΔbudBA-1 and all of ΔbudBA-2.

[0294] In the second step, the two-step homologous recombination fragment for inactivating the acetyl lactate synthase encoding gene budB and the acetyl lactate decarboxylase encoding gene budA in YY-P020 was constructed.

[0295] The DNA fragment ΔbudBA-I was obtained by PCR amplification using primers ΔbudBA up arm up (SEQ ID NO: 79) and ΔbudBA down arm down (SEQ ID NO: 82) with equimolar mixture of fragments ΔbudBA-1, ΔbudBA-2 and ΔbudBA-3 as template. The DNA fragment was 3889 bp in size, which was the splicing product of ΔbudBA-1, ΔbudBA-2 and ΔbudBA-3, and was the DNA fragment containing chloramphenicol resistance gene (cat) and levan sucrose transferase gene (sacB) and the upstream and downstream homologous arms for inactivating genes budB and budA.

[0296] The DNA fragment ΔbudBA-II was obtained by PCR amplification using primers ΔbudBA up arm up (SEQ ID NO: 79) and ΔbudBA down arm down (SEQ ID NO: 82) with equimolar mixture of fragments ΔbudBA-1 and ΔbudBA-4 as template. The DNA fragment was 1271 bp in size, which was the splicing product of ΔbudBA-1 and ΔbudBA-4, and was the DNA fragment containing the upstream and downstream homologous arms for inactivating genes budB and budA.

[0297] In the third step, the construction of recombinant strain YY-P021

[0298] The fragment ΔbudBA-I was used for the first homologous recombination: First, the pKD46-km plasmid was transformed into strain YY-P020 by calcium chloride transformation method, then the DNA fragment was electroporated into YY-P020 with pKD46-km. The specific transformation steps are as follows:

[0299] First, prepare the electroporation competent cells of Michigan Klebsiella YY-P020 with pKD46-km plasmid; 50 μl of competent cells were placed on ice, 50 ng of DNA fragment ΔbudBA-I was added, and the ice was placed for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electric shock cup. The MicroPulser (Bio-Rad Company) electroporation instrument was used, and the electric shock parameters were voltage 2kv. After electric shock, 1 ml of LB medium was quickly transferred to the electric shock cup, and after 5 times of blowing, it was transferred to a test tube, and incubated at 75 rpm, 30°C for 2h. 200 μl of bacteria were taken and spread on LB plates containing chloramphenicol (final concentration 34 μg / ml) and kanamycin (final concentration 50 μg / ml), and after overnight culture at 37°C, 8 single colonies were selected and verified by PCR using primers ΔbudBA up arm up (SEQ ID NO: 79) and ΔbudBA down arm down (SEQ ID NO: 82). The correct colony amplified a product of 3889 bp in size, and one correct single colony was selected and named YY-P021.

[0300] Fourth, the construction of recombinant bacteria YY-P022

[0301] The fragment ΔbudBA-II was used for the second homologous recombination: The DNA fragment ΔbudBA-II was electroporated into strain YY-P021. The specific transformation steps are as follows:

[0302] Firstly, prepare the electrotransformation competent cells of recombinant Klebsiella michigan YY-P021 with pKD46-km plasmid; place 50 μl of the competent cells on ice, add 50 ng of DNA fragment ΔbudBA-II, and place on ice for 2 min, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporation instrument, and the voltage is 2 kv. After the electric shock, quickly transfer 1 ml of LB medium into the electroporation cuvette, and then transfer into a test tube after 5 times of blowing, and incubate at 75 rpm at 30°C for 4 h. Transfer the bacterial solution into LB liquid medium containing 10% sucrose without sodium chloride (50 ml of medium in a 250 ml flask), and incubate for 24 h, and then streak on LB solid medium containing 6% sucrose without sodium chloride. Select a single colony, and use primers ΔbudBA up arm up (SEQ ID NO: 79) and ΔbudBA down arm down (SEQ ID NO: 82) for PCR verification, and the correct colony has an amplified product of 1271 bp, and select one correct single colony, and name it as YY-P022.

[0303] 2.11 Regulate the expression of 3-phosphoglyceraldehyde dehydrogenase encoding gene GapA in YY-P022 using artificial promoter GapA-42.

[0304] From the recombinant bacteria YY-P0022, regulate the expression of 3-phosphoglyceraldehyde dehydrogenase encoding gene GapA using GapA-42 promoter by one-step homologous recombination, and the specific steps are as follows:

[0305] Firstly, amplify the basic DNA fragment for regulating the expression of GapA using GapA-42 promoter in recombinant bacteria YY-P022.

[0306] Use PDO-046 genome as a template, and use primers GapA-42up arm up (SEQ ID NO: 86) and GapA-42up arm down (SEQ ID NO: 87) for PCR amplification to obtain DNA fragment GapA-42-1. The size of the DNA fragment is 532 bp, which is the upstream homologous arm for regulating the expression of GapA.

[0307] Use PDO-046 genome as a template, and use primers GapA-42down arm up (SEQ ID NO: 88) and GapA-42down arm down (SEQ ID NO: 89) for PCR amplification to obtain DNA fragment GapA-42-2. The size of the DNA fragment is 661 bp, which is the downstream homologous arm for regulating the expression of GapA.

[0308] PCR amplification was performed with the primer GapA-42cat up (SEQ ID NO: 90) and GapA-42sacB down (SEQ ID NO: 91) using pXZ-CS as the template to obtain DNA fragment GapA-42-3. The DNA fragment was 2664 bp in size, which contained chloramphenicol resistance gene (cat), levansucrase gene (sacB), and the fragment downstream of 23 bp of GapA-42-1 and the upstream 23 bp of the promoter GapA-42.

[0309] PCR amplification was performed with the primer GapA-42up (SEQ ID NO: 92) and GapA-42down (SEQ ID NO: 93) using plasmid GapA-42 as the template to obtain gene fragment GapA-42-4. The DNA fragment was 116 in size, which contained the coding sequence of the promoter GapA-42 and the upstream 27 bp of DNA fragment GapA-42-2.

[0310] In the second step, the GapA expression regulated by GapA-42 promoter in recombinant bacteria YY-P022 was constructed by one-step homologous recombination fragment.

[0311] PCR amplification was performed with the primer GapA-42up arm up (SEQ ID NO: 86) and GapA-42down arm down (SEQ ID NO: 89) using an equimolar mixture of fragments GapA-42-1, GapA-42-2, GapA-42-3, and GapA-42-4 as the template to obtain DNA fragment GapA-42-I. The DNA fragment was 3811 bp in size, which was the splicing product of the four fragments GapA-42-1, GapA-42-2, GapA-42-3, and GapA-42-4. It was a DNA fragment containing the upstream homologous arm for GapA expression regulation, chloramphenicol resistance gene (cat), levansucrase gene (sacB), GapA-42 promoter, and downstream homologous arm for GapA expression regulation.

[0312] In the third step, the construction of recombinant bacteria YY-P023

[0313] The fragment GapA-42-I was used for the first homologous recombination: first, the pKD46-km plasmid was transformed into strain YY-P022 by calcium chloride transformation method, and then the DNA fragment was electroporated into YY-P022 with pKD46-km. The specific transformation steps were as follows:

[0314] Firstly, the electrotransformation competent cells of the recombinant bacteria YY-P022 with pKD46-km plasmid were prepared; 50 μl of the competent cells were placed on ice, 50 ng of DNA fragment GapA-42-I was added, and the mixture was placed on ice for 2 min, and then was transferred to a 0.2 cm Bio-Rad electroporation cuvette. The MicroPulser (Bio-Rad) electroporation instrument was used, and the voltage was 2 kv. After the electroporation, 1 ml of LB medium was quickly transferred to the electroporation cuvette, and then was transferred to a test tube after being blown for 5 times, and was incubated at 75 rpm and 30°C for 2 h. 200 μl of the bacterial solution was spread on an LB plate containing chloramphenicol (a final concentration of 34 μg / ml) and kanamycin (a final concentration of 50 μg / ml), and was incubated at 37°C overnight. After the incubation, 8 single colonies were selected, and PCR was performed using primers GapA-42up arm up (SEQ ID NO: 86) and GapA-42down arm down (SEQ ID NO: 89). The size of the correct colony amplification product was 3811 bp, and one correct single colony was selected and was named YY-P023.

[0315] Example 3: Fermentation of the recombinant bacteria YY-P023 in a 5L fermenter to produce 1,3-propanediol

[0316] The fermentation was performed in a 5L fermenter (Shanghai Baohong, BIOTECH-5BG) under micro-aerobic conditions, including the following steps:

[0317] The components of the seed culture medium were 10 g / L tryptone, 10 g / L sodium chloride, and 5 g / L yeast extract.

[0318] The components of the fermentation medium were glucose (170 g / L), yeast extract (1 g / L), NH4H2PO4 (1 g / L), (NH4)2HPO4 (3 g / L), MgSO4·7H2O (1 g / L), and KCl (74 mg / L), FeCl3·6H2O (2.4 mg / L), CoCl2·6H2O (0.3 mg / L), CuCl2·2H2O (0.15 mg / L), ZnCl2 (0.3 mg / L), Na2MoO4·2H2O (0.3 mg / L), H3BO3 (0.1 mg / L), and MnCl2·4H2O (0.5 mg / L). No vitamin B12 was added, and no antibiotic was added.

[0319] (1) Seed culture: the seed culture medium was 150 ml in a 500 ml flask, and was sterilized at 115°C for 15 min. After cooling, the recombinant Klebsiella michiganensis YY-P023 was inoculated into the seed culture medium at an inoculation amount of 1% (V / V), and was cultured at 37°C and 250 rpm for 12 hours to obtain a seed solution, which was used for inoculation of the fermentation medium.

[0320] (2) Fermentation culture: the volume of 5L fermentation medium is 3L, and the sterilization is 115℃ for 25min. The seed liquid is inoculated into the fermentation medium according to the final concentration OD 550 =0.1, and the inoculation amount is 37℃ culture, stirring speed 350rpm, ventilation amount 0.5vvm, and pH control 7.0.

[0321] Fermentation results: after YY-P023 is fermented in a 5L fermentation tank for 72 hours, the yield of 1,3-propanediol reaches 92g / L, and the yield reaches 0.54g / g.

[0322] The strain and plasmid used in the application

[0323]

[0324]

[0325] The primer used in the application

[0326]

[0327]

[0328]

[0329]

[0330]

[0331] The key sequence used in the application

[0332] Yqhd-2 (SEQ ID NO: 94)

[0333] TTATCTCTGGCGGTGTTGACAAGAGATAACAACGTTGATATAATTGAGCCCGTATTGTTAGCATGTACGTTTAAACCAGGAGTATAACG

[0334] Pdu-31 (SEQ ID NO: 95)

[0335] TTATCTCTGGCGGTGTTGACAAGAGATAACAACGTTGATATAATTGAGCCACGCCTGTACAGAACCACGTTTAAACCAGGAGCCCCACG

[0336] GP-28 (SEQ ID NO: 96)

[0337] TTATCTCTGGCGGTGTTGACAAGAGATAACAACGTTGATATAATTGAGCCCATTACATATTCACCACCGTTTAAACCAGGAGTAGAAAC

[0338] GapA-42 (SEQ ID NO: 97)

[0339] TTATCTCTGGCGGTGTTGACAAGAGATAACAACGTTGATATAATTGAGCCCGCGCTGCACTGACACATGTTTAAACCAGGAGCTGCGAC

[0340] GPD1 + GPP2 fusion protein coding sequence (SEQ ID NO: 98)

[0341]

[0342] Cat sacB (SEQ ID NO: 99)

[0343]

[0344] Yqhd protein coding sequence (SEQ ID NO: 100)

[0345]

[0346] pKD46-km plasmid (SEQ ID NO: 101)

[0347] SEQUENCE LISTING <110> TIANJIN INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY, CHINESE ACADEMY OF SCIENCES TIANJIN ZHIHE BIOTECHNOLOGY CO., LTD. <120> RECOMBINANT KLEBSIELLA MICHIGANENSIS FOR PRODUCING 1,3-PROPANEDIOL AND CONSTRUCTION METHOD AND APPLICATION THEREOF <130> I2022TC6758CB <160> 117 <170> PatentIn version 3.5 <210> 1 <211> 1462 <212> DNA <213> Klebsiella michiganensis <400> 1 atggcctgga gctgctgctg gctgctgctg gctgctgctg gctgctgctg gctgctgctg 60 ctcgggtgac gagtggcgga cgggtgagta atgtctggga aactgcctga tggaggggga 120 taactactgg aaacggtagc taataccgca taacgtcgca agaccaaaga gggggacctt 180 cgggcctctt gccatcagat gtgcccagat gggattagct agtaggtggg gtaacggctc 240 acctaggcga cgatccctag ctggtctgag aggatgacca gccacactgg aactgagaca 300 cggtccagac tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga 360 tgcagccatg ccgcgtgtat gaagaaggcc ttcgggttgt aaagtacttt cagcggggag 420 gaaggcgata aggttaataa ccttgtcgat tgacgttacc cgcagaagaa gcaccggcta 480​​​​​​​​​​​​​​​​​​​​ actccgtgcc agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc 540 gtaaagcgca cgcaggcggt ctgtcaagtc ggatgtgaaa tccccgggct caacctggga 600 actgcattcg aaactggcag gctggagtct tgtagagggg ggtagaattc caggtgtagc 660 ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa ggcggccccc tggacaaaga 720 ctgacgctca ggtgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780 ctgtaaacga tgtcgacttg gaggttgttc ccttgaggag tggcttccgg agctaacgcg 840 ttaagtcgac cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa ttgacggggg 900 cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctact 960 cttgacatcc agagaactta gcagagatgc tttggtgcct tcgggaactc tgagacaggt 1020 gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg caacgagcgc 1080 aacccttatc ctttgttgcc agcggtccgg ccgggaactc aaaggagact gccagtgata 1140 aactggagga aggtggggat gacgtcaagt catcatggcc cttacgagta gggctacaca 1200 cgtgctacaa tggcatatac aaagagaagc gacctcgcga gagcaagcgg acctcataaa 1260 gtatgtcgta gtccggattg gagtctgcaa ctcgactcca tgaagtcgga atcgctagta 1320 atcgtggatc agaatgccac ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380 accatgggag tgggttgcaa aagaagtagg tagcttaacc ttcgggaggg cgcttaccac 1440 tttgtgattc atgactgggg tg 1462 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R <400> 3 ggttaccttg ttacgactt 19 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ΔptsG up arm up <400> 4 cggattgcgg tggcgtac 18 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ΔptsG up arm down <400> 5 gttttcacgc attggctcgg g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ΔptsG down arm up <400> 6 ccaaatccga taacctgaaa a 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ΔptsG down arm down <400> 7 ggtaatgcct gagctgacca t 21 <210> 8 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> ΔptsG cat up <400> 8 cccgagccaa tgcgtgaaaa ctgtgacgga agatcacttc gc 42 <210> 9 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> ΔptsG sacB down <400> 9 ttttcaggtt atcggatttg gttatttgtt aactgttaat tgtccttgtt c 51 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> ΔptsG two-step up <400> 10 cccgagccaa tgcgtgaaaa cccaaatccg ataacctgaa aa 42 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd up arm up <400> 11 acgggtaaca aatcgcatca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd up arm down <400> 12 actttgcctt ccgcatagag 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd down arm up <400> 13 aagatgtcca taccgttgcc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd down arm down <400> 14 acggcggtcc aataatacac 20 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd cat up <400> 15 ctctatgcgg aaggcaaagt tgtgacggaa gatcacttcg c 41 <210> 16 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd sacB down <400> 16 cttgtcaaca ccgccagaga taattatttg ttaactgtta attgtccttg ttc 53 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd yq2 up <400> 17 ttatctctgg cggtgttgac aag 23 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd yq2 down <400> 18 cgttatactc ctggtttaaa cgtacat 27 <210> 19 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Edd:yqhd yqhd up <400> 19 atgtacgttt aaaccaggag tataacgatg aacaacttta atctgcacac cc 52 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Edd::yqhd yqhd down <400> 20​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<223> Edd::yqhd two-step down <400> 22 cctgcttgtc gcctgtggtt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Delta glpK up arm up <400> 23 gcgtcgaaag tctcgagctg 20 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Delta glpK up arm down <400> 24 agtatgagtc ccgtagtcag attacag 27 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Delta glpK down arm up <400> 25 gtttgttgag gtattcccgg c 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Delta glpK down arm down <400> 26 cgtcagtctg tcgctgttcc t 21 <210> 27 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> ΔglpK cat up <400> 27 ctgtaatctg actacgggac tcatacttgt gacggaagat cacttcgc <210> 28 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> ΔglpK sacB down <400> 28 gccgggata cctcaacaaa cttatttgtt aactgttaat tgtccttgtt c <210> 29 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> ΔglpK one up <400> 29 ctgtaatctg actacgggac tcatactgtt tgttgaggta ttcccggc <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA1 up arm up <400> 30 ggaatgga ccgaacaggc <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA1 up arm down <400> 31 tttatcgtgg tccagcccc 19 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA1 down arm up <400> 32 tccgttcacc ctcaaataag tg 22 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA1 down arm down <400> 33 cggcctctct gctgatgg 18 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA1 cat up <400> 34 ggggctggac cacgataaat gtgacggaag atcacttcgc 40 <210> 35 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA1 sacB down <400> 35 cacttatttg agggtgaacg gattatttgt taactgttaa ttgtccttgt tc 52 <210> 36 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA1 two-step up <400> 36 ggggctggac cacgataaat ccgttcaccc tcaaataagt g 41 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220><{0001044}><223> ΔgldA2 up arm up <400> 37 [[ID=3{2}]]ggtaccgtta actcaccgag ttc 23 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence [[ID=4{2}]]<220> <223> ΔgldA2 up arm down <400> 38 gcttcctgca agagtgggaa taa 23 <2{1}0> 39<00{0}1056><211> 22 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA2 down arm up <400> 39 ttctattcct cctggatgac cg 22 <210> 40 <211> 21 Note: There seems to be some inconsistent formatting in the original text which might cause some confusion. I've tried to translate it as accurately as possible while maintaining the original structure. The parts like etc. are likely some kind of identifiers in a specific system and are kept as they are without further translation as per the instructions. If you have any further clarification about the text, it would be helpful for future translations.<212> DNA <213> Artificial Sequence <220> <223> ΔgldA2 down arm down <400> 40 ctggcacgta tctatccgct g 21 <210> 41 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA2 cat up <400> 41 ttattcccac tcttgcagga agctgtgacg gaagatcact tcgc 44 <210> 42 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA2 sacB down <400> 42 cggtcatcca ggaggaatag aattatttgt taactgttaa ttgtccttgt tc 52 <210> 43 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ΔgldA2 two-step up <400> 43 ttattcccac tcttgcagga agcttctatt cctcctggat gaccg 45 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔfrdABCD up arm up <400> 44 ccgcgagtcc atagaagacc 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔfrdABCD up arm down <400> 45 gtacccacgg tctggttcag 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔfrdABCD down arm up <400> 46 gcagcttttt ctcgcccaaa 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔfrdABCD down arm down <400> 47 ccggctttca tatgctgcac 20 <210> 48 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> ΔfrdABCD cat up <400> 48 ctgaaccaga ccgtgggtac tgtgacggaa gatcacttcg c 41 <210> 49 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> ΔfrdABCD sacb down <400> 49 tttgggcgag aaaaagctgc ttatttgtta actgttaatt gtccttgttc 50 <210> 50 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ΔfrdABCD two step up <400> 50 ctgaaccaga ccgtgggtac gcagcttttt ctcgcccaaa 40 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔadhE up arm up <400> 51 aatccggcat gggtatcgtc 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔadhE up arm down <400> 52 atgacaaccg gggtgttacc 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔadhE down arm up <400> 53 gtttccgtac tggcttccga 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔadhE down arm down <400> 54 ttctcttcca caccgccttc 20 <210> 55 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> ΔadhE cat up <400> 55 ggtaacaccc cggttgtcat tgtgacggaa gatcacttcg c 41 <210> 56 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> ΔadhE sacb down <400> 56 tcggaagcca gtacggaaac ttatttgtta actgttaatt gtccttgttc 50 <210> 57 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ΔadhE two-step up <400> 57 ggtaacaccc cggttgtcat gtttccgtac tggcttccga 40 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 up arm up <400> 58 agccataacg ccacggtcac 20 <210> 59 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 up arm down <400> 59 tgtttatgac ggattaaatt ttatctgc 28 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 down arm up <400> 60 atgcaacaag aagcactagg aatg 24 <210> 61 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 down arm down <400> 61 ccgagaatgc cgatggagc 19 <210> 62 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 cat up <400> 62 gcagataaaa tttaatccgt cataaacatg tgacggaaga tcacttcgc 49 <210> 63 <211 > 53 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 sacB down <400> 63 cttgtcaaca ccgccagaga taattatttg ttaactgtta attgtccttg ttc 53 <210> 64 <211 > 23 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 up <400> 64 ttatctctgg cggtgttgac aag 23 <210> 65 <211 > 44 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 down <400> 65 cattcctagt gcttcttgtt gcatcgtggg gctcctggtt taaa 44 <210> 66 <211 > 51 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 two-step up <400> 66 gcagataaaa tttaatccgt cataaacatt atctctggcg gtgttgacaa g 51 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP up arm up <400> 67 cgcctgttcg ttgattaccc 20 <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP up arm down <400> 68 catttcattc cacattttgg cc 22 <210> 69 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP down arm up <400> 69 gaccctgttg atatcctgat ccc 23 <210> 70 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP down arm down <400> 70 aatgagcggt tatgtcaaac gag 23 <210> 71 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP cat up <400> 71 ggccaaaatg tggaatgaaa tgtgtgacgg aagatcactt cgc 43 <210> 72 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP sacB down <400> 72 cttgtcaaca ccgccagaga taattatttg ttaactgtta attgtccttg ttc 53 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP GP28 up <400> 73 ttatctctgg cggtgttgac aag 23 <210> 74 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP GP28 down <400> 74 gtttctactc ctggtttaaa cggtg 25 <210> 75 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP GP up <400> 75 caccgtttaa accaggagta gaaacatgag cgcggcggcg gatcg 45 <210> 76 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP GP down <400> 76 gggatcagga tatcaacagg gtcttaccat ttcagcaggt catccttc 48 <210> 77 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP two-step up <400> 77 ggccaaaatg tggaatgaaa tgttatctct ggcggtgttg acaag 45 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mgsA:GP two-step down <400> 78 tggtttcgct ccagtgctcc 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔbudBA up arm up <400> 79 aaaagcgtcg gagctggtaa 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔbudBA up arm down <400> 80 atcaccgtgg gtgagcaaat 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔbudBA down arm up <400> 81 tcgagctggt cggcaatatc 20 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ΔbudBA down arm down <400> 82 ttatcgcggt aatccacggg 20 <210> 83 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> ΔbudBA cat up <400> 83 atttgctcac ccacggtgat tgtgacggaa gatcacttcg c 41 <210> 84 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> ΔbudBA sacb down <400> 84 gatattgccg accagctcga ttatttgtta actgttaatt gtccttgttc 50 <210> 85 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ΔbudBA two step up <400> 85 atttgctcac ccacggtgat tcgagctggt cggcaatatc 40 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 up arm up <400> 86 ggaatacgtg cccaaggtga 20 <210> 87 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 up arm down <400> 87 tcgatttatc ccactttccc ttt 23 <210> 88 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 down arm up <400> 88 atgactatca aagtaggtat caacggt 27 <210> 89 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 down arm down <400> 89 ctggcaggac tttacctaca gct 23 <210> 90 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 cat up <400> 90 aaagggaaag tgggataaat cgatgtgacg gaagatcact tcgc 44 <210> 91 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 sacB down <400> 91 cttgtcaaca ccgccagaga taattatttg ttaactgtta attgtccttg ttc 53 <210> 92 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 up <400> 92 ttatctctgg cggtgttgac aag 23 <210> 93 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 down <400> 93 accgttgata cctactttga tagtcatgtc gcagctcctg gtttaaac 48 <210> 94 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Yqhd-2 <400> 94 ttatctctgg cggtgttgac aagagataac aacgttgata taattgagcc cgtattgtta 60 gcatgtacgt ttaaaccagg agtataacg 89 <210> 95 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Pdu-31 <400> 95 ttatctctgg cggtgttgac aagagataac aacgttgata taattgagcc acgcctgtac 60 agaaccacgt ttaaaccagg agccccacg 89 <210> 96 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> GP-28 <400> 96 ttatctctgg cggtgttgac aagagataac aacgttgata taattgagcc cattacatat 60 tcaccaccgt ttaaaccagg agtagaaac 89 <210> 97 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> GapA-42 <400> 97 ttatctctgg cggtgttgac aagagataac aacgttgata taattgagcc cgcgctgcac 60 tgacacatgt ttaaaccagg agctgcgac 89 <210> 98 <211> 1941 <212> DNA <213> Artificial Sequence <220> <223> GPD1+GPP2 <400> 98 atgagcgcgg cggcggatcg tctgaatctg accagcggcc atctgaatgc gggtcgtaag 60 cgtagcagca gcagcgttag cctgaaggcg gcggagaagc cgttcaaagt taccgtgatt 120 ggtagcggca actggggcac caccatcgcg aaggtggttg cggagaactg caaaggttac 180 ccggaagttt tcgcgccgat tgttcagatg tgggtgtttg aggaagagat caacggtgaa 240 aagctgaccg agatcattaa cacccgtcac caaaacgtta aatatctgcc gggtattacc 300 ctgccggaca acctggtggc gaacccggac ctgatcgata gcgttaagga cgtggatatc 360 attgtgttca acattccgca ccagtttctg ccgcgtatct gcagccaact gaaaggccac 420 gttgatagcc acgtgcgtgc gatcagctgc ctgaagggtt tcgaggttgg cgcgaaaggt 480 gtgcagctgc tgagcagcta cattaccgaa gagctgggta tccaatgcgg tgcgctgagc 540 ggtgcgaaca ttgcgaccga agttgcgcag gagcactgga gcgaaaccac cgtggcgtac 600 cacatcccga aggactttcg tggtgagggc aaagacgttg atcacaaggt gctgaaagcg 660 ctgttccacc gtccgtattt tcacgttagc gtgatcgaag atgttgcggg cattagcatc 720 tgcggtgcgc tgaaaaacgt ggttgcgctg ggttgcggct tcgttgaggg tctgggttgg 780 ggtaacaacg cgagcgcggc gattcagcgt gtgggcctgg gtgaaatcat tcgttttggt 840 caaatgttct ttccggagag ccgtgaagaa acctactatc aagaaagcgc gggcgtggcg 900 gacctgatca ccacctgcgc gggtggccgt aacgttaagg tggcgcgtct gatggcgacc 960 agcggcaagg atgcgtggga atgcgagaaa gaactgctga acggccagag cgcgcaaggt 1020 ctgatcacct gcaaagaggt tcacgaatgg ctggaaacct gcggtagcgt ggaggacttc 1080 ccgctgtttg aagcggttta ccagattgtg tacaacaact atccgatgaa gaacctgccg 1140 gatatgatcg aagagctgga cctgcacgag gatggtggcg gtggcagcat gggtctgacc 1200 accaagccgc tgagcctgaa agttaacgcg gcgctgttcg acgtggatgg caccatcatt 1260 attagccagc cggcgattgc ggcgttctgg cgtgactttg gcaaggataa accgtatttc 1320 gacgcggaac acgttattca ggtgagccac ggctggcgta cctttgacgc gatcgcgaag 1380 ttcgcgccgg attttgcgaa cgaagagtac gttaacaaac tggaggcgga aattccggtg 1440 aagtatggcg agaaaagcat cgaagttccg ggtgcggtga aactgtgcaa cgcgctgaac 1500 gcgctgccga aggagaaatg ggcggttgcg accagcggca cccgtgacat ggcgcagaag 1560 tggttcgaac acctgggtat tcgtcgtccg aaatacttta tcaccgcgaa cgatgtgaag 1620 caaggcaaac cgcacccgga gccgtacctg aagggtcgta acggcctggg ttatccgatt 1680 aacgagcagg acccgagcaa gagcaaagtg gttgtgtttg aggatgcgcc ggcgggtatt 1740 gcggcgggca aggcggcggg ttgcaaaatt atcggcatcg cgaccacctt cgacctggat 1800 tttctgaagg aaaaaggttg cgacattatc gttaagaacc acgagagcat ccgtgtgggt 1860 ggctataacg cggaaaccga cgaagttgag tttatctttg acgactatct gtatgcgaag 1920 gatgacctgc tgaaatggta a 1941 <210> 99 <211> 2618 <212> DNA <213> Artificial Sequence <220> <223> Cat sacB <400> 99 tgtgacggaa gatcacttcg cagaataaat aaatcctggt gtccctgttg ataccgggaa 60 gccctgggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag gttccaactt 120 tcaccataat gaaataagat cactaccggg cgtatttttt gagttatcga gattttcagg 180 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca 240 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca 300 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt 360 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat 420 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt 480 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca 540 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc 600 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag 660 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa 720 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt 780 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg 840 gcagggcggg gcgtaatttt tttaaggcag ttatggtgc ccttaaacgc ctggtgctac 900 gcctgaataa gtgataataa gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg 960 tcgtcggttc agggcagggt cgttaaatag ccgcgagctc aagtaaatcg cgcgggtttg 1020 ttactgataa agcaggcaag acctaaaatg tgtaaagggc aaagtgtata ctttggcgtc 1080 accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca tcttcaaaca 1140 ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca tgaacgatga 1200 acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca ctgctggcag 1260 gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag gaaacatacg 1320 gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa aaaaatgaaa 1380 aatatcaagt tcctgaattc gattcgtcca caattaaaaa tatctcttct gcaaaaggcc 1440 tggacgtttg ggacagctgg ccattacaaa acgctgacgg cactgtcgca aactatcacg 1500 gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac acatcgattt 1560 acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac gctggccgcg 1620 tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac caaacacaag 1680 aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc tacactgatt 1740 tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac gtatcagcat 1800 cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt gacggtgacg 1860 gaaaaacgta tcaaaatgta cagcagttca tcgatgaagg caactacagc tcaggcgaca 1920 accatacgct gagagatcct cactacgtag aagataaagg ccacaaatac ttagtatttg 1980 aagcaaacac tggaactgaa gatggctacc aaggcgaaga atctttattt aacaaagcat 2040 actatggcaa aagcacatca ttcttccgtc aagaaagtca aaaacttctg caaagcgata 2100 aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat gattgagcta aacgatgatt 2160 acacactgaa aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca gatgaaattg 2220 aacgcgcgaa cgtctttaaa atgaacggca aatggtacct gttcactgac tcccgcggat 2280 caaaaatgac gattgacggc attacgtcta acgatattta catgcttggt tatgtttcta 2340 attctttaac tggcccatac aagccgctga acaaaactgg ccttgtgtta aaaatggatc 2400 ttgatcctaa cgatgtaacc tttacttact cacacttcgc tgtacctcaa gcgaaaggaa 2460 acaatgtcgt gattacaagc tatatgacaa acagaggatt ctacgcagac aaacaatcaa 2520 cgtttgcgcc aagcttcctg ctgaacatca aaggcaagaa aacatctgtt gtcaaagaca 2580 gcatccttga acaaggacaa ttaacagtta acaaataa 2618 <210> 100 <211> 1164 <212> DNA <213> Artificial Sequence <220> <223> Yqhd protein coding sequence <400> 100 atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60 ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120 gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180 gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240 gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300 accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360 caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420 gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480 caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540 tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600 gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660 ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720 cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780 ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840 cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900 cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960 gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020 acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080 gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140 cgtatatacg aagccgcccg ctaa 1164 <210> 101 <211> 6263 <212> DNA <213> Artificial Sequence <220> <223> pKD46‑km <400> 101 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac 60 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat 120 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca 180 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct 240 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga 300 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat 360 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct 420 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 480 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg 540 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 600 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc 660 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca 720 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 780 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 840 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac 900 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg 960 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt 1020 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca 1080 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat 1140 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 1200 acccgttttt ttgggaattc gagctctaag gaggttataa aaaatggata ttaatactga 1260 aactgagatc aagcaaaagc attcactaac cccctttcct gttttcctaa tcagcccggc 1320 atttcgcggg cgatattttc acagctattt caggagttca gccatgaacg cttattacat 1380 tcaggatcgt cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga 1440 gaaagaggca gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc 1500 gctatgcatc gatcatttgc aacgccacgg ggccagcaaa aaatccatta cccgtgcgtt 1560 tgatgacgat gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac 1620 cattgctcac caccaggttg atattgattc agaggtataa aacgaatgag tactgcactc 1680 gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg attctgtcga cccacaggaa 1740 ctgatcacca ctcttcgcca gacggcattt aaaggtgatg ccagcgatgc gcagttcatc 1800 gcattactga tcgttgccaa ccagtacggc cttaatccgt ggacgaaaga aatttacgcc 1860 tttcctgata agcagaatgg catcgttccg gtggtgggcg ttgatggctg gtcccgcatc 1920 atcaatgaaa accagcagtt tgatggcatg gactttgagc aggacaatga atcctgtaca 1980 TGCCGGATTA CC GCAAGGACC GTAATCATCC GATCTGC GTTACC GAA TGGATGGATGAA 2040 TGCCGCCGCG AACC ATTC A A AACTCGCGAAGGCAGAGAAATCACGGGGCCGTGGCAGTCG 2100 CATCCCAAAC GGATGTTACG TCATAAAGCC ATGATTCAGT GTGCCCCTCT GGCCTTCGGA 2160 TTTGCTGGTA TCTATGACAA GGATG AAGCC GAGCGCATTGTCGAAAATAC TGC AT AC ACT 2220 GCAGA ACGTC AGCCGGAACG CGAC ATC ACTCCGGTT AACG ATGAAACCAT GCAGGAGATT 2280 AACACTCTGCT GATCGCCCTG GATAAAACAT GGGATGACG ACTTATTGCC GCTCTGTTCC 2340 CAGATATTT CGCCGCGAC ATTCGTGCATCG TCAGA ACTGAC AGGCCC GAAGCAGTAAA 2400 GCTCTTGGAT TCCTGAAACA GAAAGCCGCA GAGCAGAAGGTGGCAGCATG ACACCGGACA 2460 TTATCCTGCA GC GTACCGGATCGATGTGAGAGCTGTCGAACAGGGGGAT GATGC GTGGC 2520 ACAAATTACG GCTCGGCGTC ATCACCGCTT CAGAAGTTCA CAACGTGATAGCAA AACCCC 2580 GCTCCGGAAG AAGTGGCCT GACATGAAAA TGTCTACTT CCACACCCTG CTTGCTGAGG 2640 TTTGCACCGG TGGCTCCGGA AGT TAACGCTA AAGC ACTGGCCTGGG SAAACAGTACG 2700 agaacgacgc cagaaccctg tttgaattca cttccggcgt gaatgttact gaatccccga 2760 tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc cgatggttta tgcagtgacg 2820 gcaacggcct tgaactgaaa tgcccgttta cctcccgggga tttcatgaag ttccggctcg 2880 gtggtttcga ggccataaag tcagcttaca tggcccaggt gcagtacagc atgtgggtga 2940 cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg tatgaagcgt gaaggcctgc 3000 attatgcgt gattgagcgg gatgaaaagt acatggcgag ttttgacgag atcgtgccgg 3060 agttcatcga aaaaatggac gaggcactgg ctgaaattgg ttttgtattt ggggagcaat 3120 ggcgatgacg catcctcacg atatatccg ggtaggcgca atcactttcg tctactccgt 3180 tacaaagcga ggctgggtat ttcccggcct ttctgttatc cgaaatccac tgaaagcaca 3240 gcggctggct gaggagataa ataataaacg aggggctgta tgcacaaagc atcttctgtt 3300 gagttaagaa cgagtatcga gatggcacat agccttgctc aaattggaat caggtttgtg 3360 ccaataccag tagaaacaga cgaagaatcc atgggtatgg acagttttcc ctttgatatg 3420 taacggtgaa cagttgttct acttttgttt gttagtctlg atgcttcact gatagataca 3480 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg 3540 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact 3600 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt 3660 ttttcttagt ccgttacgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc 3720 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc 3780 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt 3840 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa 3900 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat 3960 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat 4020 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa 4080 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa 4140 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc 4200 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct 4260 actgatgttc atcatctgag cgtattggtt ataagtgaac gataccgtcc gttctttcct 4320 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc 4380 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc 4440 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag 4500 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac 4560 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg 4620 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa 4680 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta 4740 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa 4800 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata 4860 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc 4920 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg 4980 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt 5040 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc 5100 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa 5160 tgcacccagt aaggcagcgg tatcatcaac ggggtctgac gctcagtgga acgaaaactc 5220 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5280 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtca 5340 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc 5400 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt 5460 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc 5520 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac 5580 GAGATCCTCG CCgtcGGGCA TGCgcgcctt GAGCCTGGCG AACAGttcGG CTGGCGCGAG 5640 CCCCTGATGC TCTTCgtcca GATCATCCTG ATCGACaAGA CCggcTTcca TCCGAgtacG 5700 TGCTCGCTCG ATGcgatgtT TCgctTGGTG GTCGaatGGG CAGGTAGccG GATCAAGCGT 5760 ATGCAGCCGC CGATTGcatC AGCCATGAT GGATACTTTc TCGGCAGGAG CAAGGTGAGA 5820 TGACAGGAGA TCCTGCCCCG GCACttcGCC CAATAGCAGC CAGTCCCTTC CCGCTTCAGT 5880 GACAACgtcg AGCACAGCTG CGCAAGGAAC GCCCgtcGTG GCCAGCCACG ATAGCCGCgc 5940 TGCCTCgtcc TGCAGTTcat TCAGGGCACC GGACAGGTcg GTCTTGAcaa AAGAACCggG 6000 GCGCCCCTGC GCTGACAGCC GGAACACGGC GGcatCAGAG CAGCCGATTG TCTgttgtgc 6060 CCAGTcatAG CCgaatagcc TCTCCACCAa GCggccggaG AACCTGCgtg CAATCCATc 6120 TTGTTcaatC ATACTCTTcc TTTTTcaata TTATTgaagc ATTTATcagg GTTATTGTCT 6180 CATGAGCGGA tacATATTtg AATGTATTTa GAAAAATAAA CAAATAGGGG TTCCGCgcAC 6240 ATTTCCCCGA AAAGTGCCAC CTG 6263 <210> 102 <211> 1434 <212> DNA <213> Artificial Sequence <220> <223> ptsG <400> 102 atgtttaaga atgcatttgc taacctgcag aaggtcggta aatcgctgat gctgccggta 60 tccgtactgc ctatcgcagg tatcctgctg ggcgtcggtt ccgcaaactt cagctggctg 120 ccagccgtag tttcccacgt catggcggaa gcgggcggtt cggtcttcgc taacatgccg 180 ctgatctttg ctatcggtgt cgcacttggc ttcaccaaca acgacggcgt atccgctctg 240 gcatcggtcg tcgcttacgg catcatggta aaaaccatgt ccgtggttgc acctctggtc 300 ctgcatttac ctgctgaaga gattgcggct aaacacctgg cggataccgg cgtactcggc 360 ggtattatct ccggtgccat cgcagcgtac atgttcaacc gcttctaccg catcaagctg 420 cctgagtatc tgggcttctt tgcgggcaag cgttttgtgc caatcatctc cggcctggca 480 gcgatcttca ctggcgtgat cctgtccttt atctggccgc cgatcggtac cgcaatccag 540 actttctccc agtgggctgc ttaccagaac ccggttgtgg cgttcggtat ctacggcttc 600 attgaacgct gcctggtgcc gtttggtctg caccacatct ggaacgttcc tttccagatg 660 cagattggtg aatacaccaa cgcagccggt caggtcttcc acggcgatat tccgcgctac 720 atggcaggcg acccgaccgc gggcaaactg tccggcggct tcctgttcaa aatgtacggt 780 ctgccggccg ctgctatcgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg 840 ggcggtatca tgatctccgc agcgctgacc tcgttcctga ccggtatcac cgagccgatc 900 gagttctcct tcatgttcgt tgcgccgatc ctgtacatta tccatgcgat tctggcaggc 960 ctggccttcc cgatctgtat cctgctgggt atgcgtgacg gtacttcgtt ctctcacggt 1020 ctgatcgact ttatcgttct gtccggcaac agcagcaaac tgtggctgtt cccgatagtc 1080 ggcatctgct atgcgatcgt ttactacgtg gtgttccgcg tgctgatcaa agcgctggat 1140 ctgaaaaccc cgggtcgtga agatgcaacc gaagacagca aagctggcgc taccagcgaa 1200 atggccccgg cactgattgc cgctttcggc ggtaaagaga acattactaa ccttgacgca 1260 tgtatcaccc gtctgcgcgt cagcgtggcg gatgtggcga aagttgatca ggctggcctg 1320 aaaaaactgg gtgccgcagg cgtggttgtt gcaggttcag gcgttcaggc tattttcggt 1380 accaaatccg ataacctgaa aactgaaatg gatgaataca tccgcagcaa ctaa 1434 <210> 103 <211> 1512 <212> DNA <213> Artificial Sequence <220> <223> glpK <400> 103 atgaccgaca aaaaatatat cgtagcgctc gaccagggca ccaccagctc ccgcgccgtg 60 gtaatggatc acgatgccaa tatcgtcagc gtctctcagc gagaattcga acaaatttat 120 ccgcgcccag gctgggttga acacgacccg atggagatct gggcatcgca aagctctacg 180 ctggtcgaag tgctggcaaa agccgatatt agctccgatg aaatcgcggc gatcggtatc 240 accaaccagc gtgaaacagc catcgtgtgg gagcgtgaaa ccggcaagcc gatttataac 300 gctatcgtct ggcagtgccg ccgtaccgcc gaaatctgcg agcagctcaa gcgcgacggc 360 atggaagagt acatccgcaa ggccaccggc ctggtggttg acccctactt ctccggcacc 420 aaagtgaagt ggatccttga ccacgtcgaa ggcgcgcgcg agcgcgctaa acgcggcgag 480 CTGCTGTTTC GGCACCGTAG ATACCTGGCT TATCTGGAAG AATGACCCAG GGACGCCTTC AC 540 GTGACCgACT ACACC A ACGC CTCGC GTACC ATGCT GTTCA ATATCCACG A GCTGGACTGG 600 GACGACAAAA TGC TC GACGC GCTGGAT ATCCCGC GC CCA TGC TGC C GGA AGTGC GCAAA 660 TCTTCCG AAGTGTACGGC CAGACCAAT ATCGGC GGT AAGGGC GGGACGC GTATTCCGATC 720 GCCGGTATCGCCGGCGACCAGCAGGCTGC GCTGTT CGGC CAGCTGTGC GTTAAAGAAGGA 780 ATGGCGAAAA ACACCTACGGTACCGGCTGC TTTATGCTGATGAATACC GGC GAGAAAGCG 840 GTGACGTCAACGCACGGCCTGCTGACCACCATCGCCTGC GGTCCGC GC GGTAGGTGAAC 900 TATGCGCTGGAAGGC GCGGT GTTTATGGCGGGC GCCTCCATTCAGTGGCTGC GCGATGAG 960 ATGAAGCTGATTAGCGACGC GTTCGATTC C G AATATTC G CCAC C A AAGT GAAGGATACC 1020 AACGGC GTGTACGTC GTTCCGGCCTTTACCGGCCTC GGC GCGCCGTACTGGACCCGTAC 1080 GCCC GC GGC GCGATCTTCGGCCTGACCCGC GGC GTAAACT CCAACCACATTATT CGC GCC 1140 ACGCTGGAGTCGATTGCCTATCAGACCCGC GACGTGCTGGAAGCGATGCAGGCTGACTCG 1200 gggatccgac tgcacgcctt gcgcgttgat ggcggagcgg tcgccaacaa cttcctgatg 1260 cagttccagt ccgatattct cggcacccgc gtggagcgcc cggaagtccg tgaagtcacg 1320 gcgctgggcg cagcctatct ggccggtctg gcggtcggtt tctggcagaa tctggatgag 1380 ctgcaggaga aagccgttat cgagcgcgag ttccgtccgg gaattgaaac caccgagcgt 1440 aactatcgct acagcggctg gaagaaagcg gtaaaacgcg cgctggcgtg ggaagagcac 1500 gacgaagcgt aa 1512 <210> 104 <211> 1098 <212> DNA <213> Artificial Sequence <220> <223> gldA1 <400> 104 atgctaaaag ttattcaatc tccagccaaa tatctccagg gaccagatgc atccacgctg 60 tttgggcaat acgccaaaaa cctggcggac agctttttcg tgattgccga tgacttcgta 120 atgaagctgg cgggagagaa agtgctggaa ggtttgcaca gccacaacat tagctgccat 180 gcggagcgtt tcaacggcga gtgcagccac gttgagatca atcgcctgat cgccattctg 240 aagcagcacg gctgccgcgg cgtggtgggt atcggcggcg gtaagacgct cgatacggcg 300 aaagccatcg gctactacca gaagctgccg gtggtggtga tcccgacgat cgcctccacc 360 gatgcgccaa ccagcgcgct gtcggttatc tacaccgaag ccggtgagtt tgaagagtat 420 ctgatctacc cgaaaaaccc ggatatggtg gtgatggata cggcgattat cgccaaagcg 480 ccggtgcgcc tgctggtggc cgggatgggg gatgcgctgt caacctggtt tgaagccaag 540 gcctgctacg acgccagagc caccagcatg gcgggcggcc agtcgacggc ggcggcgctg 600 agcctggcgc acctgtgcta tgatacgctg ctggcggaag gcgaaaaagc ccgcctcgcc 660 gcccaggccg gggtggtgac cgatgcgctg gaacgcatcg ttgaagcgaa tacctacctc 720 agcggtatcg gctttgaaag tagcggtctg gccgccgcgc acgccattca caacggtttc 780 accatcctcg aagagtgtca ccatctgtac catggcgaga aagtggcctt tggtaccctg 840 gcgcagctgg tgctgcaaaa tagcccgatg gaagagatcg aaacggtgct gaacttctgc 900 cataccgtcg gcctgccggt cacgctggcg caaatgggcg ttaaagaggg catcgatgag 960 AAAATTCAGG CAGTCGCTAA AGCGACCGTG CGGAAGGTG AAACCATTCA TAAATATGC C 1020 TTCCCGGTCA GCgcgcagag CGCGCATGCG GCGATTCTGA CCGCTGACCT GCTCGGACAG 1080 CAGTGGCTGG CGCGCTAA 1098 <210> 105 <211> 1104 <212> DNA <213> Artificial Sequence <220> <223> gldA2 <400> 105 ATGGATCGCA TTATTCAATC ACCCGGTAAG TATATTCAGG GCgcggacgc AATGACTCGC 60 CTTGGCGGCT ACCTCAAACC GCTGGCTGAA CGCTGGCTCA TCgttggcga TAAATTTGTC 120 CTCGGTtttG CCgaagaaaa ACTGCGtaaa AGCCTGGCGG ATGCTGGGCT GGTcGGCGAA 180 ATCGCTCCAT TTGGCGGTGA ATGCTCACAC AATGAAATCA ATCGCTTGCG TGGTATCGCC 240 GATTCTGCCC AATGCACAGC CGTGCTCGGT ATCGGCGGCG GCAAGACGCT GGATAccgCA 300 AAAGCATTAG CACATTttAT GAATCTTCCG GTGttATTGC GCcgacgatt GCCTCTACC 360 GATGCgcCGT GCAGCgcACT CTCGgtCATC TATTCCGATG ATGGCGAGTT TGATAGCTAT 420 ctgatgctgc cgcgcaaccc gaacatggtg attgttgaca cgcaaatcgt tgctggggcc 480 ccggcacgcc tgctggcagc cggtatcggc gatgcgctgg cgacctggtt tgaagcgcgt 540 gcctgctccc gcagcggcgc gaccaccatg gcgggcggta aatgcaccca ggcggcgctg 600 gcgctggctg aactgtgcta caacacgctg attgaagagg gtgaaaaagc gatgctggcg 660 gcagagcagc acgttgtgac gccggcgctg gaacgggtga ttgaagccaa cacctacctg 720 agcggcgtcg ggtttgaaag cggcggcctg gcggctgcgc acgctatcca caacggcctg 780 accgcgattc cggatgccca tcattactac catggtgaaa aagtcgcctt cggcacgctc 840 acgcagctgg ttctggaaaa cgcgccggtg gaagagattg aaacggtggc agcgctgtgc 900 catagcgtcg ggctgccgat caccctggcg cagctggata ttaaagccga tatcccgggc 960 aaaatgcgta ccgtggcgga agccgcctgc gcggaaggtg aaaccatcca caatatgccc 1020 ggcggcgcga cgccggatca ggtctatgcg gcgcttctgg tggccgacca gtacggccag 1080 cgcttcctgc aagagtggga ataa 1104 <210> 106 <211> 2676 <212> DNA <213> Artificial Sequence <220> <223> adhE <400> 106 atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgcgtaaa aaaagcccag 60 cgtgaatatg ccagtttcac tcaagaacaa gtcgacaaaa tcttccgcgc cgccgctctg 120 gccgctgctg atgctcgaat ccctctcgcg aagatggccg ttgccgaatc cggcatgggt 180 atcgtcgaag ataaagtgat caaaaaccac tttgcttctg aatacatcta taacgcctat 240 aaagatgaaa agacctgcgg tgtcctgtct gaagatgaca ccttcggtac gatcaccatc 300 gctgaaccga ttggtattat ctgcggtatc gtcccaacca ctaacccgac ttcaactgcg 360 atctttaaat ctctgatcag cctgaagacg cgtaacgcca tcatcttctc tccgcatccg 420 cgtgctaaag atgcaaccaa taaagctgct gacatcgtcc tgcaggctgc gatcgctgcc 480 ggtgcgccga aagatctgat tggttggatt gaccagcctt ccgtagaact gtcgaacgcg 540 ctgatgcacc acccggatat caacctgatc ctcgcgaccg gcggtccagg catggttaaa 600 gcggcgtaca gctccggtaa acctgccatc ggcgttggcg cgggtaacac cccggttgtc 660 atcgacgaaa cggctgatat caagcgtgct gtcgcttccg tactgatgtc aaaaaccttc 720 gataacggcg ttatttgtgc ttctgagcag tccgttgtgg ttgttgattc cgtatacgat 780 gccgttcgcg aacgtttctc cagccacggc ggctatctgc tgcagggcca ggaactgaag 840 gctgtgcaga acatcatcct gaaaaacggc gcgctgaacg ccgcgatcgt tggtcaacca 900 gcgtataaaa tcgctgagct ggctggcttc accgtccctg tttccaccaa aattctgatt 960 ggtgaagtta ccgacgttga tgaaagcgaa ccgtttgccc acgaaaaact gtctccgacg 1020 ctggcgatgt atcgtgcgaa gaatttcgaa gacgcagttg ataaagctga aaaactggta 1080 gcaatgggcg gcatcggcca cacctcctgc ctgtataccg accaggacaa ccagccagaa 1140 cgcgttgctt acttcggcca gttgatgaaa accgcgcgta tcctgatcaa caccccggct 1200 tctcagggtg gtatcggtga cctgtataac ttcaaactcg caccttccct gactctgggt 1260 tgtggttcat ggggtggtaa ctccatctcg gaaaacgttg gtcctaagca cctgatcaac 1320 aagaaaaccg ttgctaagcg agctgaaaac atgttgtggc acaaacttcc gaaatctatc 1380 tacttccgtc gtggctcact gccaatcgcg ctggatgaag tgattactga tggtcacaaa 1440 cgcgcgctga tcgtgaccga ccgcttcctg ttcaacaacg gctatgcaga ccagatcact 1500 tctgtgctga aagccgctgg cgtagaaacg gaagttttct ttgaagttga agctgacccg 1560 accctgacca tcgtacgtaa aggcgctgag ctggccaact ccttcaaacc agacgtgatt 1620 atcgcgctgg gcggcggctc cccgatggat gctgcgaaaa ttatgtgggt catgtacgaa 1680 catccggaaa cccacttcga agaactggcg ctgcgcttta tggatatccg taaacgtatc 1740 tacaagttcc cgaaaatggg cgtcaaagcg aagatggtgg cgatcaccac gacttccggc 1800 accggttctg aagttacgcc gttcgccgtg gttaccgatg acgcaaccgg tcagaaatat 1860 ccgctggcag actatgcgct gactccggac atggcgattg ttgacgccaa cctggttatg 1920 gaaatgccga agtcactgtg tgctttcggt ggtctggatg ccgtgacgca tgccctggaa 1980 gcttacgttt ccgtactggc ttccgagttc tctgacggtc aggctctgca ggcgcttaag 2040 ctgctgaaag aaaacctgcc ggcgtcctac catgaagggt ctaaaaaccc ggtagcgcgt 2100 gagcgtgtac acagtgctgc gaccatcgcc ggtatcgcgt ttgcgaacgc cttcctgggc 2160 gtatgtcact caatggcgca caaactgggc tctcagttcc acattcctca cggtctggcg 2220 aacgccctgt tgatcagcaa cgtgattcgt tataacgcta acgacaaccc gaccaaacag 2280 acggccttca gccagtacga ccgtccgcag gctcgtcgtc gctacgctga aatcgctgac 2340 catctgggtc tgactgctcc gggtgaccgc accgcagcga aaatcgaaaa actgcttggc 2400 tggttggacg aaattaaagc tgaactgggt attcctaagt ctatccgcga agctggcgtg 2460 caggaagctg acttcctggc ccacgttgat aagctgtctg aagacgcttt tgacgaccag 2520 tgcaccggcg caaacccgcg ctacccgctg atcgccgagc tgaaacagat tctgctggat 2580 accttctacg gacgtaacta cgttgaaggc ggtgtggaag agaaaaaaga agccgcaccg 2640 gctaaagcag agaagaaagc gaaaaaatcc gcttaa 2676 <210> 107 <211> 1637 <212> DNA <213> Artificial Sequence <220> <223> budB <400> 107 gtggataatc aacatcaacc gcgccagtgg gcgcacggcg ccgacctcat cgtcagccag 60 cttgaggccc agggagtacg ccaggtgttc ggcattccgg gggccaaaat cgataaagtc 120 ttcgattcgc tgctcgactc ctccattcgc attatcccgg tgcgccacga agccaacgcc 180 gcctttatgg ccgccgcggt tggccgcatc accggcaaag cgggcgtcgc gctggtcacg 240 tccggaccgg gctgctctaa cctgattacc gggatggcaa cggccaatag cgagggcgac 300 ccggtggtgg cgctgggcgg cgcggtaaaa cgcgccgata aggccaaaca ggtgcatcag 360 agtatggata cggtggcgat gtttagcccg gtgaccaaat actcggtgga agtcaccgcg 420 ccggaagcgc tggcggaggt tgtctccaac gcgtttcgcg cagccgagca gggacgcccc 480 ggcagcgcct tcgtcagcct gccgcaggac gtggtcgacg ggccggtcca cgccagggtt 540 ctgcccgccg gcgatgcgcc gcagaccggc gcggcgccgg acgaagccat tgcgcgagtc 600 gcgaagatga ttgccggcgc gaaaaatccg atatttctgc tcggcctgat ggccagccag 660 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC GCGCGCGCGC 60 gtacggctga aagcgaacgt cctgcatatc atctgggtcg ataacggcta caacatggtg 1440 gcgattcagg aggagaaaaa ataccagcgg ctctccggcg ttgagttcgg cccggtggat 1500 tttaaagttt atgccgaagc cttcggcgcc aaagggtttg cggtagagag cgccgaagcc 1560 cttgagccga cgctgcgggc ggcgatggac gttgacggcc ccgccgtcgt ggccatcccc 1620 gtggattacc gcgataa 1637 <210> 108 <211> 780 <212> DNA <213> Artificial Sequence <220> <223> budA <400> 108 atgaaccatt ctgttgaatg ctcttgtgaa gagagcctgt gtgaaactct acgaggattt 60 tccgcgcaac atcccgatag cgtcatctac cagacctctc tgatgagcgc gctgcttagc 120 ggcgtttatg aaggtaatac gaccatcgcc gatttgctca cccacggtga tttcggcctc 180 gggaccttta atgaactgga cggcgagctg atcgcgttta gcagcgaagt ttaccagctg 240 cgcgccgacg gcagcgcccg caaagcccgt atggaacagc gtacgccgtt cgcggtgatg 300 acctggtttc agccgcagta tcgcaaaacg ttcgataaac cggtcagccg cgaacagctg 360 cacaacatca tcgaccagca aatcccgtcg gacaatctgt tctgcgccct gcgtattaac 420 ggccattttc gccacgccca tacccgcacg gtaccgcgcc agacgccgcc ctaccgggcg 480 atgaccgacg tactcgacga ccagccggtt tttcgcttca accagcgcga gggggtcctg 540 gttgggttca gaacgccgca gcatatgcag ggcattaacg tggccggcta ccacgaacac 600 ttcatcaccg atgaccgcca gggcggcggc catctgctcg attatcagct cgaccacggc 660 gtgctgacct ttggcgagat ccacaaattg atgattgacc ttcctgccga tagcgccttc 720 ctgcaggcgg atctgcatcc agacaatctt gatgccgcca ttcgctcagt cgaaaactaa 780 <210> 109 <211> 2777 <212> DNA <213> Artificial Sequence <220> <223> Pdu <400> 109 atgcaacaag aagcactagg aatggtagaa accaaaggct taaccgcagc catagaggcc 60 gctgatgcaa tggtgaagtc agccaatgtg atgttagtgg gctatgaaaa gattggctcc 120 gggctggtca ccgtcatcgt gcgcggcgac gttggcgcgg taaaagcggc caccgatgcc 180 ggtgccgccg ccgcacgtaa cgtgggagaa gtgaaagccg tacacgtcat cccacgccct 240 cacaccgatg tagaaaaaat cttaccgaag ggaattagcc aatgagcagc aatgagctgg 300 ttgaacagat catggctcag gtgattgccc gtgtggcaac gccggaacaa caggccatcc 360 ctactgaaaa ccatccaata cgagagacgg ctatggcaga aaaaagctgc agtttaacgg 420 aatttgtcgg gaccgccatt ggcgacaccc tgggcctggt gatcgccaac gtcgataccg 480 ccctgctgga cgcgatgaag ctggaaaaac gctatcgctc catcggcatt ctcggcgccc 540 gcaccggcgc tggcccgcac attatggcgg cggatgaagc ggtgaaggcc accaacaccg 600 aagtggtcag cattgagctt ccgcgcgata ccaaaggcgg cgcgggccac ggttcgctga 660 ttattttagg cggcaacgac gtttccgacg ttaagcgcgg tatcgaagtc gcgctgaaag 720 agctcgaccg caccttcggc gatgtttacg gcaacgaagc cgggcatatc gagctgcagt 780 acaccgctcg cgccagctac gccctggaaa aagcgttcgg cgcgccgatt ggccgcgcct 840 gcggcatcat cgttggcgct ccggcctccg ttggcgtgct gatggccgat accgcgctga 900 agtcagccaa cgttgaagtc gtggcctata gctccccggc gcacggcacc agcttcagta 960 acgaagccat tctggtgatt tccggcgact ccggcgcggt ccgccaggcg gtcatctcgg 1020 cgcgcgaaat cggcaaaacc gttcttgcga ccctcggtgc tgaaccgaaa aacgatcgcc 1080 cgtcctacat ctgataccca cgaggctgat tcatgagatc gaaaagattt gaagcactgg 1140 cgaaacgcccc tgtgaatcag gacggcttcg ttaaggagtg gatcgaagaa ggctttatcg 1200 caatggaaag cccgaacgac ccgaaaccgt cgattaaaat cgttaacggc gcggtaaccg 1260 agctggacgg gaaccggta agcgaattcg acctgatcga ccactttatc gcccgctacg 1320 gcatcaatct gaaccgcgct gaaagtga tggcgatgga ttcggtcaag ctggccaata 1380 tgctgtgcga tccgaacgtc aaacgcagcg aaatcgtccc gctgaccacc gcgatgacgc 1440 cggcgaaaat cgtcgaagtg gtttcgcata tgaacgtggt cgagatgatg atggcgatgc 1500 agaaaatgcg cgcccgccgc accccgtccc agcaggccca cgtcaccaac gtcaaagata 1560 acccggtaca aattgccgcc gacgccgccg aaggcgcatg gcgcggattt gacgaacagg 1620 aaaccaccgt tgcggtagcg cgctatgcgc cgttcaacgc catcgcgctg ctggtgggct 1680 cgcaggtagg ccgtccgggc gtgctgactc agtgctcgct ggaagaagcc accgagctga 1740 agcttggcat gctgggccat acctgctacg ccgaaaccat ctccgtctac ggcaccgagc 1800 cggtctttac cgatggcgac gacacgccgt ggtcgaaggg cttcctggcc tcgtcctacg 1860 cctctcgcgg cctgaaaatg cgcttcacct ccggctccgg ctcggaagtg cagatgggct 1920 acgccgaagg caaatccatg ctttatctgg aagcgcgctg catctacatc accaaagccg 1980 ccggcgttca ggggctgcaa aacggctcgg taagctgcat cggcgtgccg tccgcggtgc 2040 cgtcaggcat tcgcgcggtg ctggcggaaa acctgatctg ctcgtcgctg gatctggaat 2100 gcgcctccag taacgaccag acctttaccc actccgatat gcgtcgcacc gcgcgtctgc 2160 tgatgcagtt cctgccggga accgacttca tctcttccgg ttattccgcg gtgccgaact 2220 acgacaacat gttcgctggc tccaacgaag atgccgaaga ctttgacgac tacaacgtta 2280 tccagcgcga cctgaaggtg gacggcggtc tgcgtccggt gcgtgaagag gacgtcatcg 2340 ccatccgtaa caaagccgcc cgcgcgctgc aggccgtatt tgccggaatg gggctgccgc 2400 cgattaccga tgaagaagtt gaagccgcga cctatgccca cggttcgaaa gatatgccgg 2460 agcgcaatat cgtcgaagac atcaagttcg cccaggaaat catcaatcaa aaccgcaacg 2520 gtctggaagt ggtgaaagcg ctggcgcagg gcgggtttac cgacgtggcc caggacatgc 2580 tcaacatcca gaaggccaag ctgaccgggg actatctgca tacctccgcg attatcgtcg 2640 gcgacggaca ggtgctgtca gccgtcaacg acgttaacga ttatgccggt ccggcaacgg 2700 gctatcgcct gcagggcgaa cgctgggaag agattaaaaa catccctggc gctcttgatc 2760 ccaacgagat tgattaa 2777 <210> 110 <211> 1176 <212> DNA <213> Artificial Sequence <220> <223> GPD1 <400> 110 atgagcgcgg cggcggatcg tctgaatctg accagcggcc atctgaatgc gggtcgtaag 60 CGTAGCAGCA GCAGCGTTAG CCTGAAGGCG GCAGAAGACC GTTCAAAGTT ACCGTGATT 120 GGTAGCGGCA ACTGGGGCAC CACC ATCGCG AAGGTGGTTG CGGAGAAGTG CAAAGGTTAC 180 CCGGAAGTTT TC GC CCGATTT TTC AGATGTGGGTGTTTG AGGAAGAGAT CAACGGTGAA 240 AAGCTGACCG AGATC ATTAACCCCGTCACCAAAACGTT AATATCTGCCGGGTATTA CC 300 CTGCCGGACA CCTGGTGGCGAACC CGACCTGATCGATAGC GTTAAGACGTGGATATC 360 ATTGTGTTCA AC ATTC CGCACC AGTTTCTGCCGC GTATCTGCAGCC AACTGAAAGGC CAC 420 GTTGATAGCC AC GTGC GTGC GATC AGCTGCCTGAAGGGTTTCGAGGTTGGCGCGAAAGGT 480 GTGCAGCTGCTGAGCAGCTAC ATTCG AAGAGCTGGGTATCCAATGCGGTGC GCTGAGC 540 G GTGC GAAC ATT GC GACC GAAGTT GC GC AG G AGC ACTGGAGCGAAACC ACC GTGGC GT AC 600 CAC ATCCCGAAGGACTTTCGTGGTGAGGGCAAAGACGTTGATCACAAGGTGCTGAAAGCG 660 CTGTTCCACC GTCCGTATTTTCACGTTAGCGTGATCGAAGATGTTGCGGGC ATTAGCATC 720 TGCGGTGCGCTGAAAAACGTGGTTGC GCTGG GTT GC G GCTTC GTT G AGGGTCTGG GTT GG 780 ggtaacaacg cgagcgcggc gattcagcgt gtgggcctgg gtgaaatcat tcgttttggt 840 caaatgttct ttccggagag ccgtgaagaa acctactatc aagaaagcgc gggcgtggcg 900 gacctgatca ccacctgcgc gggtggccgt aacgttaagg tggcgcgtct gatggcgacc 960 agcggcaagg atgcgtggga atgcgagaaa gaactgctga acggccagag cgcgcaaggt 1020 ctgatcacct gcaaagaggt tcacgaatgg ctggaaacct gcggtagcgt ggaggacttc 1080 ccgctgtttg aagcggttta ccagattgtg tacaacaact atccgatgaa gaacctgccg 1140 gatatgatcg aagagctgga cctgcacgag gattaa 1176 <210> 111 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> GPP2 <400> 111 atgggtctga ccaccaagcc gctgagcctg aaagttaacg cggcgctgtt cgacgtggat 60 ggcaccatca ttattagcca gccggcgatt gcggcgttct ggcgtgactt tggcaaggat 120 aaaccgtatt tcgacgcgga acacgttatt caggtgagcc acggctggcg tacctttgac 180 GCGATCGCGA AGTTCGCGCC GGATTTTGCg AACGAAGAGT ACGTTCACAA CTGGAGGCG 240 GAAATTCGGT GAAGTATGGC GAGAAAAGC ATCGAAGTTC CGGgtGCgGT GAAACTGTGC 300 AACGCGCTGA ACgcgCTGCC GAAGGAGAAA TGGGCGGTTG CGACCAGCGG CACCCGTGAC 360 ATGGCGCAGA AGTGGTTCGA ACACCTGGGT ATTCTCgtcc GAAATACTTT ATCACCGCG 420 AACGATGTGA AGCAAGGCAA ACCGCACCCG GAGCCGTACC TGAAGGGTCG TAACGGCCTG 480 GgtTATCCGAT TAACGAGCAG GACCCGAGC AAGAGCAAAG TGgtTGTGTT TGAGGATGCG 540 CCGGCGGGTA TTGCggCGGG CAAGGCggCG GGTGCAAAAT TATCGGcat CgcgACCACC 600 TTcgACCTGG ATTTTCTGAA GGAAAAAGGT TGCgACATTA TCGTTAAGAA CCACGAGAGC 660 ATCCGTGTGG GTGGCTATAC GCggAAACC GACGAAGTTG AGTTTATCTT TGACGACTAT 720 CTGTATGCgA AGGATGACCT GCTGAAATGG TAA 753 <210> 112 <211> 996 <212> DNA <213> Artificial Sequence <220> <223> GapA <400> 112 atgactatca aagtaggtat caacggtttt ggccgtatcg gtcgcattgt tttccgtgct 60 gctcaggaac gttctgacat cgagatcgtt gcaatcaacg acctgttaga cgctgactac 120 atggcttaca tgctgaagta tgactccact cacggccgtt tcaacggtac cgttgaagtg 180 aaagacggtc atctgatcgt taacggtaaa aaaatccgtg ttaccgctga gcgtgacccg 240 gctaacctga agtgggacga agtgggtgtt gacgttgttg ctgaagcaac cggtatcttc 300 ctgaccgacg aaaccgctcg taaacacatc accgctggcg cgaaaaaagt cgtactgact 360 ggcccgtcca aagacaacac cccgatgttt gttaaaggcg ctaactttga caaatacgaa 420 ggccaggaca tcgtttccaa cgcatcctgc accaccaact gcctggctcc gctggcaaaa 480 gttatcaacg acaacttcgg catcatcgaa ggtctgatga ctaccgttca cgcaaccacc 540 gcaactcaga aaaccgttga tggcccgtct cacaaagact ggcgcggcgg ccgcggcgca 600 gctcagaaca tcatcccgtc ttctaccggc gctgctaaag ctgtaggtaa agtcctgcca 660 gaactgaatg gcaaactgac cggtatggcg ttccgcgttc cgactccgaa cgtatccgtt 720 gttgacctga ccgttcgtct ggaaaaagct gcttcttatg aagaaattaa gaaagcaatc 780 aaagctgctt ctgaaggcgc aatgaaaggc gttctgggct acaccgaaga cgacgttgta 840 tccaccgatt tcaacggtga aatctgcact tccgtattcg acgccaaagc gggtatcgct 900 ctgaacgaca actttgtgaa actggtttcc tggtacgata acgaaactgg ttactccaac 960 aaggttctgg acctgattgc tcacatctcc aaataa 996 <210> 113 <211> 3294 <212> DNA <213> Artificial Sequence <220> <223> frdABCD <400> 113 gtgcaaactt ttcaagccga tcttgccgta ataggcgcag gcggagcagg tcttcgcgcc 60 gcaatagccg ccgcgcaagc taatccaaat gctaaaatcg ctctaatttc aaaagtctat 120 cctatgcgca gccataccgt tgctgctgaa gggggttccg ctgccgtcgc gcaggatcat 180 gatagctttg agtaccattt ccacgacacc gttgcaggcg gcgactggct gtgtgagcag 240 gacgtcgttg actacttcgt tcatcactgc ccgacggaaa tgacccagct cgaacagtgg 300 ggatgcccgt ggagtcgccg cccggacggc agcgtcaacg tccggcgatt cggcggcatg 360 aagatcgagc gaacctggtt cgctgccgat aaaaccggct ttcatatgct gcacaccctg 420 ttccagactt cgcttcaatt cccgcaaatc cagcgcttcg atgagcattt tgttctggat 480 attttggtgg atgatggcca ggctcgcggt ctggtcgcga tgaatatgat ggaaggtacg 540 ctggtgcaaa tccgcgccaa cgcggtcatt ctggcgaccg gcggcgcagg acgcgtgtat 600 cgctacaata ccaacggcgg tatcgtcacc ggcgacggta tgggtatggc gctcagccac 660 ggcgtaccgc tgcgcgatat ggagttcgtg caatatcacc ccaccggtct cccgggttcc 720 ggaattctga tgacggaagg ctgccgcggc gaaggcggta ttctggtcaa caagaacggc 780 taccgctatc tgcaggatta cggcatgggt ccggaaactc cgctgggcga accgaaaaat 840 aaatatatgg agctgggccc gcgcgataaa gtctcccagg ctttctggca cgaatggcgt 900 aagggcaaca cgatccccac gccgcgaggc gatgtggtct atctcgatct gcgacatttg 960 ggcgagaaaa agctgcttga acgtctgccg tttatctgcg aactctcgaa ggcctacgtt 1020 ggcgtcgatc cggtgaaaga tcccattccg gtacgtccga ccgcgcacta caccatgggc 1080 gggatcgaaa ccgaccagca gtgtgaaacc cgcatcaaag ggctatttgc cgtgggcgaa 1140 tgttcttcgg tcggcctgca cggcgccaac cgccttggct ccaactccct ggcggagctg 1200 gtggtgtttg gccgtctggc cggagaacag gccatgcagc gcgcaaccga agcgggcgaa 1260 gccaatagcg ccgcgctgga cgcgcaggtc gtcgatatcg agaaccgcct gaaagatctg 1320 gttaatcagg aaggtaacga gaactgggcc aagattcgtg acgaaatggg cctgtcaatg 1380 gaagaaggct gcggtatcta ccgtacgccg gagcttatgc agaaaacggt tgataagctg 1440 gccgagctgc aggagcgctt caagcgcgtg cgtatcaccg acacctccag cgtgttcaat 1500 accgacctgc tgtacaccat cgaactgggc catggcctga acgtcgccga atgtatggcg 1560 cattccgccc tcgcgcgtaa agagtcccgc ggcgcgcatc agcgtctgga cgaaggctgc 1620 accgagcgtg acgacgtcaa cttcctcaag cataccctcg ccttccgcga tgccgacggt 1680 aataccagtc tggagtacag tgaggtgaaa atcaccactc tgccgccggc aaaacgcgtg 1740 taccgtgcgg aagcggaagc agccgagaag aaggacga cgaatggctg agatgaaaaa 1800 tatgaaagtc gaagtggtgc gctacaaccc ggaagtggat atcgcaccgc atagcgcttt 1860 ctatgaagtt ccctacgatg agcaaacctc actgcttgat gctctgggct acatcaagga 1920 caacctcgcg ccagacctca gctaccgctg gtcctgccgc atggcgattt gcggttcctg 1980 cggcatgatg gtgaacaagg tgccaaagct ggcctgtaaa actttcctgc gcgaatacac 2040 caaaggtatt aaagtcgagg cgctggccaa cttcccaatc gagcgcgatc tggtggtcga 2100 tatgacccac tttatcgaaa gcctggaagc cattaagccg tacattattg gcaacccgcg 2160 cacgcccgat cagggcccga acacgcagac gccggcgcaa atggccaaat accatcagtt 2220 ctccggctgc atcaactgcg gcctgtgcta tgcggcctgt ccgcagtttg gtcttaatcc 2280 ggagtttatc ggcccggcgg cgatcaccct cgcccatcgc tacaacgaag acagccgcga 2340 ccacggtaaa aaagcacgta tggcgcagct taacggccag aatggcgtct ggacatgtac 2400 tttcgtgggc tactgctccg aagtctgccc gaaaacgtc gacccggcgg ccgcgatcca 2460 gcagggcaag gtagaaagct cgaaagactt tcttatcgcc accctgaaac cacgctaagg 2520 agtgctgtat gacgactaaa cgcaaaccct acgtgcggcc gatgccgtcc acctggtgga 2580 aaaagctgcc gtttatcgc ttctatatgc tgcgtgaagg aacggcggta cccacggtct 2640 ggttcagcat cgtgctgatt tacgggctgt tcgcactgaa gcatggcgcg gagagctgggg 2700 cgggctatat cggtttcctg caaaacccga ttgtcgtgat cctgaacctg atcacgcttg 2760 cagccgcgct gctgcatacc aaaacctggt ttgagctggc gccgaaagcc gccaatatta 2820 tccttaaagg cgagaaaatg gggccagagc caattattaa agggctttgg gtcgtcactg 2880 cggtcgtcac cgtggtgatc ctgtttgtcg cgctgttctg gtaaggaggc tggaatgatt 2940 aatcccaatc caaaacgttc ggatgagccg gtattctggg ggctgtttgg cgctggcggc 3000 atgtggggag cgattatcgc ccccgtcatt gtgctgctgg taggtattat gctgccgctg 3060 ggcctcgcgc ctgctgacgc attcagctat gaacgcgtac tggcttttgc ccatagttt 3120 attggccgtg cattcatttt cctgatgatt gttctgccgc tatggtgcgg gcttcaccgt 3180 attcaccatg cgatgcacga tttgaaaatt cacgtaccca acggtaagtg ggtcttctat 3240 ggactcgcgg cgattctgac cgtcgtcacc ctcgtcggca tattgttcat ctaa 3294 <210> 114 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> Edd <400> 114 atgaattcag aaatgttacg ggtaacaaat cgcatcatcg aacgctcgcg cgatacccgc 60 gaagcctacc tcgcccggat taaccaggcc aaaaccgata ccgttcatcg cgcccagctg 120 gcgtgcggta acctggccca cggctttgcc gcctgccagg cggatgacaa agcctcgctg 180 aaaagcatgc tgcgcaataa catcgccatc attacctcct acaacgatat gctctccgct 240 caccagccct atgagcgcta tccggacatt atccgcaaag cgctgcactc cgccaacgcg 300 gtaggccagg tcgccggcgg cgttccggcg atgtgcgatg gcgtgacgca ggggcaagat 360 gggatggagc tatcgctgct gagtcgcgaa gtgattgcca tgtccgccgc gattggcctg 420 tcgcacaata tgtttgacgg ggcgctctac cttggcgtct gcgataaaat cgtccccggc 480 CTGGCGATGG CTGCGCTATC GTTCGGCCAT CTGCCGTCGA TCTTTATTCCTTCGGGCCG 540 ATGGCCAGCG GCCTGGCGAA TAAAGAAAAA GTGCGCATTC GTCA GCTCATGC GGAAGGC 600 AAAGTGGATC GTATGGCGCT GCTGGAGTCC GAGGCCGCTC TTACC ATGC GCCTGGCAC 660 TGTACCTTCT ACGGTACCGC CAATACCAAC CAGATGGTGG TTGAGTTTAT GGGGATGCAG 720 CTGCCC GGCTCGTCATTC GTGCATCCGG ATGC GCCGCTGC GC GAAGCGCTG ACCGCCGCC 780 GCCGCCC GTCAGGTGACCC GCCTGACCGGC AACGGTAACG AGTGGATGCC GCTGGGCAA 840 ATGTTTGATG AAAAAGTGGT GGTCAACGGG ATTGTCGCGC TGCTGGCAAC CGGC GGCTCC 900 ACCAACCATA C CATGCACCT GGTGGCGATG GC GC GGCGGC AGGGATTAT CATT AACTGG 960 GATGATTTCT CCGATCTCTC AGACGTGGTG CCGCTGCTGG C GC CCTCTACC CGAACGGC 1020 CCGGCGGATA TTAACCAC TTCCAGGC GGC GGC GGC GTGCCGGTG CTGGTGC GC GAGCTG 1080 CTGAAAGGC GGCCTGCTGC ATGAAGATGTC ATACCCTTGCCGGTTTCGGCCTCTCTCGC 1140 TACACC ATGG AGCCCTGGCT TAAT AACGGT GAAC TTGACTGGCGAAGGGGC GGCCGCT 1200 ccgcttgatg aagcggttat cgccaccttt gacaagccgt tctctcgcca cggcggcact 1260 aaagtgctga gcggcaatct cggccgcgcg gtgatgaaaa cctccgccgt gccggttgag 1320 aaccagatta ttgaagcgcc ggcggtggtt ttcgagagcc agcatgacgt gctgcccgcc 1380 tttgaggcgg gactgctcga caaagattgc gtggttgtgg tccgtcatca gggaccaaaa 1440 gcgaacggca tgccagaatt acataaactt atgccgccac ttggtgtatt attggaccgc 1500 cgtttcaaaa ttgcgctggt gaccgatggt cgactctccg gcgcatcagg caaagtgccg 1560 tcggctatcc acgtgacgcc cgaagcgtac gatggtggtc tgctggcgaa ggtacgcgat 1620 ggcgacatta ttcgcgtcaa cggccagacg ggcgagctga cgctgcgggt cgatgacgcc 1680 gagctggccg cgcgtcagcc gcatattcct gacctgagcg gttcgcgcgt gggaaccggc 1740 cgggaaatgt tcggtgcgct gcgtgagaaa ctctccggag cagaacaggg cgcaacctgc 1800 atcacatttt aa 1812 <210> 115 <211> 459 <212> DNA <213> Artificial Sequence <220> <223> mgsA <400> 115 atggaactaa caacacgtac attgccagcg cgcaagcata ttgcactggt cgctcacgat 60 cactgtaaag atatgctgat gaaatgggtc gagcgccacc agcaactgct ggcgcagcac 120 acgttatcgg caacaggaac gaccggaaat ctggtgtcac gcgcaaccgg gctggaggtg 180 caggcgatgc tgagtggtcc catgggcggc gaccagcagg taggagcgct gatttcagaa 240 ggtaaaatcg atgtattgat ttttttctgg gatccgctga atgccgtgcc gcacgatccg 300 gatgtgaaag cgttattacg tttggcaacg gtatggaata ttcccgtcgc caccaacgtt 360 tcgaccgccg actttattat ccagtccccg cacttcaacg accctgttga tatcctgatc 420 cctgattatc aacgctacct tgcagagcgt ctgaaataa 459 <210> 116 <211> 1422 <212> DNA <213> Artificial Sequence <220> <223> sacB <400> 116 atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 60 gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca 120 tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 180 gaaaaatatc aagttcctga attcgattcg tccacaatta aaaatatctc ttctgcaaaa 240 ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 300 cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg 360 atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc 420 cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca 480 caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact 540 gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca 600 gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt 660 gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc 720 gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta 780 tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa 840 gcatactatg gcaaagcac atcattctc cgtcaagaaa gtcaaaact tctgcaaagc 900 gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat 960 gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctacacagt aacagatgaa 1020 attgaacgcg cgaacgtctt taaaatgaac ggcaatggt acctgttcac tgactcccgc 1080 ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt 1140 tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg 1200 gatcttgatc ctaacgatgt aaccttact tactcacact tcgctgtacc tcaagcgaaa 1260 ggaaaacaatg tcgtgattac aagctatatg aaaacagag gattctacgc agaaaaaaaaaa 1320 tcaacgtttg cgccaagctt cctgctgaac atcaaggca agaaacacc tgttgtcaa 1380 gapcatcc ttgacaagg acattaca gttaacaat aa 1422 <210> 117 <211> 660 <212> DNA <213> Artificial Sequence <220> <223> Paint <400> 117 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 60 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 120 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 180 cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt 240 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 300 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 360 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 420 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 480 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 540 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat 600 gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa 660

Claims

1. A genetically engineered recombinant Klebsiella michiganensis (K. michiganensis) Klebsiella michiganensis ) having an inactivated glucose-specific PTS transport system IIBC component, a glycerol kinase, a glycerol dehydrogenase, an alcohol dehydrogenase, an acetolactate synthase, an acetolactate decarboxylase, and a fumarate reductase, a reduced activity 3-phosphoglyceraldehyde dehydrogenase, E. coli NADPH-dependent alcohol dehydrogenase activity, enhanced diol dehydratase, 3-phosphoglycerate dehydrogenase, and 3-phosphoglycerate phosphatase activities.

2. The recombinant Klebsiella michiganensis of claim 1, further having an inactivated phosphogluconate dehydratase and / or an acetolactate synthase, and / or further having levan sucrose transferase activity and / or chloramphenicol acetyltransferase activity.

3. The recombinant Klebsiella michiganensis of claim 1, which (1) lacks ptsG, glpK, gldAl, gldA2, adhE, budB, budA genes and frdABCD gene cluster: (2) has overexpressed yqhd gene from Escherichia coli, (3) has overexpressed pdu gene cluster, GPD1 and GPP2 genes; and / or (4) has attenuated GapA gene.

4. The recombinant Klebsiella michiganensis of claim 3, wherein (1) the ptsG gene is as set forth in the nucleotide sequence of SEQ ID NO: 102 or a degenerate sequence thereof; and / or (2) the glpK gene is as set forth in the nucleotide sequence of SEQ ID NO: 103 or a degenerate sequence thereof; and / or (3) the gldAl gene is as set forth in the nucleotide sequence of SEQ ID NO: 104 or a degenerate sequence thereof; and / or (4) the gldA2 gene is as set forth in the nucleotide sequence of SEQ ID NO: 105 or a degenerate sequence thereof; and / or (5) the adhE gene is as set forth in the nucleotide sequence of SEQ ID NO: 106 or a degenerate sequence thereof; and / or (6) the budB gene is as set forth in the nucleotide sequence of SEQ ID NO: 107 or a degenerate sequence thereof; and / or (7) the budA gene is as set forth in the nucleotide sequence of SEQ ID NO: 108 or a degenerate sequence thereof; and / or (8) the yqhd gene is as set forth in the nucleotide sequence of SEQ ID NO: 100 or a degenerate sequence thereof; and / or (9) the pdu gene cluster is as set forth in the nucleotide sequence of SEQ ID NO: 109 or a degenerate sequence thereof; and / or (10) the GPD1 gene is as set forth in the nucleotide sequence of SEQ ID NO: 110 or a degenerate sequence thereof; and / or (11) the GPP2 gene is as set forth in the nucleotide sequence of SEQ ID NO: 111 or a degenerate sequence thereof; and / or (12) the GapA gene is as set forth in the nucleotide sequence of SEQ ID NO: 112 or a degenerate sequence thereof; and / or (13) the frdABCD gene cluster is as set forth in the nucleotide of SEQ ID NO: 113 or a degenerate sequence thereof.

5. The recombinant Klebsiella michiganensis of claim 3, wherein the recombinant Klebsiella michiganensis further lacks edd and / or mgsA genes, and / or expresses sacB gene and / or cat gene.

6. The recombinant Klebsiella michiganensis of claim 5, wherein: (1) the edd gene is as set forth in the nucleotide sequence of SEQ ID NO: 114 or a degenerate sequence thereof; and / or (2) the mgsA gene is as set forth in the nucleotide sequence of SEQ ID NO: 115 or a degenerate sequence thereof; and / or (3) the sacB gene is as set forth in the nucleotide sequence of SEQ ID NO: 116 or a degenerate sequence thereof; and / or (4) the cat gene is as set forth in the nucleotide sequence of SEQ ID NO: 117 or a degenerate sequence thereof. (2) the mgsA gene is a nucleotide sequence as shown in SEQ ID NO: 115 or a degenerate sequence thereof; and / or (3) the sacB gene is a nucleotide sequence as shown in SEQ ID NO: 116 or a degenerate sequence thereof; and / or (4) the cat gene is a nucleotide sequence as shown in SEQ ID NO: 117 or a degenerate sequence thereof.

7. The recombinant K. michiganensis of claim 3, wherein: (1) the pdu gene cluster, GPD1, GPP2 and / or yqhd gene is under the control of a strong promoter Pdu-31, GP-28 or Yqhd-2, and / or (2) the GapA gene is under the control of a weak promoter GapA-42, and / or (3) the yqhd gene is located at the site of edd gene, and / or (4) the GPD1 and GPP2 are located at the site of mgsA gene.

8. The recombinant K. michiganensis of any one of claims 1-7, which is the strain deposited in CGMCC with the deposit number of CGMCC No. 24576.

9. A method of making a recombinant Klebsiella michiganensis that produces 1,3-propanediol, comprising: Inactivating the activities of glucose-specific PTS transporter system components IIBC, glycerol kinase, glycerol dehydrogenase, alcohol dehydrogenase, acetolactate synthase, acetolactate decarboxylase and fumarate reductase, weakening the activity of 3-phosphoglyceraldehyde dehydrogenase, conferring E. coli NADPH-dependent alcohol dehydrogenase activity, and enhancing the activities of diol dehydratase, 3-phosphoglycerate dehydrogenase and 3-phosphoglycerate phosphatase in an initial K. michiganensis strain to obtain a genetically engineered recombinant K. michiganensis.

10. The method of claim 9, further comprising: Inactivating the activities of phosphogluconate dehydratase and / or acetolactate synthase, and / or conferring levansucrose transferase activity and / or chloramphenicol acetyltransferase activity.

11. The method of claim 9 or 10, comprising: Deleting ptsG, glpK, gldA1, gldA2, adhE, budB, budA genes and frdABCD gene cluster, overexpressing yqhd, GPD1, GPP2 genes and pdu gene cluster, and weakening GapA gene in an initial K. michiganensis strain.

12. The method of claim 11, wherein: (1) the ptsG gene is a nucleotide sequence as shown in SEQ ID NO: 102 or a degenerate sequence thereof; and / or (2) the glpK gene is a nucleotide sequence as shown in SEQ ID NO: 103 or a degenerate sequence thereof; and / or (3) the gldA1 gene is a nucleotide sequence as shown in SEQ ID NO: 104 or a degenerate sequence thereof; and / or (4) the gldA2 gene is a nucleotide sequence as shown in SEQ ID NO: 105 or a degenerate sequence thereof; and / or (5) the adhE gene is a nucleotide sequence as shown in SEQ ID NO: 106 or a degenerate sequence thereof; and / or (6) the budB gene is a nucleotide sequence as shown in SEQ ID NO: 107 or a degenerate sequence thereof; and / or (7) the frdABCD gene cluster is a nucleotide sequence as shown in SEQ ID NO: 108 or a degenerate sequence thereof; and / or (8) the yqhd gene is a nucleotide sequence as shown in SEQ ID NO: 109 or a degenerate sequence thereof; and / or (9) the GPD1 gene is a nucleotide sequence as shown in SEQ ID NO: 110 or a degenerate sequence thereof; and / or (10) the GPP2 gene is a nucleotide sequence as shown in SEQ ID NO: 111 or a degenerate sequence thereof; and / or (11) the pdu gene cluster is a nucleotide sequence as shown in SEQ ID NO: 112 or a degenerate sequence thereof; and / or (12) the GapA gene is a nucleotide sequence as shown in SEQ ID NO: 113 or a degenerate sequence thereof. (7) the budA gene is as shown in the nucleotide sequence of SEQ ID NO: 108 or a degenerate sequence thereof; and / or (8) the yqhd gene is as shown in the nucleotide sequence of SEQ ID NO: 100 or a degenerate sequence thereof; and / or (9) the pdu gene cluster is as shown in the nucleotide sequence of SEQ ID NO: 109 or a degenerate sequence thereof; and / or (10) the GPD1 gene is as shown in the nucleotide sequence of SEQ ID NO: 110 or a degenerate sequence thereof; and / or (11) the GPP2 gene is as shown in the nucleotide sequence of SEQ ID NO: 111 or a degenerate sequence thereof; and / or (12) the GapA gene is as shown in the nucleotide sequence of SEQ ID NO: 112 or a degenerate sequence thereof; and / or (13) the frdABCD gene cluster is as shown in the nucleotide sequence of SEQ ID NO: 113 or a degenerate sequence thereof.

13. The method of claim 11, further comprising deleting the edd and / or mgsA genes, and / or expressing the sacB gene and / or the cat gene.

14. The method of claim 13, wherein (1) the edd gene is as shown in the nucleotide sequence of SEQ ID NO: 114 or a degenerate sequence thereof; and / or (2) the mgsA gene is as shown in the nucleotide sequence of SEQ ID NO: 115 or a degenerate sequence thereof; and / or (3) the sacB gene is as shown in the nucleotide sequence of SEQ ID NO: 116 or a degenerate sequence thereof; and / or (4) the cat gene is as shown in the nucleotide sequence of SEQ ID NO: 117 or a degenerate sequence thereof.

15. The method of claim 11, wherein: (1) the pdu gene cluster, GPD1, GPP2 and / or yqhd gene is placed under the control of a strong promoter Pdu-31, GP-28 or Yqhd-2, and / or (2) the GapA gene is placed under the control of a weak promoter GapA-42, and / or (3) the yqhd gene is placed at the edd gene site, and / or (4) the gene encoding the fusion protein of GPD1 and GPP2 is placed at the mgsA gene site.

16. Use of the recombinant K. michiganensis of any one of claims 1 to 8 or obtained by the method of any one of claims 9 to 15 for the production of 1,3-propanediol.

17. The use of claim 16, wherein 1,3-propanediol is produced without the addition of additional vitamin B12 and / or antibiotics.

18. A method for producing 1,3-propanediol, comprising culturing the recombinant K. michiganensis of any one of claims 1 to 8 or obtained by the method of any one of claims 9 to 15.

19. The method of claim 18, wherein the method is performed without the addition of additional vitamin B12 and / or antibiotics in the production of 1,3-propanediol.

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