WO2018010698A1 - 一种菌及其获取方法和应用 - Google Patents

一种菌及其获取方法和应用 Download PDF

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WO2018010698A1
WO2018010698A1 PCT/CN2017/093235 CN2017093235W WO2018010698A1 WO 2018010698 A1 WO2018010698 A1 WO 2018010698A1 CN 2017093235 W CN2017093235 W CN 2017093235W WO 2018010698 A1 WO2018010698 A1 WO 2018010698A1
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bacterium
gene
pdl
strain
fermentation
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French (fr)
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许平
辛波
陶飞
王钰
唐鸿志
马翠卿
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上海交通大学
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Priority to US16/317,790 priority Critical patent/US11248206B2/en
Publication of WO2018010698A1 publication Critical patent/WO2018010698A1/zh
Priority to US17/671,001 priority patent/US11946036B2/en

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Definitions

  • the invention belongs to the field of bioengineering and relates to a bacterium and a method and application thereof.
  • the methods of obtaining here include methods for constructing artificial bacteria and screening methods for wild bacteria.
  • Petroleum-based polymers bring great convenience to people's production and life.
  • Bio-based materials have the advantages of easy biodegradation, wide source of raw materials and easy chemical improvement, making them important applications in many fields.
  • PTT polytrimethylene terephthalate
  • PLA polylactic acid
  • PTT is a new polyester fiber with excellent properties. It is a polycondensation of terephthalic acid and 1,3-propanediol (1,3-PD). It combines polyethylene terephthalate (PET).
  • PLA also known as polylactide
  • LAC lactic acid
  • PLA products have good biodegradability and can be completely degraded by microorganisms in nature after use, and are recognized as environmentally friendly materials.
  • the huge market demand for bio-based materials PTT and PLA has driven the development of bio-based production of bio-based material monomers 1,3-PD and optically pure LAC.
  • the methods for the production of 1,3-PD by biological methods are mainly divided into two categories: the first is the use of genetically engineered Escherichia coli, using glucose as a substrate to produce 1,3-PD (eg CN201110093628; ZL200710104008); The second type is the production of 1,3-PD (ZL200410100479; CN201180064621) using intestinal bacteria and glycerol as a substrate.
  • glycerol as a by-product of its gradual decline in price is an ideal substrate for 1,3-PD production.
  • the strains which can produce 1,3-PD using glycerol as a substrate mainly include Klebsiella peneumoniae, Klebsiella oxytoca, Citrobacter freundii and Ding Clostridium butyricum and the like.
  • the LAC used to produce PLA must have high optical purity. Therefore, the researchers screened many bacteria that produce optically pure LAC, such as Lactobacillus rhamnosus, Bacillus coagulans, and soil spore milk.
  • Wild bacteria such as Sporolactobacillus terrae can produce high concentrations of optically pure L-lactic acid (L-LAC) or D-lactic acid (D-LAC) from sugars. (ZL200710176057; ZL201210115365; CN201410022868).
  • by-products such as acetic acid and formic acid
  • the two strains ferment glycerol in the fermentation broth, both 1,3-PD and LAC, but because of the large concentration difference between the two compounds (generally not in the same order of magnitude) leads to one of the compounds It can only be treated as a by-product, and there are many kinds of alcohol products and acid products, and the composition is complicated. It is difficult to separate high-purity LAC, and simultaneous production of 1,3-PD and LAC cannot be achieved. In industrial large-scale production, factors such as product concentration, conversion rate, extraction cost, etc. must be considered. If a strain can be used, glycerol is used as substrate, 1,3-PD and optically pure LAC (L-LAC or D-LAC). As the main product, the high conversion rate can simultaneously produce 1,3-PD and optical pure LAC (L-LAC or D-LAC), which will greatly reduce production costs and improve production efficiency.
  • the present invention provides a bacterium having the characteristics of co-production of 1,3-propanediol and D-lactic acid.
  • Co-production refers to simultaneous production.
  • bacteria are artificial bacteria obtained by transformation of wild bacteria.
  • wild bacteria has the following metabolic pathways:
  • the wild strain also has one or more of the following metabolic pathways:
  • pyruvic acid ⁇ ⁇ -acetolactate, ⁇ -acetolactate synthase (budB) is an enzyme that catalyzes this metabolic pathway;
  • ⁇ -acetolactate ⁇ acetoin, ⁇ -acetolactate decarboxylase is an enzyme that catalyzes this metabolic pathway
  • pyruvic acid ⁇ acetic acid, pyruvate oxidase (poxB) is an enzyme that catalyzes this metabolic pathway;
  • acetyl-CoA ⁇ acetyl phosphate, acetyl phosphotransferase (pta) is an enzyme that catalyzes this metabolic pathway;
  • acetyl phosphate ⁇ acetic acid, acetyl kinase (ackA) is an enzyme that catalyzes this metabolic pathway;
  • acetyl-CoA ⁇ acetaldehyde, aldehyde dehydrogenase (adhE) is an enzyme that catalyzes this metabolic pathway;
  • fumaric acid ⁇ succinic acid, fumarate reductase (frdA) is an enzyme that catalyzes this metabolic pathway;
  • the modification comprises: blocking one or more of 3)-9) of the metabolic pathways.
  • the wild mushroom is Klebsiella oxytoca.
  • the wild mushroom is Klebsiella oxytoca PDL-0
  • the Klebsiella oxytoca PDL-0 is deposited in China on April 8, 2016.
  • Typical Culture Collection, the deposit registration number is CCTCC M 2016184.
  • the engineering comprises blocking one or more of 3)-9) of the metabolic pathway by inhibiting or removing the activity of the enzyme.
  • the engineering comprises inhibiting or removing the activity of the enzyme by altering the gene of the enzyme.
  • the transformation comprises: altering the gene of the enzyme by genetic recombination.
  • sequence encoding the ⁇ -acetolactate decarboxylase gene is as shown in SEQ ID NO: 1;
  • the sequence encoding the aldehyde dehydrogenase gene is set forth in SEQ ID NO:3;
  • sequence encoding the acetate kinase and acetylphosphotransferase genes is set forth in SEQ ID NO:4;
  • sequence encoding the pyruvate oxidase gene is set forth in SEQ ID NO:5;
  • the sequence encoding the fumarate reductase gene is shown in SEQ ID NO: 6.
  • one of the budA, budB, adhE, ackA-pta, poxB and frdA of the artificial bacteria or a plurality of gene defects are included in the budA, budB, adhE, ackA-pta, poxB and frdA of the artificial bacteria or a plurality of gene defects.
  • the budA and budB genes of the artificial bacteria are defective.
  • the budA, budB and adhE genes of the artificial bacteria are defective.
  • the budA, budB, adhE, and ackA-pta genes of the artificial bacteria are defective.
  • the gene defect is produced by a method of homologous recombination.
  • the genotype of the artificial bacteria includes ⁇ budA ⁇ budB ⁇ adhE ⁇ ackA-pta ⁇ poxB ⁇ frdA.
  • ⁇ budA ⁇ budB ⁇ adhE ⁇ ackA-pta ⁇ poxB ⁇ frdA indicates ⁇ -acetolactate decarboxylase gene (budA), ⁇ -acetolactate synthase gene (budB), aldehyde dehydrogenase gene (adhE), acetate kinase and acetylphosphotransferase gene (ackA) -pta), pyruvate oxidase gene (poxB) and fumarate reductase gene (frdA) deficiency or mutation.
  • the genotype of the artificial bacteria is ⁇ budA ⁇ budB ⁇ adhE ⁇ ackA-pta ⁇ poxB ⁇ frdA.
  • said artificial bacteria are obtained from budA, budB, adhE, ackA-pta, poxB and frdA gene defects of said wild bacteria; said artificial bacteria produce 1,3-PD and D-LAC, and The total conversion of 1,3-PD and D-LAC exceeds 90%.
  • the budA, budB, adhE, ackA-pta, poxB and frdA gene defects are obtained by homologous recombination, and there is no fixed sequence for obtaining each gene defect;
  • the method of recombination refers to amplifying an upstream homologous fragment and a downstream homologous fragment of the above-mentioned gene by PCR, and constructing the upstream homologous fragment and the downstream homologous fragment into a suicide plasmid and transforming into Escherichia (Escherichia) In coli), a donor strain is obtained; the donor strain is crossed with the corresponding recipient strain, and the upstream homologous fragment and the downstream homologous fragment are identical to the genome of the recipient strain.
  • the source is recombined to obtain the strain of the above-described gene defect, that is, the artificial bacteria.
  • the wild mushroom is Klebsiella oxytoca PDL-0
  • the Klebsiella oxytoca PDL-0 is preserved in China on April 8, 2016.
  • Culture Collection, the deposit registration number is CCTCC M 2016184.
  • the DNA sequence of the budA is as shown in SEQ ID NO: 1; and/or the DNA sequence of the budB is as shown in SEQ ID NO: 2; and/or the DNA sequence of the adhE is as SEQ ID NO: And/or the DNA sequence of the ackA-pta is as set forth in SEQ ID NO: 4; and/or the DNA sequence of the poxB is set forth in SEQ ID NO: 5; and/or the DNA of the frdA The sequence is shown in SEQ ID NO: 6.
  • the engineering comprises introducing an exogenous 1,3-PD synthesis pathway and/or a D-LAC synthesis pathway into the wild strain.
  • the introduction of the exogenous 1,3-PD synthesis pathway is to convert the glycerol dehydratase encoding gene dhaB and the 1,3-PD oxidoreductase encoding gene dhaT into the wild bacteria.
  • the introduction of the exogenous D-LAC synthesis pathway is to perform the codon optimization of the D-lactate dehydrogenase gene dldh Bc for the wild-type expression and replace it with the shuttle plasmid to the wild mushroom D-lactate dehydrogenase position in the genome, thereby enabling constitutive expression of dldh Bc on the wild-type genome.
  • D- introduced the exogenous lactate dehydrogenase gene derived from aggregated dldh Bc Bacillus (Bacillus coagulans) 2-6.
  • Bacillus coagulans 2-6 is Bacillus coagulans CASH in CN101173242A, and its accession number is CGMCC No 2184.
  • the wild mushroom is Escherichia coli.
  • the wild mushroom is Escherichia coli K12.
  • the transformation further comprises: altering the budA, budB, adhE, ackA-pta, poxB and/or frdA genes by genetic recombination.
  • the bacteria claimed in the present invention are artificial bacteria.
  • the bacteria claimed in the present invention may also be wild bacteria, that is, the bacteria are wild bacteria.
  • the bacterium is Klebsiella oxytoca.
  • the bacterium is Klebsiella oxytoca PDL-0, and the Klebsiella oxytoca PDL-0 is preserved in a typical Chinese culture on April 8, 2016.
  • the Conservation Depository, the deposit registration number is CCTCC M 2016184.
  • the bacterium is Klebsiella oxytoca PDL-5, and the Klebsiella oxytoca PDL-5 is on April 8, 2016. It is deposited at the China Center for Type Culture Collection with the registration number CCTTC M 2016185.
  • the bacteria provided by the present invention have the following metabolic pathways:
  • the bacteria provided by the invention have the characteristics of co-production of 1,3-propanediol and D-lactic acid, wherein the molar conversion of 1,3-propanediol is ⁇ 36.5%; the molar conversion of D-lactic acid is ⁇ 39.0 %.
  • the molar conversion of D-lactic acid obtained by co-production is ⁇ 54.0%.
  • the molar conversion of 1,3-propanediol obtained by co-production is ⁇ 42.6%; the molar conversion of D-lactic acid is ⁇ 52.8%.
  • the bacterium provided by the present invention has the characteristics of co-production of 1,3-propanediol and D-lactic acid, wherein the total conversion of 1,3-propanediol and D-lactic acid obtained by co-production exceeds 90%.
  • the bacterium provided by the present invention has the characteristics of co-production of 1,3-propanediol and D-lactic acid, wherein the mass ratio of 1,3-propanediol to D-lactic acid obtained by co-production is 1:0.1-10. Further, the mass ratio of 1,3-propanediol and D-lactic acid obtained by co-production is 1:0.2-5. Preferably, the mass ratio of 1,3-propanediol to D-lactic acid obtained by co-production is 1:0.5-2.
  • the bacterium provided by the present invention is derived from a fungus of Aspergillus, Saccharomyces, Mycobacterium, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces Genus, Mucor, Saccharomyces or Streptomyces; or from the genus Methyl, Salmonella, Bacillus, Pseudomonas, Klebsiella, Lactobacillus, Enterobacter Genus, Citrobacter, genus, genus, or Clostridium.
  • the invention also discloses a method for constructing the bacteria as described above, comprising the following steps:
  • Step 1 Screening soil samples for K. acidophilus producing 1,3-propanediol and lactic acid;
  • Step 2 Defecting one of the budA, budB, adhE, ackA-pta, poxB and frdA of the acid-producing Klebsiella bacterium obtained in the first step or a plurality of genes to obtain the bacterium.
  • Klebsiella oxytoca PDL-0 obtained in the first step is Klebsiella oxytoca PDL-0
  • Klebsiella oxytoca PDL-0 is Deposited with the China Type Culture Collection on April 8, 2016, the deposit registration number is CCTCC M2016184.
  • the DNA sequence of the budA is as shown in SEQ ID NO: 1; the DNA sequence of the budB is shown in SEQ ID NO: 2; the DNA sequence of the adhE is shown in SEQ ID NO: 3; The DNA sequence of ackA-pta is shown in SEQ ID NO: 4; the DNA sequence of poxB is shown in SEQ ID NO: 5; the DNA sequence of frdA is shown in SEQ ID NO: 6.
  • step 2 includes the following steps:
  • Step 2-1 the ⁇ -acetolactate decarboxylase gene and the ⁇ -acetolactate synthase gene of the Klebsiella acid-producing PDL-0CCTCC M 2016184 are deficient, and ⁇ -acetolactate decarboxylase and ⁇ -acetolactate are obtained.
  • Step 2-2 the aldehyde dehydrogenase gene defect of the Klebsiella pneumoniae PDL-1 is obtained, and the strain in which the aldehyde dehydrogenase activity is lost is named as Klebsiella pneumoniae PDL-2;
  • Step 2-3 Defecting the acetic acid kinase and acetylphosphotransferase gene of the Klebsiella oxytocin PDL-2 to obtain a strain in which the activity of the acetate kinase and the acetylphosphotransferase is lost, and the acid-producing Krebs is named.
  • Step 2-4 the pyruvate oxidase gene of the Klebsiella pneumoniae PDL-3 is deficient, and the strain having lost pyruvate oxidase activity is named, and the Klebsiella pneumoniae PDL-4 is named;
  • Step 2-5 the fumarate reductase gene defect of the Klebsiella acid-producing PDL-4 is obtained, and a strain in which fumarate reductase activity is lost is obtained, and the strain is named Klebsiella pneumoniae PDL-5. ;
  • the ⁇ -acetolactate decarboxylase gene sequence is represented by SEQ ID NO. 1; the ⁇ -acetolactate synthase gene sequence is represented by SEQ ID NO. 2; and the aldehyde dehydrogenase gene sequence is SEQ ID NO.
  • the sequence of the acetate kinase and acetylphosphotransferase gene is shown in SEQ ID NO. 4; the pyruvate oxidase gene sequence is shown in SEQ ID NO. 5; the fumarate reductase gene The sequence is shown in SEQ ID NO.
  • the ⁇ -acetolactate decarboxylase gene, the ⁇ -acetolactate synthase gene, the aldehyde dehydrogenase gene, the acetate kinase and the K. pneumoniae obtained in the step 1 are obtained.
  • An acetylphosphotransferase gene, a pyruvate oxidase gene, and a fumarate reductase gene are defective by PCR amplifying an upstream homologous fragment and a downstream homologous fragment of the above-described gene, and the upstream homologous fragment and the The downstream homologous fragment is constructed on a suicide plasmid and transformed into E.
  • the donor bacterium is hybridized with the corresponding recipient bacterium to make the upstream homologous fragment and the downstream
  • the homologous fragment is homologously recombined with the genome of the recipient bacterium, thereby obtaining a strain having the above-described gene defect, that is, the bacterium.
  • the suicide plasmid comprises a suicide plasmid pKR6K
  • the E. coli comprises a large intestine rod Escherichia coli S17-1 ( ⁇ pir)
  • the donor bacteria include Klebsiella pneumoniae PDL-0CCTCC M 2016184, Klebsiella pneumoniae PDL-1, Klebsiella oxytosus PDL -2 and Klebsiella pneumoniae PDL-3
  • the recipient bacteria include Klebsiella pneumoniae PDL-1, Klebsiella pneumoniae PDL-2, and Klebsiella pneumoniae PDL -3 and Klebsiella pneumoniae PDL-4.
  • the present invention also provides another method for constructing a bacterium as described above, characterized in that an exogenous 1,3-PD synthesis pathway and/or an exogenous D-LAC synthesis pathway are introduced into the strain by genetic engineering.
  • the strain may be a strain producing 1,3-PD and/or D-LAC, or a strain not producing 1,3-PD and/or D-LAC. Exogenously added to the 1,3-PD synthetic pathway and/or the D-LAC synthetic pathway for the purpose of allowing the engineered strain to produce 1,3-PD and/or D-LAC, or to produce 1,3-PD and/or The amount of D-LAC is higher.
  • the introduction of the exogenous 1,3-PD synthesis pathway means that the glycerol dehydratase encoding gene dhaB and the 1,3-PD oxidoreductase encoding gene dhaT are transformed into the strain by a plasmid to obtain strain A, Strain A produced 1,3-PD.
  • the introduction of the exogenous 1,3-PD synthesis pathway refers to: selecting a glycerol dehydratase encoding gene dhaB and 1,3-PD redox in the 1,3-PD synthesis pathway in Klebsiella acid-producing bacteria.
  • the enzyme-encoding gene dhaT was subjected to PCR cloning, ligated to plasmid DNA pet-Duet, and transformed into the strain to obtain strain A, which produced 1,3-PD.
  • the introduction of the exogenous D-LAC synthesis pathway means that the gene dldh Bc of D-lactate dehydrogenase is transformed into the strain by a plasmid to obtain strain B, which produces D-LAC.
  • the introduction of the exogenous D-LAC synthesis pathway means: selecting a gene dldh Bc derived from D-lactate dehydrogenase from Bacillus aeruginosa 2-6, and performing codon optimization suitable for expression of the strain, and This was replaced with a shuttle plasmid to the D-lactate dehydrogenase position in the genome of the strain to obtain strain B, which produced D-LAC.
  • the method further comprises the steps of: generating a budA, budB, adhE, ackA-pta by homologous recombination to a strain introducing an exogenous 1,3-PD synthesis pathway and/or introducing an exogenous D-LAC synthesis pathway; A strain deficient in poxB and/or frdA gene.
  • the present invention also provides a screening method of the above-mentioned bacteria, comprising the steps of: adding a soil sample to a first liquid medium, and then transferring the solid medium; and growing a single colony on the solid medium. Inoculation into a second liquid medium; after the end of the culture, the yield of 1,3-propanediol and D-lactic acid in the second liquid medium is detected, and the strains satisfying the requirements of 1,3-propanediol and D-lactic acid are picked. .
  • first liquid medium and the second liquid medium are both glycerol liquid medium; the solid medium is a glycerol solid medium.
  • the single colony refers to a single colony with an acid-producing transparent ring.
  • the invention further relates to the use of a bacterium as described above for the production of 1,3-propanediol or D-lactic acid or the co-production of 1,3-propanediol and D-lactic acid.
  • Klebsiella oxytoca PDL-5CCTCC M 2016185 in the production of 1,3-propanediol or D-lactic acid or the co-production of 1,3-propanediol and D-lactic acid.
  • the present invention provides a method for producing 1,3-propanediol and D-lactic acid by fermentation of the above-mentioned bacteria.
  • the method of producing 1,3-propanediol and D-lactic acid by fermentation of the above-mentioned bacteria actually means the application of the above-mentioned bacteria.
  • the method for co-producing 1,3-propanediol and D-lactic acid as described above is characterized in that the bacterium is Klebsiella oxytoca PDL-5CCTCC M 2016185.
  • the amount of ventilation during fermentation is 0-2.0 vvm.
  • the amount of ventilation during fermentation is 1.0 vvm.
  • a mixed emulsion of calcium hydroxide and water is used as a neutralizing agent to adjust the pH of the fermentation broth during fermentation.
  • Step (1) strain selection: selection of Klebsiella oxytoca PDL-5CCTCC M 2016185;
  • Step (2) seed culture
  • Step (3) fermentation: During the fermentation, the neutralizing agent is used to adjust the pH of the fermentation liquid to 5.5-7.5.
  • Step (1) strain selection: selection of Klebsiella oxytoca PDL-5CCTCC M 2016185;
  • Step (2) seed culture: the strain of the step (1) is selected and inoculated into a glycerol culture medium under sterile conditions, the culture temperature is 25-40 ° C, the shaking speed of the shaker is 100-300 rpm, and the culture time is 6-24 hours to prepare a seed culture solution;
  • Step (3) fermentation: inoculation of the seed culture solution prepared in the step (2) into a fermenter containing glycerol medium, the inoculum amount v/v is 0.5-10%, and the fermentation temperature is 25-40. °C, the aeration rate is 0.3-2.0vvm, the stirring speed is 50-400rpm, during the fermentation process, the neutralization agent is used to adjust the pH of the fermentation liquid to 5.5-7.5, and the fermentation mode is batch fermentation or fed-batch fermentation, batching During fermentation, when the glycerol in the glycerol medium is exhausted, the fermentation is stopped; when the fed-batch fermentation is carried out, when the glycerin in the glycerin medium is exhausted, 400-800 g/L of glycerin is added to the fermenter. The solution controls the concentration of glycerin in the fermentation broth to be 5-40 g/L. When the concentration of 1,3-propanediol or D-lactic acid in the fermentation broth is no longer increased,
  • the culture temperature is 30-37 ° C
  • the shaking speed of the shaker is 150-250 rpm
  • the culture time is 10-16 hours
  • the inoculum amount v/v in the step (3) is 2- 6%
  • the fermentation temperature is 30-37 ° C
  • the aeration is 0.7-1.5 vvm
  • the stirring speed is 150-300 rpm
  • the pH of the fermentation broth is adjusted to 6.0-7.0
  • the fermentation mode is fed-batch fermentation
  • the added glycerin solution Glycerol concentration is 500-700 g/L, controlling the concentration of glycerin in the fermentation broth to be 10-30 g/L
  • the neutralizing agent in the step (3) comprises an aqueous solution of sodium hydroxide, an aqueous solution of potassium hydroxide, an aqueous solution of ammonia, and a mixture of calcium hydroxide and water. Any one or more of the emulsions.
  • the obtained fermentation broth has a high concentration of the target product, a small amount of by-products and a low concentration. It can produce 1,3-PD with a concentration of more than 70g/L and D-LAC with a concentration of more than 110g/L. The total conversion of 1,3-PD and D-LAC exceeds 90%, and the D-LAC produced has high optical purity. (Purity greater than 99.9%). Only a small amount of by-product acetic acid (2.3 g/L) and succinic acid (4.1 g/L) were detected in the fermentation broth.
  • L-LAC or D-LAC optically pure LAC
  • Klebsiella metabolizes glycerol as a glycerol disproportionation pathway, it is divided into a glycerol oxidation pathway and a glycerol reduction pathway.
  • the conversion of glycerol to 1,3-propanediol belongs to the reduction pathway of glycerol, and the conversion process requires the cofactors NADH and ATP produced in the oxidation pathway to provide reducing power and energy.
  • Lactic acid was selected by the US Department of Energy as one of the top 30 key development compounds in 2004. In 2010, it was selected as one of the most promising platform compounds in view of its broad use.
  • Lactic acid is an important monomer that can be used to synthesize a biodegradable, biocompatible, and environmentally friendly biopolymer, polylactic acid (PLA). Adding an isomer D-lactic acid of lactic acid to PLA can significantly improve the performance of PLA and its application in polymeric materials. In addition to the economic value of lactic acid, the net yield of lactic acid is 1mol. NADH can provide a reducing power for the synthesis of 1,3-PD to achieve a state of reducing the equilibrium of the reducing power; from the conversion of glycerol to lactic acid, it is a three-carbon to three-carbon conversion, without carbon loss, which can ensure the recovery rate of carbon.
  • lactic acid has very good biocompatibility, which is essential for industrial high-yield lactic acid; since the biosynthesis of lactic acid has achieved industrial-grade production, its downstream purification technology is relatively mature. All of this makes lactic acid the best choice for co-production with 1,3-PD.
  • the facts also prove that the 1,3-PD and D-LAC have achieved a very high total conversion rate (more than 90%). According to existing reports (Biotechnol Bioeng. Metabolism in 1,3-propanediol fed-batch fermentation by a D-lactate deficient mutant of Klebsiella pneumoniae. 2009; 104(5): 965-72.; Bioresour Technol.
  • Klebsiella pneumoniae PDL-5CCTCC M 2016185 is easy to centrifuge and filter, which is beneficial to the efficient biological production and extraction of biomaterial monomers 1,3-PD and D-LAC.
  • Klebsiella pneumoniae PDL-5CCTCC M 2016185 has important practical application value.
  • the concentration of 1,3-PD and D-LAC obtained by co-production of Klebsiella pneumoniae PDL-0, PDL-1, PDL-2, PDL-3, PDL-4 and PDL-5 is not much different.
  • the mass ratio is in the range of 1:0.1-10, especially in the range of 1:0.2-5, further in the range of 1:0.5-2.
  • Figure 1 is a schematic representation of the genetic modification of Klebsiella oxytoca.
  • Figure 2 is a bar graph of the effect of different neutralizers on the fermentation product.
  • Figure 3 is a bar graph of the effect of different aeration rates on fermentation products.
  • the Klebsiella oxytocin PDL-5CCTCC M 2016185 of the present invention causes ⁇ -acetolactate decarboxylase, ⁇ -acetolactate synthase, due to defects in budA, budB, adhE, ackA-pta, poxB and frdA genes,
  • the activities of aldehyde dehydrogenase, acetate kinase, acetylphosphotransferase, pyruvate oxidase and fumarate reductase are lost, so the metabolic pathways of 2,3-butanediol, ethanol, acetic acid and succinic acid are inactivated, as shown in Fig. 1. Shown.
  • glycerol can be converted to 1,3-propanediol and D-lactic acid, and the following metabolic pathways are inactivated: ⁇ -acetolactate synthase catalyzes the production of ⁇ -acetolate from pyruvate; ⁇ -acetolactate decarboxylase Catalyzed ⁇ -acetolactate to form acetoin; pyruvate oxidase catalyzes the production of acetic acid by pyruvate; acetylphosphotransferase catalyzes the formation of acetyl phosphate by acetyl-CoA; acetyl-kinase catalyzes the production of acetic acid by acetyl-phosphate; aldehyde dehydrogenase catalyzes the production of acetyl-CoA An aldehyde; fumarate reductase catalyzes the formation of succin
  • Klebsiella oxysporum PDL-5CCTCC M 2016185 is used to produce high concentrations of 1,3-PD and high concentrations of D-LAC using glycerol as a substrate.
  • L-LAC is not detected in the fermentation broth, therefore, produced D-LAC has high optical purity (purity greater than 99.9%).
  • 2,3-butanediol, ethanol and formic acid were not detected in the fermentation broth. Only a small amount of acetic acid and succinic acid could be detected.
  • Table 1 The test results of the products in the fermentation broth are shown in Table 1.
  • Table 1 Klebsiella pneumoniae PDL-5CCTCC M 2016185 Composition of glycerol as a substrate fermentation broth
  • Klebsiella pneumoniae PDL-5CCTCC M 2016185 using glycerol as a substrate, can produce 76.2 g/L of 1,3-PD and 111.9 g/L of D-LAC, of which 1 The molar conversion of 3-PD reached 42.6%, the molar conversion of D-LAC reached 52.8%, and the total conversion of the two main products 1,3-PD and D-LAC exceeded 90%. Only a small amount of by-product acetic acid and succinic acid can be detected in the fermentation broth.
  • Example 1 Screening and identification of strains with 1,3-PD and LAC as main products
  • the above strains were streaked and purified on glycerol solid medium several times, and then subjected to 10 cycles of culture test.
  • the yield and molar conversion of 1,3-PD and LAC produced in 10 cycles of culture were basically maintained at the original level. It was confirmed that the above strain was the target strain, and the strain was named PDL-0.
  • the ratio of D-LAC to L-LAC in the culture medium of the strain PDL-0 was determined. The results showed that the ratio of D-LAC in the LAC produced by the strain PDL-0 was greater than 99.9%, and the ratio of L-LAC was less than 0.01%.
  • the whole genome of the strain PDL-0 was extracted, and then the gene sequence of the 16S rRNA of the strain PDL-0 was amplified by PCR, and the PCR product was sequenced.
  • the sequence of the 16S rRNA gene sequenced was as shown in SEQ ID NO: 7.
  • the 16S rRNA gene sequence of the strain PDL-0 has 99% homology with other 16S rRNA gene sequences of K. oxytosus in the NCBI database (http://www.ncbi.nlm.nih.gov/). The analysis showed that the strain PDL-0 was Klebsiella oxytosus.
  • the formula of the glycerin liquid medium is: yeast powder 3g/L, K 2 HPO 4 ⁇ 3H 2 O 10g/L, KH 2 PO 4 2g/L, NH 4 Cl 1g/L, MgSO 4 ⁇ 7H 2 O 0.5 g/L, FeCl 3 ⁇ 6H 2 O 20 mg/L, CoCl 2 ⁇ 6H 2 O 50 mg/L, and glycerin 20 g/L; sterilized at 121 ° C for 20 minutes.
  • the formulation of the glycerin solid medium is: yeast powder 3g/L, K 2 HPO 4 ⁇ 3H 2 O 10g/L, KH 2 PO 4 2g/L, NH 4 Cl 1g/L, MgSO 4 ⁇ 7H 2 O 0.5 g/L, FeCl 3 ⁇ 6H 2 O 20 mg/L, CoCl 2 ⁇ 6H 2 O 50 mg/L, glycerin 20 g/L and agar powder 15 g/L; sterilized at 121 ° C for 20 minutes.
  • the screening method for co-production of 1,3-PD and D-LAC strains is given in Example 1, and the numerical ranges involved are not to be construed as limiting the invention, and variations within a reasonable range can achieve similar effects.
  • the strain obtained by the present screening method is not limited to PDL-0, and may be a strain of another species or a different strain of the same genus.
  • Example 2 Defects of ⁇ -acetolactate decarboxylase gene (budA) and ⁇ -acetolactate synthase gene (budB) in Klebsiella pneumoniae PDL-0
  • Primers were designed based on the budA gene sequence (shown as SEQ ID NO: 1) and the upstream and downstream homologous fragments of the budA gene were PCR amplified. Using the genomic DNA of K. oxytosus PDL-0 as a template, primer budA-1: 5'-ACATGATTACGAATTCATGAACCATTCTGCTGAATG-3' (as shown in SEQ ID NO: 8) and primer budA-2: 5'-AACGGGCTGGCATCACCGCGAAGGGCGTGC were used.
  • PCR amplification of -3' (as shown in SEQ ID NO: 9) to obtain an upstream homologous fragment; using primer budA-3: 5'-CGCGGTGATGCCAGCCCGTTTTCCGCTTCA-3' (as shown in SEQ ID NO: 10) and primer budA -4:5'-TACCGAGCTCGAATTCTTAGTTTTCGACTGAGCGAA-3' (as shown in SEQ ID NO: 11) was subjected to PCR amplification to obtain a downstream homologous fragment.
  • the PCR amplification conditions were: 95 ° C for 5 minutes; 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute for a total of 30 cycles; 72 ° C for 5 minutes.
  • the PCR amplification product was subjected to 1.0% agarose gel electrophoresis, recovered and purified to obtain upstream and downstream homologous fragments.
  • the suicide plasmid pKR6K (Wang et al., J. Biol. Chem. 2014, 289: 6080-6090) was digested with restriction endonuclease EcoRI, and the digested product was subjected to 1.0% agarose gel electrophoresis, recovered and purified. The linearized plasmid pKR6K was obtained.
  • the suicide plasmid pKR6K can be obtained by replacing the replicon of the plasmid pK18mobsacB (Youbo Bio Inc.) with the replicon of the plasmid pCAM140.
  • the sequence of the replicon of plasmid pCAM140 can be obtained by gene synthesis.
  • the upstream homologous fragment, the downstream homologous fragment and the linearized plasmid pKR6K were ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ budA of the budA gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ budA of the budA gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • pKR6K- ⁇ budA was transformed into Escherichia coli S17-1 ( ⁇ pir) (Beijing Quanjin Biotechnology Co., Ltd.) to obtain a donor strain Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ budA).
  • the donor bacterium Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ budA) was crossed with the recipient strain Klebsiella pneumoniae PDL-0, and the homologous fragment upstream and downstream of the budA gene on pKR6K- ⁇ budA was generated.
  • the homologous fragment is homologously recombined with the genome of Klebsiella pneumoniae PDL-0, so that the budA gene of Klebsiella pneumoniae PDL-0 is deleted by 200 bp, and the budA gene defect is achieved. :
  • step b Pick a single colony grown on the M9 solid medium plate in step b to 5 mL LB liquid medium supplemented with 50 ⁇ g/mL kanamycin, and incubate at 37 ° C, 200 rpm for 12 hours.
  • the bacterial solution was transferred to fresh 5 mL LB liquid medium (without addition of kanamycin), and cultured at 37 ° C, 200 rpm for 12 hours.
  • the above bacterial solution was appropriately diluted, applied to a LAS solid medium plate, and cultured at 25 ° C for 24 hours.
  • the LB liquid medium formula is: peptone 10 g / L, yeast powder 5 g / L and NaCl 10 g / L, and sterilized at 121 ° C for 20 minutes.
  • the LB solid medium formula is: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L and agar powder 15 g / L, and sterilized at 121 ° C for 20 minutes.
  • the M9 solid medium formula is: Na 2 HPO 4 ⁇ 12H 2 O 1.7 g / L, KH 2 PO 4 0.3 g / L, NaCl 0.05 g / L, NH 4 Cl 0.1 g / L, citric acid three Sodium 0.5 g/L and agar powder 15 g/L were sterilized at 121 ° C for 20 minutes.
  • the LAS solid medium formula is: peptone 10 g / L, yeast powder 5 g / L, sucrose 150 g / L and agar powder 15 g / L, sterilized at 115 ° C for 20 minutes.
  • Primers were designed based on the budB gene sequence (shown as SEQ ID NO: 2) to amplify upstream and downstream homologous fragments of the budB gene.
  • SEQ ID NO: 2 the genomic DNA of K. oxytosus PDL-0 as a template
  • primer budB-1 5'-ACGCGAATTCGTGGATAATCAACATCAACCGCGCC-3' (as shown in SEQ ID NO: 12)
  • primer budB-2 5'-ACGCGGATCCGGGGCGTCCCTGCTCGGC were used.
  • PCR amplification of -3' (as shown in SEQ ID NO: 13) to obtain an upstream homologous fragment; using primer budB-3: 5'-ACGCGGATCCATCGCCCGCTATCTCTACAGCTTCC-3' (as shown in SEQ ID NO: 14) and primer budB -4:5'-ACGCCTGCAGATTTGACTGAGATGAAGCTGGCCCA-3' (as shown in SEQ ID NO: 15) was subjected to PCR amplification to obtain a downstream homologous fragment.
  • the PCR amplification conditions were: 95 ° C for 5 minutes; 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute for a total of 30 cycles; 72 ° C for 5 minutes.
  • the PCR amplification product was subjected to 1.0% agarose gel electrophoresis, recovered and purified to obtain upstream and downstream homologous fragments.
  • the upstream homologous fragment was digested with restriction endonucleases EcoRI and BamHI, and the downstream homologous fragment was digested with restriction endonucleases BamHI and PstI, and the suicide plasmid pKR6K was digested with the restriction enzymes EcoRI and PstI (Wang et al J. Biol. Chem. 2014, 289: 6080-6090), the digested product was subjected to 1.0% agarose gel electrophoresis, recovered and purified to obtain a linearized plasmid pKR6K and upstream and downstream with sticky ends. Source arm fragment.
  • the upstream homologous fragment with the cohesive terminus, the downstream homologous fragment and the linearized plasmid pKR6K were ligated using T4 ligase (NEB) to obtain a suicide plasmid pKR6K- ⁇ budB which partially deleted the budB gene.
  • NEB T4 ligase
  • pKR6K- ⁇ budB was transformed into E. coli S17-1 ( ⁇ pir) to obtain a donor strain Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ budB).
  • the donor strain Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ budB) was crossed with the strain of the recipient budA gene, and the upstream and downstream homologous fragments of budB gene and budA on pKR6K- ⁇ budB were made.
  • the genome of the gene-deficient strain is homologously recombined, and the budB gene of the budA gene-deficient strain is deleted by 722 bp, thereby achieving the purpose of making the budB gene defective.
  • step (2) The specific method is the same as step (2) except that the primer budB-1: 5'-ACGCGAATTCGTGGATAATCAACATCAACCGCGCC-3' (such as SEQ ID NO: 12) is used for PCR verification. PCR validation was performed with primer budB-4: 5'-ACGCCTGCAGATTTGACTGAGATGAAGCTGGCCCA-3' (shown as SEQ ID NO: 15).
  • the obtained budA gene and the budB gene-deficient strain were named Klebsiella pneumoniae PDL-1.
  • Example 3 Defects of aldehyde dehydrogenase gene (adhE) in Klebsiella pneumoniae PDL-1
  • Primers were designed based on the adhE gene sequence (shown as SEQ ID NO: 3) and the upstream and downstream homologous fragments of the adhE gene were PCR amplified. Using the genomic DNA of Klebsiella pneumoniae PDL-1 as a template, primers adhE-1: 5'-ACATGATTACGAATTCATGGCTGTTACTAATGTCGC-3' (as shown in SEQ ID NO: 16) and primer adhE-2: 5'-TGCTGTCTGTTGGCGTTACGGGTCTTCAGG were used.
  • the suicide plasmid pKR6K (Wang et al., J. Biol. Chem. 2014, 289: 6080-6090) was digested with restriction endonuclease EcoRI, and the digested product was subjected to 1.0% agarose gel electrophoresis, recovered and purified. The linearized plasmid pKR6K was obtained.
  • the upstream homologous fragment, the downstream homologous fragment and the linearized plasmid pKR6K were ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ adhE of the adhE gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the pKR6K- ⁇ adhE was transformed into E. coli S17-1 ( ⁇ pir) to obtain the donor bacterium Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ adhE).
  • the donor bacterium Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ adhE) was crossed with the recipient strain Klebsiella pneumoniae PDL-1, and the homologous fragment upstream and downstream of the adhE gene on pKR6K- ⁇ adhE was generated.
  • the homologous fragment was homologously recombined with the genome of Klebsiella pneumoniae PDL-1, so that the adhE gene of Klebsiella pneumoniae PDL-1 was deleted by 1876 bp, which achieved the purpose of making the adhE gene defective.
  • the specific method was the same as in Example 2 except that the primer was used for PCR verification using primer adhE-1: 5'-ACATGATTACGAATTCATGGCTGTTACTAATGTCGC-3' (as shown in SEQ ID NO: 16) and primer adhE-4: 5'-TACCGAGCTCGAATTCTTAAGCGGATTTTTTCGCTT-3' ( As SEQ ID NO: 19 Shown) for PCR validation.
  • the obtained adhE gene-deficient strain was named Klebsiella pneumoniae PDL-2.
  • Example 4 Defects of the acetate kinase and acetylphosphotransferase genes (ackA-pta) in Klebsiella pneumoniae PDL-2
  • Primers were designed based on the ackA-pta gene sequence (shown as SEQ ID NO: 4) and the upstream and downstream homologous fragments of the ackA-pta gene were PCR amplified. Using the genomic DNA of K.
  • primers ackA-pta-1 5'-ACATGATTACGAATTCATGTCGAGTAAGTTAGTACT-3' (as shown in SEQ ID NO: 20) and primer ackA-pta-2 were used: 5'-CACGCGCGGTCCTCAGCGATACCGATCAGG-3' (as shown in SEQ ID NO: 21) was subjected to PCR amplification to obtain an upstream homologous fragment; using primer ackA-pta-3: 5'-ATCGCTGAGGACCGCGCGTGGCCATGCTCT-3' (eg SEQ ID NO: 22) PCR amplification was performed by primer ackA-pta-4:5'-TACCGAGCTCGAATTCTTATGCTTGCTGCTGGGACG-3' (as shown in SEQ ID NO: 23) to obtain a downstream homologous fragment.
  • the PCR amplification conditions were: 95 ° C for 5 minutes; 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute for a total of 30 cycles; 72 ° C for 5 minutes.
  • the PCR amplification product was subjected to 1.0% agarose gel electrophoresis, recovered and purified to obtain upstream and downstream homologous fragments.
  • the suicide plasmid pKR6K (Wang et al., J. Biol. Chem. 2014, 289: 6080-6090) was digested with restriction endonuclease EcoRI, and the digested product was subjected to 1.0% agarose gel electrophoresis, recovered and purified. The linearized plasmid pKR6K was obtained.
  • the upstream homologous fragment, the downstream homologous fragment and the linearized plasmid pKR6K were ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ ackA-pta of the ackA-pta gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ ackA-pta of the ackA-pta gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ ackA-pta of the ackA-pta gene was ligated using a seamless cloning and
  • pKR6K- ⁇ ackA-pta was transformed into E. coli S17-1 ( ⁇ pir) to obtain a donor strain Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ ackA-pta).
  • the donor bacterium Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ ackA-pta) was hybridized with the recipient strain Klebsiella pneumoniae PDL-2 to make the ackA-pta gene on pKR6K- ⁇ ackA-pta
  • the upstream homologous fragment and the downstream homologous fragment are homologously recombined with the genome of Klebsiella pneumoniae PDL-2, so that the ackA-pta gene of Klebsiella pneumoniae PDL-2 is deleted by 2749 bp, so that ackA is achieved.
  • Example 5 Defects of pyruvate oxidase gene (poxB) in Klebsiella pneumoniae PDL-3
  • Primers were designed based on the poxB gene sequence (shown as SEQ ID NO: 5) to amplify upstream and downstream homologous fragments of the poxB gene.
  • SEQ ID NO: 5 The genomic DNA of Klebsiella pneumoniae PDL-3 as a template, primers poxB-1: 5'-ACATGATTACGACATCATGAAACAGACCGTGGCGGC-3' (as shown in SEQ ID NO: 24) and primer poxB-2: 5'-AAAATCCCCCGGGTTGAGACCAGTTCACAG were used.
  • PCR amplification of -3' (as shown in SEQ ID NO: 25) to obtain an upstream homologous fragment; using primer poxB-3: 5'-GTCTCAACCCGGGGGATTTTCTCTCGCTGG-3' (as shown in SEQ ID NO: 26) and primer poxB -4:5'-TACCGAGCTCGAATTCTTACCTTAGCCAGTTAGTTT-3' (as shown in SEQ ID NO: 27) was subjected to PCR amplification to obtain a downstream homologous fragment.
  • the PCR amplification conditions were: 95 ° C for 5 minutes; 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute for a total of 30 cycles; 72 ° C for 5 minutes.
  • the PCR amplification product was subjected to 1.0% agarose gel electrophoresis, recovered and purified to obtain upstream and downstream homologous fragments.
  • the suicide plasmid pKR6K (Wang et al., J. Biol. Chem. 2014, 289: 6080-6090) was digested with restriction endonuclease EcoRI, and the digested product was subjected to 1.0% agarose gel electrophoresis, recovered and purified. The linearized plasmid pKR6K was obtained.
  • the upstream homologous fragment, the downstream homologous fragment and the linearized plasmid pKR6K were ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ poxB of the poxB gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ poxB of the poxB gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ poxB of the poxB gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit
  • the pKR6K- ⁇ poxB was transformed into Escherichia coli S17-1 ( ⁇ pir) to obtain the donor bacterium Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ poxB).
  • the parental strain Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ poxB) was crossed with the recipient strain Klebsiella pneumoniae PDL-3, and the homologous fragment upstream and downstream of the poxB gene on pKR6K- ⁇ poxB was generated.
  • the homologous fragment is homologously recombined with the genome of Klebsiella pneumoniae PDL-3, so that the poxB gene of Klebsiella pneumoniae PDL-3 is deleted by 919 bp, and the poxB gene defect is achieved.
  • the specific method is the same as in Example 2 except that the primer poxB-1 is used for PCR verification: 5'-ACATGATTACGAATTCATGAAACAGACCGTGGCGGC-3' (as shown in SEQ ID NO: 24) and primer poxB-4: 5'-TACCGAGCTCGAATTCTTACCTTAGCCAGTTAGTTT-3' (as shown in SEQ ID NO: 27) were subjected to PCR verification.
  • the obtained strain defective in the poxB gene was named Klebsiella pneumoniae PDL-4.
  • Example 6 Defects of fumarate reductase gene (frdA) in Klebsiella pneumoniae PDL-4
  • Primers were designed based on the frdA gene sequence (shown as SEQ ID NO: 6) and the upstream and downstream homologous fragments of the frdA gene were PCR amplified. Using the genomic DNA of Klebsiella pneumoniae PDL-4 as a template, primer frdA-1: 5'-ACATGATTACGAATTCGTGCAAACTTTTCAAGCCGA-3' (as shown in SEQ ID NO: 28) and primer frdA-2: 5'-GTAGATGCCGAGCCGGTTTTATCGGCAGCG were used.
  • the PCR amplification conditions were: 95 ° C for 5 minutes; 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute for a total of 30 cycles; 72 ° C for 5 minutes.
  • the PCR amplification product was subjected to 1.0% agarose gel electrophoresis, recovered and purified to obtain upstream and downstream homologous fragments.
  • the suicide plasmid pKR6K (Wang et al., J. Biol. Chem. 2014, 289: 6080-6090) was digested with restriction endonuclease EcoRI, and the digested product was subjected to 1.0% agarose gel electrophoresis, recovered and purified. The linearized plasmid pKR6K was obtained.
  • the upstream homologous fragment, the downstream homologous fragment and the linearized plasmid pKR6K were ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ frdA of the frdA gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ frdA of the frdA gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit) to obtain a partial deletion.
  • the suicide plasmid pKR6K- ⁇ frdA of the frdA gene was ligated using a seamless cloning and assembly kit (PEASY-Uni Seamless Cloning and Assembly Kit
  • pKR6K- ⁇ frdA was transformed into E. coli S17-1 ( ⁇ pir) to obtain a donor strain Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ frdA).
  • the donor strain Escherichia coli S17-1 ( ⁇ pir) (pKR6K- ⁇ frdA) was crossed with the recipient strain Klebsiella pneumoniae PDL-4, and the homologous fragment and downstream of the frdA gene on pKR6K- ⁇ frdA were generated.
  • the homologous fragment is homologously recombined with the genome of Klebsiella pneumoniae PDL-4, so that the frdA gene of Klebsiella pneumoniae PDL-4 is deleted by 991 bp, and the frdA gene defect is achieved.
  • the specific method was the same as in Example 2 except that the primers frdA-1: 5'-ACATGATTACGAATTCGTGCAAACTTTTCAAGCCGA-3' (as shown in SEQ ID NO: 28) and the primer frdA-4 were used for PCR verification: 5'-TACCGAGCTCGAATTCTCAGCCATTCGTCGTCTCCT-3' (as shown in SEQ ID NO: 31) was subjected to PCR verification.
  • the obtained strain lacking the frdA gene was named Klebsiella pneumoniae PDL-5.
  • Examples 2-6 show methods for constructing budA, budB, adhE, ackA-pta, poxB and frdA enzyme inactivating strains, ie, genetic homologous recombination to achieve gene defects leading to inactivation of the enzyme encoded thereby .
  • the way in which the enzyme is inactivated is not limited to the gene-approved recombination, but may also be: small RNA interference, point mutation, addition of an inhibitor of the relevant enzyme, and the like.
  • Example 7 Production of 1,3-PD and D-LAC by batch fermentation using Klebsiella pneumoniae PDL-5CCTCC M 2016185
  • strain selection selection of Klebsiella pneumoniae PDL-5CCTCC M 2016185;
  • Seed culture the strain of step (1) is selected and inoculated into glycerol culture medium under aseptic conditions, the culture temperature is 30 ° C, the shaking speed of the shaker is 200 rpm, and the culture time is 15 hours, and the seed culture liquid is prepared. ;
  • the seed culture liquid prepared in the step (2) is inoculated into a fermenter containing a glycerin medium, the inoculum amount is 5% (v/v), the fermentation temperature is 30 ° C, and the aeration amount is 0.5. Vvm, stirring speed is 200 rpm.
  • the pH of the fermentation broth is adjusted to 7.0 by using 25% (w/v) sodium hydroxide aqueous solution as a neutralizing agent.
  • the fermentation method is batch fermentation, when the glycerol in the glycerol medium is depleted. At the time, the fermentation is stopped.
  • the formula of the glycerin medium is: yeast powder 2g/L, K 2 HPO 4 ⁇ 3H 2 O 5g/L, KH 2 PO 4 1g/L, NH 4 Cl 2g/L, MgSO 4 ⁇ 7H 2 O 0.1 g/L, FeCl 3 ⁇ 6H 2 O 10 mg/L, CoCl 2 ⁇ 6H 2 O 10 mg/L, and glycerin 60 g/L; sterilized at 121 ° C for 20 minutes.
  • the glycerol in the glycerol medium was consumed, the fermentation was stopped, and the product composition and concentration in the fermentation broth were examined.
  • the main products of fermentation were 1,3-PD and D-LAC, the concentration of 1,3-PD was 23.1g/L, the molar conversion was 46.6%; the concentration of D-LAC was 27.2g/L, and the molar conversion was 46.3%. .
  • the by-product was only able to detect a small amount of acetic acid and succinic acid, the acetic acid concentration was 0.4 g/L, the molar conversion was 1.0%, the succinic acid concentration was 0.6 g/L, and the molar conversion was 0.8%. 2,3-butanediol, ethanol and formic acid were not detected in the fermentation broth.
  • Example 8 Production of 1,3-PD and D-LAC using Klebsiella pneumoniae PDL-5CCTCC M 2016185 fed-batch fermentation
  • strain selection selection of Klebsiella pneumoniae PDL-5CCTCC M 2016185;
  • step (1) Seed culture: the strain of step (1) is selected and inoculated into glycerol culture medium under aseptic conditions, the culture temperature is 37 ° C, the shaking speed of the shaker is 150 rpm, and the culture time is 12 hours, and the seed culture liquid is prepared. ;
  • Fermentation inoculation of the seed culture liquid prepared in the step (2) to the hair containing the glycerin medium
  • the inoculum was 2.5% (v/v)
  • the fermentation temperature was 37 ° C
  • the aeration was 1 vvm
  • the stirring speed was 250 rpm
  • 25% (w/v) of calcium hydroxide and water were used.
  • the mixed emulsion is used as a neutralizing agent to adjust the pH of the fermentation liquid to 6.5, and the fermentation method is fed-batch fermentation.
  • the fermentation broth is controlled by adding 700 g/L glycerin solution to the fermenter.
  • the concentration of glycerol in the medium is 5-30 g/L, and when the concentration of 1,3-PD or D-LAC in the fermentation broth is no longer increased, the fermentation is stopped.
  • the formula of the glycerin medium is: yeast powder 5g/L, K 2 HPO 4 ⁇ 3H 2 O 10g/L, KH 2 PO 4 2g/L, NH 4 Cl 1g/L, MgSO 4 ⁇ 7H 2 O 0.1 g/L, FeCl 3 ⁇ 6H 2 O 20 mg/L, CoCl 2 ⁇ 6H 2 O 15 mg/L, and glycerin 20 g/L; sterilized at 121 ° C for 20 minutes.
  • the main products of fermentation were 1,3-PD and D-LAC, the concentration of 1,3-PD was 76.2g/L, the molar conversion was 42.6%; the concentration of D-LAC was 111.9g/L, and the molar conversion was 52.8%. .
  • the by-product was only able to detect a small amount of acetic acid and succinic acid, the acetic acid concentration was 2.3 g/L, the molar conversion was 1.6%, the succinic acid concentration was 4.1 g/L, and the molar conversion was 1.5%. 2,3-butanediol, ethanol and formic acid were not detected in the fermentation broth.
  • Embodiments 7 and 8 are only two preferred embodiments of the present invention in the application method, and the numerical values defined therein can achieve the same purpose within a reasonable range, and therefore cannot be given the values given in Examples 7 and 8. The invention is limited.
  • Examples 7 and 8 were tested for the fermentation product of Klebsiella pneumoniae PDL-5, it was demonstrated that Klebsiella pneumoniae PDL-5 can produce high molar conversion of 1,3-PD and D. - LAC, but it is not only understood that the technical solution for genetically modifying K. oxysporum disclosed in the present invention is only applicable to Klebsiella pneumoniae PDL-5, but should be understood as long as it is through the present invention.
  • the disclosed artificial strains obtained by genetically engineering Klebsiella oxytosus have the ability to simultaneously increase the molar conversion of 1,3-PD and D-LAC and reduce by-products.
  • Examples 1-8 were genetically engineered with Klebsiella pneumoniae PDL-0 as a wild strain, and the same was carried out by other bacteria having the same metabolic pathway as Klebsiella pneumoniae PDL-0. Genetic modification should be understood to also increase the molar conversion of 1,3-PD and D-LAC and reduce by-products.
  • Klebsiella oxysporum PDL-5CCTCC M 2016185 was used to carry out fed-batch fermentation with glycerol as a substrate.
  • the obtained product had high concentration of 1,3-PD and D-LAC and low by-products. And the concentration is low.
  • Klebsiella pneumoniae PDL-5CCTCC M 2016185 is easy to centrifuge and filter. These advantages are beneficial to the efficient bio-production of 1,3-PD and D-LAC, and facilitate the extraction of products.
  • the Klebsiella oxytocin PDL-5CCTCC M 2016185 of the present invention has important practical application value.
  • the neutralization agent NaOH which is commonly used in the production of 1,3-PD, was used as a neutralizing agent to adjust the pH.
  • the optical density (OD) of the cells at 620 nm can reach 5.3.
  • the cellular OD appearing herein refers to the optical density at 620 nm of the cells.
  • the yield of 1,3-PD and D-LAC was not significantly improved, and the yield of 1,3-PD was 34.7 g/L. And 34.8 g / L; D-Lac yield was 61.6g / L and 73.5g / L; cell OD were 5.2 and 6.1, respectively.
  • the lactic acid synthesized by the bacteria involved in the present invention is calibrated with NaOH or ammonia water, a large amount of sodium salt or ammonia salt is formed, and the two salts are easily dissociated, causing excessive osmotic pressure and severely inhibiting cells. Growth and metabolism. Calcium lactate is not easily dissociated and has little effect on cell osmotic pressure, so cells can be synthesized to produce higher yields of lactic acid.
  • Example 10 Optimization of dissolved oxygen content during fermentation
  • the glycerol dehydratase Since the key enzyme for the synthesis of 1,3-PD, the glycerol dehydratase, is an oxygen-sensitive enzyme, too high dissolved oxygen inhibits the synthesis of 1,3-PD. Therefore, the air that is introduced has an effect on its production. It is also reflected in other reports on the metabolism of glycerol to 1,3-PD.
  • the optimum fermentation conditions for the synthesis of 1,3-PD from fermented glycerol are generally microaerobic fermentation. In the biosynthesis of LAC, anaerobic fermentation is usually used.
  • the inventors used anaerobic conditions and microaerobic conditions for the co-production fermentation in the experiment.
  • the specific implementation method is reflected in the change in ventilation rate.
  • the ventilation rates were 0, 0.5 vvm, 1.0 vvm, 2.0 vvm, respectively.
  • the stirring speed was 250 rpm.
  • the fermentation data is shown in Table 2.
  • Klebsiella oxysporum PDL-0 was used to carry out fed-batch fermentation with glycerol as the substrate.
  • the obtained product had high concentration of 1,3-PD and D-LAC in the fermentation broth, and 6M NaOH was used as the fermentation.
  • the neutralizing agent was used, the yields were 35.0 g/L and 44.9 g/L, respectively, and the molar amount was close to 1:1.
  • the by-product types are organic acids such as acetic acid, succinic acid, and formic acid, and organic alcohols such as 2,3-butanediol and ethanol. The concentration of each by-product is relatively low.
  • the wild type PDL-0 was used as a neutralizing agent to adjust the pH of the fermentation broth with a 25% (w/v) mixed emulsion of calcium hydroxide and water
  • the final yield of 1,3-PD and D-LAC was 50.7 g, respectively.
  • /L and 64.2g/L the molar conversions were 36.5% and 39.0%, respectively, and the molar amount was still close to 1:1.
  • the enzymatic properties of the natural D-lactate dehydrogenase of the bacterium provide convenience for genetic engineering of the bacterium.
  • the bacteria are easy to centrifuge and filter. These advantages are beneficial to the efficient bio-production of 1,3-PD and D-LAC after genetic engineering, and are beneficial to the extraction of products, which has important practical application value.
  • strain selection selection of Klebsiella pneumoniae PDL-1;
  • step (1) Seed culture: the strain of step (1) is selected and inoculated into glycerol culture medium under aseptic conditions, the culture temperature is 37 ° C, the shaking speed of the shaker is 150 rpm, and the culture time is 12 hours, and the seed culture liquid is prepared. ;
  • the seed culture liquid prepared in the step (2) is inoculated into a fermenter containing a glycerin medium, the inoculum amount is 2.5% (v/v), the fermentation temperature is 37 ° C, and the aeration amount is 1 vvm.
  • the stirring speed is 250 rpm.
  • 6M sodium hydroxide is used as a neutralizing agent to adjust the pH of the fermentation liquid to 6.5.
  • the fermentation mode is fed-batch fermentation.
  • the glycerol in the glycerol medium is exhausted, the fermentation is carried out.
  • a 700 g/L glycerin solution was added to the tank to control the concentration of glycerol in the fermentation broth to be 5-30 g/L.
  • the concentration of 1,3-PD or D-LAC in the fermentation broth was no longer increased, the fermentation was stopped.
  • the formula of the glycerin medium is: yeast powder 5g/L, K 2 HPO 4 ⁇ 3H 2 O 10g/L, KH 2 PO 4 2g/L, NH 4 Cl 1g/L, MgSO 4 ⁇ 7H 2 O 0.1 g/L, FeCl 3 ⁇ 6H 2 O 20 mg/L, CoCl 2 ⁇ 6H 2 O 15 mg/L, and glycerin 20 g/L; sterilized at 121 ° C for 20 minutes.
  • the main products of PDL-1 fermentation were 1,3-PD and D-LAC, the concentration of 1,3-PD was 32.1 g/L, and the concentration of D-LAC was 60.4 g/L.
  • the by-products were able to detect ethanol, acetic acid, formic acid and succinic acid with an ethanol concentration of 1.7 g/L, an acetic acid concentration of 0.9 g/L, a formic acid concentration of 2.2 g/L, and a succinic acid concentration of 1.5 g/L.
  • the yield of D-LAC increased from 48.2 g/L to 60.1 g/L, and the molar conversion rate increased from 39.1% of the wild type to 52.7%.
  • the fermentation method was the same as that in Example 12, but the strain selected was Klebsiella pneumoniae PDL-2.
  • the fermentation results showed that the main products were 1,3-PD and D-LAC, the concentration of 1,3-PD was 43.5 g/L, and the concentration of D-LAC was 59.3 g/L.
  • By-products were able to detect acetic acid, formic acid and succinic acid with an acetic acid concentration of 3.9 g/L, a formic acid concentration of 1.1 g/L and a succinic acid concentration of 1.5 g/L.
  • the synthesis of ethanol was not detected in the fermentation broth, the yield of D-LAC was basically unchanged, the yield of 1,3-PD increased from 32.1g/L to 43.5g/L, and the molar conversion rate was from PDL- 33.2% of 1 increased to 40.0%.
  • the fermentation method was the same as that in Example 12, but the strain selected was Klebsiella pneumoniae PDL-3.
  • the fermentation results showed that the main products were 1,3-PD and D-LAC, the 1,3-PD concentration was 44.5 g/L, and the D-LAC concentration was 71.9 g/L.
  • By-products were able to detect small amounts of acetic acid, formic acid and succinic acid with an acetic acid concentration of 1.5 g/L, a formic acid concentration of 0.3 g/L and a succinic acid concentration of 1.5 g/L.
  • Formic acid is a very important metabolic pathway in microorganisms in microaerobic and anaerobic fermentation. Catalyzed by pyruvate-formate lyase, pyruvate cleaves to form formic acid and acetyl-CoA. The synthesis of formic acid was detected in the wild type. Therefore, after knocking out the butanediol pathway and the ethanol pathway, the inventors knocked out the gene pflB encoding pyruvate formate lyase.
  • formic acid can be metabolized to H 2 and CO 2 by formate dehydrogenase and hydrogenase, as long as the fermentation conditions are controlled under certain conditions, the formic acid synthesized in the intermediate metabolism can be decomposed, thereby achieving no effect on the growth of the cells. At the same time, it does not accumulate formic acid at the end. This can be reflected in the fermentation process of PDL-5 during the construction of engineering bacteria.
  • Example 16 Exogenous introduction of new pathways to achieve joint production of 1,3-PD and D-LAC
  • strain selection Escherichia coli K12
  • the glycerol dehydratase-encoding gene dhaB and the 1,3-PD oxidoreductase-encoding gene dhaT in the 1,3-PD synthesis pathway of K. acid-producing bacteria were selected for PCR cloning and ligated to plasmid DNA pet-Duet. And converted to E. coli K12. This bacterium was identified as K12-dhaBdhaT.
  • the dldh Bc gene encoding D-lactate dehydrogenase from Bacillus aeruginosa 2-6 was used to perform codon optimization suitable for E. coli expression, and the D-lactic acid was replaced with a shuttle plasmid into the E. coli K12 genome. Dehydrogenase position, thereby achieving constitutive expression of dldh Bc on the E. coli genome.
  • This bacterium was identified as K12-dhaBdhaTdldh Bc .
  • Seed culture the strain of step (3) is selected and inoculated into glycerol culture medium under aseptic conditions, the culture temperature is 37 ° C, the shaking speed of the shaker is 200 rpm, and the culture time is 15 hours, and the seed culture liquid is prepared. ;
  • Fermentation The seed culture liquid prepared in the step (4) is inoculated into a fermenter containing a glycerin medium, the inoculum amount is 5% (v/v), the fermentation temperature is 37 ° C, and the aeration amount is 0.5. Vvm, stirring speed is 200 rpm.
  • the aqueous solution of calcium hydroxide is used as a neutralizing agent to adjust the pH of the fermentation liquid to 7.0, and the fermentation mode is batch fermentation. When the glycerol in the glycerin medium is exhausted, the fermentation is stopped.
  • the glycerin medium is formulated as: 10 mM ammonium sulfate, 50 mM MOPS/KOH buffer, pH 7.5, 5 mM potassium phosphate buffer, pH 7.5, 2 mM magnesium chloride, 0.7 mM calcium chloride, 50 uM manganese chloride, 1 uM chlorine Zinc, 1.72 uM copper sulfate, 2.53 uM cobalt chloride, 2.4 uM sodium molybdate, 2 uM thiamine hydrochloride, 0.8 ug/mL vitamin B12, and 50 ng/ul ampicillin.
  • the carbon source was selected from glycerin 60 g/L as needed; and sterilized at 121 ° C for 20 minutes.
  • the OD of the bacteria in the medium for 3 hours of fermentation was 1.0, and IPTG was induced to induce the synthesis of 1,3-PD.
  • IPTG was induced to induce the synthesis of 1,3-PD.
  • the glycerol in the glycerol medium was consumed, the fermentation was stopped, and the product composition and concentration in the fermentation broth were examined.
  • the main products of the fermentation were 1,3-PD and D-LAC, the concentration of 1,3-PD was 14.5 g/L, and the concentration of D-LAC was 17.2 g/L.
  • the by-products were able to detect ethanol, formic acid, acetic acid and succinic acid with an ethanol concentration of 2.1 g/L, a formic acid concentration of 0.6 g/L, an acetic acid concentration of 4.4 g/L, and a succinic acid concentration of 0.8 g/L.
  • E. coli K12 is used as an example to demonstrate that the exogenous 1,3-PD synthesis pathway can be introduced by genetic engineering to strains that do not have a 1,3-PD synthesis pathway and/or a D-LAC synthesis pathway. Exogenous D-LAC synthesis pathway. Combining the concept given in the present embodiment with the inventive examples 1-8, it is possible to further obtain a strain of 1,3-PD and D-LAC which co-produce high molar conversion, which is also within the scope of protection of the present invention. Inside.

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Abstract

提供一种菌及其获取方法和应用。该菌具有联产1,3-丙二醇和D-乳酸的特性。进一步地该菌为产酸克雷伯氏菌(Klebsiella oxytoca)，包括产酸克雷伯氏菌PDL-5 CCTCC M 2016185。该菌的获取方法可以是通过直接从环境中筛选满足条件的野生菌，或者是对野生菌进行基因工程改造。

Description

一种菌及其获取方法和应用 技术领域
本发明属于生物工程领域，涉及一种菌及其获取方法和应用。这里的获取方法包括对人工菌的构建方法和对野生菌的筛选方法。
背景技术
石油基聚合物为人们的生产生活带来了极大的便利。但是，随着石化资源的日益减少，以及使用石化资源带来的环境问题，寻找新的环境友好的非石油基聚合物受到越来越多的重视。生物基材料具有易生物降解、原料来源广和易化学改良等优点，使其在很多领域中都有重要的应用。目前，聚对苯二甲酸丙二醇酯(PTT)和聚乳酸(PLA)是受关注度较高的两种生物基材料。PTT是一种性能优异的聚酯类新型纤维，由对苯二甲酸和1,3-丙二醇(1,3-PD)缩聚而成，它综合了聚对苯二甲酸乙二醇酯(PET)和聚对苯二甲酸丁二醇酯(PBT)的优势，具有很好的柔软性、蓬松性、抗污性、弹性和可以常温染色等特点，可以广泛用于服装、装饰和工程塑料等各个领域。PLA也称为聚丙交酯，是以乳酸(LAC)为主要原料聚合得到的聚合物，它具有较好的热稳定性、抗溶剂性和生物相容性，可进行多种方式的加工，可用于制造包装材料、纤维和非织造物等在服装、建筑、农业和医疗卫生等领域用途广泛。此外，PLA制品具有良好的生物可降解性，使用后能被自然界中微生物完全降解，是公认的环境友好材料。
生物基材料PTT和PLA巨大的市场需求，推动了生物法生产生物基材料单体1,3-PD和光学纯LAC的发展。目前生物法生产1,3-PD的方法主要分为两类：第一类是利用基因工程大肠杆菌，以葡萄糖等糖类为底物，生产1,3-PD(例如CN201110093628；ZL200710104008)；第二类是利用肠道细菌，以甘油为底物，生产1,3-PD(ZL200410100479；CN201180064621)。最近，由于生物柴油产业的发展，甘油作为其副产物，价格逐渐下降，是1,3-PD生产的理想底物。可利用甘油为底物生产1,3-PD的菌株主要包括肺炎克雷伯氏菌(Klebsiella peneumoniae)、产酸克雷伯氏菌(Klebsiella oxytoca)、弗氏柠檬酸杆菌(Citrobacter freundii)和丁酸梭菌(Clostridium butyricum)等。用于生产PLA的LAC必须具有高光学纯度，因此，研究者筛选得到了很多可生产光学纯LAC的细菌，例如鼠李糖乳杆菌(Lactobacillus rhamnosus)、凝结芽孢杆菌(Bacillus coagulans)和土芽孢乳杆菌(Sporolactobacillus terrae)等野生细菌，都可以以糖类为原料生产高浓度的光学纯L-乳酸(L-LAC)或D-乳酸(D-LAC) (ZL200710176057；ZL201210115365；CN201410022868)。
利用上述的菌株和生产方法，可实现1,3-PD和LAC中一种化合物的生物法生产，但是，目前尚无可同时生产1,3-PD和光学纯LAC(L-LAC或D-LAC)的菌株。肺炎克雷伯氏菌和产酸克雷伯氏菌在以甘油为底物，生产1,3-PD的过程中，还会生产少量的2,3-丁二醇、乙醇、LAC、琥珀酸、乙酸和甲酸等副产物，虽然这两种菌株发酵甘油的发酵液中，同时具有1,3-PD和LAC，但由于两种化合物的浓度差距大(一般不在同一数量级)导致其中一种化合物只能被当做副产物处理，且醇类产物和酸类产物种类多，成分复杂，难以分离得到高纯度的LAC，无法实现同时生产1,3-PD和LAC。在工业化大规模生产中，必须考虑产物浓度、转化率，提取成本等因素，如果能够利用一个菌株，以甘油为底物，以1,3-PD和光学纯LAC(L-LAC或D-LAC)为主要产物，高转化率的实现同时生产1,3-PD和光学纯LAC(L-LAC或D-LAC)，将大大降低生产成本，提高生产效率。
发明内容
针对现有技术的不足，本发明提供一种菌，所述菌具有联产1,3-丙二醇和D-乳酸的特性。联产是指同时生产。
进一步地，所述菌是由野生菌改造获得的人工菌。
进一步地，所述野生菌具有如下代谢途径：
1)甘油→1,3-丙二醇；和/或
2)甘油→丙酮酸→D-乳酸；
所述野生菌还具有如下代谢途径中一个或多个：
3)丙酮酸→α-乙酰乳酸，α-乙酰乳酸合成酶(budB)是催化该代谢途径的酶；
4)α-乙酰乳酸→乙偶姻，α-乙酰乳酸脱羧酶(budA)是催化该代谢途径的酶；
5)丙酮酸→乙酸，丙酮酸氧化酶(poxB)是催化该代谢途径的酶；
6)乙酰辅酶A→乙酰磷酸，乙酰磷酸转移酶(pta)是催化该代谢途径的酶；
7)乙酰磷酸→乙酸，乙酰激酶(ackA)是催化该代谢途径的酶；
8)乙酰辅酶A→乙醛，醛脱氢酶(adhE)是催化该代谢途径的酶；
9)富马酸→琥珀酸，富马酸还原酶(frdA)是催化该代谢途径的酶；
所述改造包括：阻断所述代谢途径中的3)－9)中的一个或多个。
进一步地，所述野生菌为产酸克雷伯氏菌(Klebsiella oxytoca)。
进一步地，所述野生菌为产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0于2016年4月8日保藏于中国 典型培养物保藏中心，保藏登记号为CCTCC M 2016184。
进一步地，所述改造包括：通过抑制或去除酶的活性来阻断所述代谢途径中的3)－9)中的一个或多个。
进一步地，所述改造包括：通过改变酶的基因来抑制或去除酶的活性。
进一步地，所述改造包括：通过基因重组的办法来改变酶的基因。
进一步地，编码α-乙酰乳酸脱羧酶基因的序列如SEQ ID NO:1所示；
编码α-乙酰乳酸合成酶基因的序列如SEQ ID NO:2所示；
编码醛脱氢酶基因的序列如SEQ ID NO:3所示；
编码乙酸激酶和乙酰磷酸转移酶基因的序列如SEQ ID NO:4所示；
编码丙酮酸氧化酶基因的序列如SEQ ID NO:5所示；
编码富马酸还原酶基因的序列如SEQ ID NO:6所示。
进一步地，所述人工菌的budA、budB、adhE、ackA-pta、poxB和frdA中的一种基因或者多种基因缺陷。
在一个较佳实施方式中，所述人工菌的budA、budB基因缺陷。
在另一个较佳实施方式中，所述人工菌的budA、budB和adhE基因缺陷。
在又一个较佳实施方式中，所述人工菌的budA、budB、adhE和ackA-pta基因缺陷。
进一步地，所述基因缺陷通过同源重组的方法产生。
进一步地，所述人工菌的基因型包括ΔbudA ΔbudB ΔadhE ΔackA-pta ΔpoxBΔfrdA。ΔbudA ΔbudB ΔadhE ΔackA-pta ΔpoxB ΔfrdA表示α-乙酰乳酸脱羧酶基因(budA)、α-乙酰乳酸合成酶基因(budB)、醛脱氢酶基因(adhE)、乙酸激酶和乙酰磷酸转移酶基因(ackA-pta)、丙酮酸氧化酶基因(poxB)和富马酸还原酶基因(frdA)缺陷或突变。
优选地，所述人工菌的基因型是ΔbudA ΔbudB ΔadhE ΔackA-pta ΔpoxBΔfrdA。
从另一角度描述，所述人工菌是由所述野生菌的budA、budB、adhE、ackA-pta、poxB和frdA基因缺陷获得；所述人工菌产1,3-PD和D-LAC，且1,3-PD和D-LAC的总转化率超过90％。总转化率计算公式为：总转化率＝1,3-丙二醇的摩尔转化率+D-乳酸的摩尔转化率；其中，1,3-丙二醇的摩尔转化率＝(1,3-PD的最终浓度×最终发酵液体积×甘油的摩尔质量92)/(消耗的甘油质量×1,3-PD的摩尔质量76)；D-乳酸的摩尔转化率＝(D-LAC的最终浓度×最终发酵液体积×甘油的摩尔质量92)/(消耗的甘油质量×D-LAC的摩尔质量90)。
进一步地，budA、budB、adhE、ackA-pta、poxB和frdA基因缺陷的获得采用同源重组的方法，且各基因缺陷的获得不存在固定的先后顺序；所述同源 重组的方法是指通过PCR扩增以上所述基因的上游同源片段和下游同源片段，并将所述上游同源片段和所述下游同源片段构建到自杀质粒中转化至大肠杆菌(Escherichia coli)中，得到供体菌；将所述供体菌与相应的受体菌进行双亲本杂交，使所述上游同源片段和所述下游同源片段与所述受体菌的基因组发生同源重组，从而获得以上所述基因缺陷的菌株，即所述人工菌。
优选地，所述野生菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0于2016年4月8日保藏于中国典型培养物保藏中心，保藏登记号为CCTCC M 2016184。
进一步地，所述budA的DNA序列如SEQ ID NO:1所示；和/或所述budB的DNA序列如SEQ ID NO:2所示；和/或所述adhE的DNA序列如SEQ ID NO:3所示；和/或所述ackA-pta的DNA序列如SEQ ID NO:4所示；和/或所述poxB的DNA序列如SEQ ID NO:5所示；和/或所述frdA的DNA序列如SEQ ID NO:6所示。
在一较佳实施方式中，所述改造包括向所述野生菌中引入外源1,3-PD合成途径和/或D-LAC合成途径。
进一步地，所述引入外源1,3-PD合成途径是将甘油脱水酶编码基因dhaB及1,3-PD氧化还原酶编码基因dhaT转化到所述野生菌中。
进一步地，所述引入外源D-LAC合成途径是将D-乳酸脱氢酶的基因dldhBc进行适用于所述野生菌表达的密码子优化，并将其利用穿梭质粒置换到所述野生菌基因组中的D-乳酸脱氢酶位置，从而实现dldhBc在所述野生菌基因组上的组成型表达。
进一步地，所述外源引入的D-乳酸脱氢酶的基因dldhBc来自于凝集芽孢杆菌(Bacillus coagulans)2-6。凝集芽孢杆菌(Bacillus coagulans)2-6为CN101173242A中的凝结芽孢杆菌(Bacillus coagulans)CASH，其保藏号为CGMCC No2184。
进一步地，所述野生菌是大肠杆菌。优选地，所述野生菌是大肠杆菌K12。
进一步地，所述改造还包括：通过基因重组的办法来改变budA、budB、adhE、ackA-pta、poxB和/或frdA基因。
以上描述了本发明所要求保护的菌是人工菌的情况，事实上，本发明所要求保护的菌也可以是野生菌，即所述菌为野生菌
进一步地，所述菌是产酸克雷伯氏菌(Klebsiella oxytoca)。
优选地，所述菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0于2016年4月8日保藏于中国典型培 养物保藏中心，保藏登记号为CCTCC M 2016184。
在一个较佳实施方式中，所述菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5于2016年4月8日保藏于中国典型培养物保藏中心，保藏登记号为CCTCC M 2016185。
无论是野生菌还是人工菌，本发明提供的所述菌具有如下代谢途径：
1)甘油→1,3-丙二醇；
2)甘油→丙酮酸→D-乳酸。
本发明提供的一种菌具有联产1,3-丙二醇和D-乳酸的特性，其中联产得到的：1,3-丙二醇的摩尔转化率≥36.5％；D-乳酸的摩尔转化率≥39.0％。
进一步地，联产得到的：D-乳酸的摩尔转化率≥52.7％。
进一步地，联产得到的：1,3-丙二醇的摩尔转化率≥40％。
进一步地，联产得到的：D-乳酸的摩尔转化率≥54.0％。
进一步地，联产得到的：1,3-丙二醇的摩尔转化率≥42.6％；D-乳酸的摩尔转化率≥52.8％。
本发明提供的一种菌具有联产1,3-丙二醇和D-乳酸的特性，其中联产得到的：1,3-丙二醇和D-乳酸的总转化率超过90％。
本发明提供的一种菌具有联产1,3-丙二醇和D-乳酸的特性，其中联产得到的1,3-丙二醇和D-乳酸的质量比为1:0.1-10。进一步地，联产得到的1,3-丙二醇和D-乳酸的质量比为1:0.2-5。优选地，联产得到的1,3-丙二醇和D-乳酸的质量比为1:0.5-2。
进一步地，本发明提供的所述菌来自于真菌中的曲霉属、酵母属、接合酵母属、毕赤酵母属、克鲁维酵母属、假丝酵母属、汉逊酵母属、德巴利酵母属、毛霉属、球拟酵母属或链霉菌属；或者来自细菌中的甲基菌属、沙门氏菌属、芽孢杆菌属、假单胞菌属、克雷伯氏菌、乳杆菌属、肠杆菌属、柠檬酸杆菌属、暗杆菌属、泥杆菌属或者梭菌属。
本发明还公开了一种如上所述的菌的构建方法，包括以下步骤：
步骤一、从土壤样品中筛选能生产1,3-丙二醇和乳酸的产酸克雷伯氏菌；
步骤二、使得所述步骤一中得到的产酸克雷伯氏菌的budA、budB、adhE、ackA-pta、poxB和frdA中的一种基因或者多种基因缺陷，得到所述菌。
进一步地，所述步骤一中得到的产酸克雷伯氏菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0于2016年4月8日保藏于中国典型培养物保藏中心，保藏登记号为CCTCC M2016184。
进一步地，所述budA的DNA序列如SEQ ID NO:1所示；所述budB的DNA序列如SEQ ID NO:2所示；所述adhE的DNA序列如SEQ ID NO:3所示；所述ackA-pta的DNA序列如SEQ ID NO:4所示；所述poxB的DNA序列如SEQ ID NO:5所示；所述frdA的DNA序列如SEQ ID NO:6所示。
进一步地，所述步骤二包括以下步骤：
步骤2-1、使所述产酸克雷伯氏菌PDL-0CCTCC M 2016184的α-乙酰乳酸脱羧酶基因和α-乙酰乳酸合成酶基因缺陷，得到α-乙酰乳酸脱羧酶和α-乙酰乳酸合成酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-1；
步骤2-2、使所述产酸克雷伯氏菌PDL-1的醛脱氢酶基因缺陷，得到醛脱氢酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-2；
步骤2-3、使所述产酸克雷伯氏菌PDL-2的乙酸激酶和乙酰磷酸转移酶基因缺陷，得到乙酸激酶和乙酰磷酸转移酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-3；
步骤2-4、使所述产酸克雷伯氏菌PDL-3的丙酮酸氧化酶基因缺陷，得到丙酮酸氧化酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-4；
步骤2-5、使所述产酸克雷伯氏菌PDL-4的富马酸还原酶基因缺陷，得到富马酸还原酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-5；
所述α-乙酰乳酸脱羧酶基因序列如SEQ ID NO.1所示；所述α-乙酰乳酸合成酶基因序列如SEQ ID NO.2所示；所述醛脱氢酶基因序列如SEQ ID NO.3所示；所述乙酸激酶和乙酰磷酸转移酶基因序列如SEQ ID NO.4所示；所述丙酮酸氧化酶基因序列如SEQ ID NO.5所示；所述富马酸还原酶基因序列如SEQ ID NO.6所示。
进一步地，所述步骤二中，使得所述步骤一中得到的产酸克雷伯氏菌的α-乙酰乳酸脱羧酶基因、α-乙酰乳酸合成酶基因、醛脱氢酶基因、乙酸激酶和乙酰磷酸转移酶基因、丙酮酸氧化酶基因和富马酸还原酶基因缺陷是通过PCR扩增以上所述基因的上游同源片段和下游同源片段，并将所述上游同源片段和所述下游同源片段构建到自杀质粒上，并转化至大肠杆菌中，得到供体菌；将所述供体菌与相应的受体菌进行双亲本杂交，使所述上游同源片段和所述下游同源片段与所述受体菌的基因组发生同源重组，从而获得以上所述基因缺陷的菌株，即所述菌。
进一步地，所述自杀质粒包括自杀质粒pKR6K；所述大肠杆菌包括大肠杆 菌(Escherichia coli)S17-1(λpir)；所述供体菌包括产酸克雷伯氏菌PDL-0CCTCC M 2016184、产酸克雷伯氏菌PDL-1、产酸克雷伯氏菌PDL-2和产酸克雷伯氏菌PDL-3；所述受体菌包括产酸克雷伯氏菌PDL-1、产酸克雷伯氏菌PDL-2和产酸克雷伯氏菌PDL-3和产酸克雷伯氏菌PDL-4。
本发明还提供另一种如上所述的菌的构建方法，其特征在于，通过基因工程改造向菌株中引入外源1,3-PD合成途径和/或外源D-LAC合成途径。该菌株可以是产1,3-PD和/或D-LAC的菌株，也可以是不产1,3-PD和/或D-LAC的菌株。外源加入1,3-PD合成途径和/或D-LAC合成途径其目的是为了使得改造后的菌株产1,3-PD和/或D-LAC，或者产1,3-PD和/或D-LAC的量更高。
进一步地，所述引入外源1,3-PD合成途径指：将甘油脱水酶编码基因dhaB及1,3-PD氧化还原酶编码基因dhaT通过质粒转化到所述菌株中，得到菌株A，所述菌株A产1,3-PD。
进一步地，所述引入外源1,3-PD合成途径指：选用产酸克雷伯氏菌中的1,3-PD合成途径中的甘油脱水酶编码基因dhaB及1,3-PD氧化还原酶编码基因dhaT，进行PCR克隆后连接到质粒DNA pet-Duet上，并转化到所述菌株中，得到菌株A，所述菌株A产1,3-PD。
进一步地，所述引入外源D-LAC合成途径指：将D-乳酸脱氢酶的基因dldhBc通过质粒转化到所述菌株中，得到菌株B，所述菌株B产D-LAC。
进一步地，所述引入外源D-LAC合成途径指：选用来自于凝集芽孢杆菌2-6中编码D-乳酸脱氢酶的基因dldhBc，进行适用于所述菌株表达的密码子优化，并将其利用穿梭质粒置换到所述菌株的基因组中的D-乳酸脱氢酶位置，得到菌株B，所述菌株B产D-LAC。
进一步地，还包括如下操作：向引入外源1,3-PD合成途径和/或引入外源D-LAC合成途径的菌株，采用同源重组的方法产生budA、budB、adhE、ackA-pta、poxB和/或frdA基因缺陷的菌株。
本发明还提供一种如上所述的菌的筛选方法，包括如下操作：将土壤样品加入第一液体培养基中培养，再转入固体培养基；将所述固体培养基上长出的单菌落接种到第二液体培养基中培养；培养结束后检测所述第二液体培养基中1,3-丙二醇和D-乳酸的产量，挑取1,3-丙二醇和D-乳酸产量满足要求的菌株。
进一步地，所述第一液体培养基和第二液体培养基均是甘油液体培养基；所述固体培养基是甘油固体培养基。
进一步地，所述单菌落是指带有产酸透明圈的单菌落。
本发明还涉及如上所述的菌在生产1,3-丙二醇或D-乳酸或联产1,3-丙二醇和D-乳酸中的应用。
尤其是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5CCTCC M 2016185在生产1,3-丙二醇或D-乳酸或联产1,3-丙二醇和D-乳酸中的应用。
本发明提供一种通过发酵如上所述的菌联产1,3-丙二醇和D-乳酸的方法。通过发酵如上所述的菌联产1,3-丙二醇和D-乳酸的方法，实际上是指如上所述的菌的应用。
进一步地，如上所述的联产1,3-丙二醇和D-乳酸的方法，其特征在于，所述菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5CCTCC M 2016185。
进一步地，发酵时通气量为0-2.0vvm。优选地，发酵时通气量为1.0vvm。
进一步地，发酵时采用氢氧化钙和水的混合乳液作为中和剂调节发酵液的pH。
进一步地，包括以下步骤：
步骤(1)、菌株选择：选用产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5CCTCC M 2016185；
步骤(2)、种子培养；
步骤(3)、发酵：发酵过程中，使用中和剂调节发酵液pH为5.5-7.5。
进一步地，包括以下步骤：
步骤(1)、菌株选择：选用产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5CCTCC M 2016185；
步骤(2)、种子培养：选用所述步骤(1)的菌株，在无菌条件下接种至甘油培养基中，培养温度为25-40℃，摇床震荡转速为100-300rpm，培养时间为6-24小时，制得种子培养液；
步骤(3)、发酵：将所述步骤(2)中制得的种子培养液接种至装有甘油培养基的发酵罐中，接种量v/v为0.5-10％，发酵温度为25-40℃，通气量为0.3-2.0vvm，搅拌转速为50-400rpm，发酵过程中，使用中和剂调节发酵液pH为5.5-7.5，发酵方式为分批发酵或分批补料发酵，进行分批发酵时，当甘油培养基中的甘油耗尽时，停止发酵；进行分批补料发酵时，当甘油培养基中的甘油耗尽时，通过向发酵罐中补加400-800g/L的甘油溶液，控制发酵液中甘油浓度为5-40g/L，当发酵液中1,3-丙二醇或D-乳酸浓度不再升高时，停止发酵。
进一步地，所述步骤(2)中培养温度为30-37℃，摇床震荡转速为150-250rpm，培养时间为10-16小时；所述步骤(3)中接种量v/v为2-6％，发酵温度为30-37℃，通气量为0.7-1.5vvm，搅拌转速为150-300rpm，调节发酵液pH为6.0-7.0，发酵方式为分批补料发酵，补加的甘油溶液中甘油浓度为500-700 g/L，控制发酵液中甘油浓度为10-30g/L；所述步骤(3)中的中和剂包括氢氧化钠水溶液、氢氧化钾水溶液、氨水水溶液，以及氢氧化钙和水的混合乳液中的任一一种或者多种。
本发明的有益效果包括：
1、使用产酸克雷伯氏菌PDL-5CCTCC M 2016185，得到的发酵液中目标产物浓度高，副产物种类少且浓度低。可生产浓度超过70g/L的1,3-PD和浓度超过110g/L的D-LAC，1,3-PD和D-LAC的总转化率超过90％，生产的D-LAC具有高光学纯度(纯度大于99.9％)。发酵液中仅能检测到少量的副产物乙酸(2.3g/L)和琥珀酸(4.1g/L)。
联产1,3-PD和光学纯LAC(L-LAC或D-LAC)的好处是：由于克雷伯氏菌代谢甘油属于甘油歧化途径，分为甘油氧化途径和甘油还原途径。甘油到1,3-丙二醇的转化属于甘油的还原途径，转化过程需要氧化途径中产生的辅因子NADH和ATP来提供还原力及能量。乳酸在2004年被美国能源部评选为前三十种重点发展的平台化合物之一，鉴于其广阔的用途，在2010年又被选为最有前途的平台化合物之一。乳酸是一种重要的单体，可以用来合成可生物降解、可生物相容且环境友好的生物多聚体－聚乳酸(PLA)。在PLA中加入乳酸的一种同分异构体D-乳酸，可以显著的提高PLA的性能以及有利于其在聚合材料中的应用；除了乳酸的经济价值外，合成乳酸的过程中净产1mol NADH，可以为合成1,3-PD提供还原力，从而达到还原力平衡的状态；从甘油到乳酸的转化过程中，是三碳到三碳的转化，没有碳损失，可以保证碳的回复率；另外，乳酸具有非常好的生物相容性，这对工业上高产乳酸至关重要；由于乳酸的生物合成已经实现了工业级别的生产，因此，其下游纯化技术也相对成熟。这一切的特征都使得乳酸成为与1,3-PD联产的最佳选择。而事实也证明联产1,3-PD和D-LAC获得了非常高的总转化率(超过90％)。根据现有报道(Biotechnol Bioeng.Metabolism in 1,3-propanediol fed-batch fermentation by a D-lactate deficient mutant of Klebsiella pneumoniae.2009；104(5):965-72.；Bioresour Technol.Production of optically pure d-lactate from glycerol by engineered Klebsiella pneumoniae strain.2014；172:269-75.)，在克雷伯氏菌中，由甘油直接转化合成1,3-PD的最高产量为95.4g/L，从甘油到1,3-PD的摩尔转化率为0.48mol/mol(即48％)；从甘油直接转化D-乳酸的最高产量为142.1g/L，转化率为0.84mol/mol(即84％)。但是根据现在已有报道，尚无可同时生产1,3-PD和光学纯LAC(L-LAC或D-LAC)的菌株。
由于在工业化大规模生产中，必须考虑产物浓度、转化率，提取成本等因素，如果能够利用一个菌株，以甘油为底物，以1,3-PD和光学纯LAC(L-LAC 或D-LAC)为主要产物，高摩尔转化率的实现同时生产1,3-PD和光学纯LAC(L-LAC或D-LAC)，将大大降低生产成本，提高生产效率。
2、产酸克雷伯氏菌PDL-5CCTCC M 2016185易于离心和过滤，有利于生物材料单体1,3-PD和D-LAC的高效生物法生产和提取。
3、产酸克雷伯氏菌PDL-5CCTCC M 2016185，具有重要的实际应用价值。
4、采用氢氧化钙作为发酵中和剂调节pH，有利于提高发酵液中1,3-PD及D-LAC的产量。分批补料数据显示，1,3-PD及D-LAC的产量分别为74.5g/L、111.9g/L，细胞OD达到了12.5，比用NaOH作为中和剂分别提高了81.2％、56.5％及135.8％。
5、产酸克雷伯氏菌PDL-0、PDL-1、PDL-2、PDL-3、PDL-4和PDL-5联产得到的1,3-PD和D-LAC的浓度差距不大，其质量比都在1:0.1-10范围内，尤其是在1:0.2-5范围内，进一步地在1:0.5-2范围内。
附图说明
图1是对产酸克雷伯氏菌(Klebsiella oxytoca)进行基因改造的示意图。
图2是不同中和剂对发酵产物的影响的柱状图。
图3是不同通气速率对发酵产物的影响的柱状图。
具体实施方式
下面结合实施例对本发明的技术内容做进一步的说明：下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明所述的产酸克雷伯氏菌PDL-5CCTCC M 2016185，由于budA、budB、adhE、ackA-pta、poxB和frdA基因缺陷，导致α-乙酰乳酸脱羧酶、α-乙酰乳酸合成酶、醛脱氢酶、乙酸激酶、乙酰磷酸转移酶、丙酮酸氧化酶和富马酸还原酶的活性丢失，因此2,3-丁二醇、乙醇、乙酸和琥珀酸代谢途径失活，如图1所示。从图1中可以看出，甘油可转化得到1,3-丙二醇和D-乳酸，并且以下代谢途径失活：α-乙酰乳酸合成酶催化丙酮酸生成α-乙酰乳酸；α-乙酰乳酸脱羧酶催化α-乙酰乳酸生成乙偶姻；丙酮酸氧化酶催化丙酮酸生成乙酸；乙酰磷酸转移酶催化乙酰辅酶A生成乙酰磷酸；乙酰激酶催化乙酰磷酸生成乙酸；醛脱氢酶催化乙酰辅酶A生成乙醛；富马酸还原酶催化富马酸生成琥珀酸。在图1中，差号表示该酶活性丢失，相应的代谢途径失活。
使用产酸克雷伯氏菌PDL-5CCTCC M 2016185，以甘油为底物，可生产高浓度的1,3-PD和高浓度的D-LAC。发酵液中检测不到L-LAC，因此，生产的 D-LAC具有高光学纯度(纯度大于99.9％)。发酵液中检测不到2,3-丁二醇、乙醇和甲酸，仅能检测到少量的乙酸和琥珀酸，发酵液中产物检测结果如表1所示。
表1：产酸克雷伯氏菌PDL-5CCTCC M 2016185以甘油为底物发酵液中产物组成
Figure PCTCN2017093235-appb-000001
如表1所示，产酸克雷伯氏菌PDL-5CCTCC M 2016185，以甘油为底物，可生产76.2g/L的1,3-PD和111.9g/L的D-LAC，其中，1,3-PD的摩尔转化率达到42.6％，D-LAC的摩尔转化率达到52.8％，两种主要产物1,3-PD和D-LAC的总转化率超过90％。发酵液中仅能检测到少量的副产物乙酸和琥珀酸。
实施例1：以1,3-PD和LAC为主要产物的菌株的筛选和鉴定
称取2g土壤样品加入50mL甘油液体培养基中，置于摇床中37℃培养24小时，摇床转速为200rpm。然后用无菌的生理盐水稀释培养液，分别稀释10倍、100倍、1000倍和10000倍后涂布到含甘油固体培养基的培养皿中，37℃静置培养24小时。待长出单菌落后，挑选菌落面积和产酸透明圈面积大的菌落，接种到甘油液体培养基中，置于摇床中37℃培养24小时，摇床转速为200rpm。对培养液进行离心，测定培养液中1,3-PD和LAC的产量，挑取一株1,3-PD和LAC产量高，同时易于离心的菌株。
将上述菌株多次在甘油固体培养基上划线分离纯化，然后再进行10个循环的培养测试，10次循环培养产生的1,3-PD和LAC产量和摩尔转化率基本保持原有水平，证明上述菌株即是目标菌株，将该菌株命名为PDL-0。测定菌株PDL-0培养液中D-LAC和L-LAC的比例，结果表明，菌株PDL-0生产的LAC中，D-LAC的比例大于99.9％，L-LAC的比例小于0.01％。
抽提菌株PDL-0的全基因组，然后PCR扩增菌株PDL-0的16S rRNA的基因序列，将PCR产物测序，测序得到的16S rRNA基因序列如SEQ ID NO:7所示。 菌株PDL-0的16S rRNA基因序列与NCBI数据库(http://www.ncbi.nlm.nih.gov/)中其他产酸克雷伯氏菌的16S rRNA基因序列具有99％的同源性，分析结果表明，菌株PDL-0为产酸克雷伯氏菌。
其中，所述甘油液体培养基的配方为：酵母粉3g/L、K2HPO4·3H2O 10g/L、KH2PO4 2g/L、NH4Cl 1g/L、MgSO4·7H2O 0.5g/L、FeCl3·6H2O 20mg/L、CoCl2·6H2O 50mg/L和甘油20g/L；121℃灭菌20分钟。
其中，所述甘油固体培养基的配方为：酵母粉3g/L、K2HPO4·3H2O 10g/L、KH2PO4 2g/L、NH4Cl 1g/L、MgSO4·7H2O 0.5g/L、FeCl3·6H2O 20mg/L、CoCl2·6H2O 50mg/L，甘油20g/L和琼脂粉15g/L；121℃灭菌20分钟。
实施例1给出了联产1,3-PD和D-LAC菌株的筛选方法，其中所涉及的数值范围不应理解为对本发明的限制，其在合理区间内的变动也能达到类似效果。通过本筛选方法获得的菌株不限于PDL-0，可以是其他种属的菌株或者同一种属的其他不同菌株。
实施例2：产酸克雷伯氏菌PDL-0中α-乙酰乳酸脱羧酶基因(budA)和α-乙酰乳酸合成酶基因(budB)的缺陷
(1)用于在产酸克雷伯氏菌PDL-0中部分缺失budA基因的载体的构建
根据budA基因序列(如SEQ ID NO:1所示)设计引物，PCR扩增budA基因的上游和下游同源片段。以产酸克雷伯氏菌PDL-0的基因组DNA为模板，使用引物budA-1：5’-ACATGATTACGAATTCATGAACCATTCTGCTGAATG-3’(如SEQ ID NO:8所示)和引物budA-2：5’-AACGGGCTGGCATCACCGCGAAGGGCGTGC-3’(如SEQ ID NO:9所示)进行PCR扩增，得到上游同源片段；使用引物budA-3：5’-CGCGGTGATGCCAGCCCGTTTTCCGCTTCA-3’(如SEQ ID NO:10所示)和引物budA-4：5’-TACCGAGCTCGAATTCTTAGTTTTCGACTGAGCGAA-3’(如SEQ ID NO:11所示)进行PCR扩增，得到下游同源片段。PCR扩增条件为：95℃5分钟；95℃30秒，60℃30秒，72℃1分钟，共30个循环；72℃5分钟。PCR反应结束后，将PCR扩增产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到上游和下游同源片段。
使用限制性内切酶EcoRI酶切自杀质粒pKR6K(Wang et al.,J.Biol.Chem.2014,289:6080-6090)，将酶切产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到线性化的质粒pKR6K。自杀质粒pKR6K可通过将质粒pK18mobsacB(优宝生物公司)的复制子替换成质粒pCAM140的复制子获得。质粒pCAM140的复制子的序列可通过基因合成的方式获得，质粒pCAM140的序列见文献报道(Wilson K J,Sessitsch A,Corbo J C,et al.β-Glucuronidase(GUS)transposons  for ecological and genetic studies of rhizobia and other Gram-negative bacteria[J].Microbiology,1995,141(7):1691-1705.)。
使用无缝克隆和组装试剂盒(pEASY-Uni Seamless Cloning and Assembly Kit，北京全式金生物技术有限公司)，将上游同源片段、下游同源片段和线性化的质粒pKR6K连接，得到可部分缺失budA基因的自杀质粒pKR6K-ΔbudA。
(2)budA基因部分缺失的产酸克雷伯氏菌的构建
将pKR6K-ΔbudA转化至大肠杆菌S17-1(λpir)(北京全式金生物技术有限公司)中，得到供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔbudA)。将供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔbudA)与受体菌产酸克雷伯氏菌PDL-0进行双亲本杂交，使pKR6K-ΔbudA上的budA基因上游同源片段和下游同源片段与产酸克雷伯氏菌PDL-0的基因组发生同源重组，从而使产酸克雷伯氏菌PDL-0的budA基因缺失200bp，达到使budA基因缺陷的目的，具体方法为：
a.分别接种活化后的供体菌和受体菌至5mL LB液体培养基中，37℃摇床中，200rpm培养2-3小时，当供体菌和受体菌同时生长到OD620nm为0.5-0.8；将5mL供体菌菌液离心，无菌生理盐水洗两遍；将1mL受体菌菌液离心，无菌生理盐水洗两遍；将上述供体菌和受体菌的菌体一共用100μL无菌生理盐水重悬，将重悬液全部滴在LB固体培养基平板中间，平板正面放置，37℃培养12-18小时。
b.将步骤a中LB固体培养基平板上的菌落用无菌生理盐水和刮刀刮下，无菌生理盐水洗两遍，进行适当稀释，涂布于加入了50μg/mL卡那霉素的M9固体培养基平板，37℃培养24-36小时。
c.挑取步骤b中M9固体培养基平板上生长的单菌落至加入50μg/mL卡那霉素的5mL LB液体培养基中，37℃，200rpm培养12小时。转接菌液至新鲜的5mL LB液体培养基中(不添加卡那霉素)，37℃，200rpm培养12小时。
d.将上述菌液适当稀释，涂布于LAS固体培养基平板，25℃培养24小时。
e.挑取步骤d中LAS固体培养基平板上生长的单菌落至5mL LB液体培养基中，37℃，200rpm培养12小时，提取基因组DNA，使用引物budA-1：5’-ACATGATTACGAATTCATGAACCATTCTGCTGAATG-3’(如SEQ ID NO:8所示)和引物budA-4：5’-TACCGAGCTCGAATTCTTAGTTTTCGACTGAGCGAA-3’(如SEQ ID NO:11所示)进行PCR验证。获得的budA基因缺陷的菌株。
其中，所述LB液体培养基配方为：蛋白胨10g/L、酵母粉5g/L和NaCl10g/L，121℃灭菌20分钟。
其中，所述LB固体培养基配方为：蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L和琼脂粉15g/L，121℃灭菌20分钟。
其中，所述M9固体培养基配方为：Na2HPO4·12H2O 1.7g/L、KH2PO4 0.3g/L、NaCl 0.05g/L、NH4Cl 0.1g/L、柠檬酸三钠0.5g/L和琼脂粉15g/L，121℃灭菌20分钟。
其中，所述LAS固体培养基配方为：蛋白胨10g/L、酵母粉5g/L、蔗糖150g/L和琼脂粉15g/L，115℃灭菌20分钟。
(3)用于在budA基因缺陷的菌株中部分缺失budB基因的载体的构建
根据budB基因序列(如SEQ ID NO:2所示)设计引物，PCR扩增budB基因的上游和下游同源片段。以产酸克雷伯氏菌PDL-0的基因组DNA为模板，使用引物budB-1：5’-ACGCGAATTCGTGGATAATCAACATCAACCGCGCC-3’(如SEQ ID NO:12所示)和引物budB-2：5’-ACGCGGATCCGGGGCGTCCCTGCTCGGC-3’(如SEQ ID NO:13所示)进行PCR扩增，得到上游同源片段；使用引物budB-3：5’-ACGCGGATCCATCGCCCGCTATCTCTACAGCTTCC-3’(如SEQ ID NO:14所示)和引物budB-4：5’-ACGCCTGCAGATTTGACTGAGATGAAGCTGGCCCA-3’(如SEQ ID NO:15所示)进行PCR扩增，得到下游同源片段。PCR扩增条件为：95℃5分钟；95℃30秒，60℃30秒，72℃1分钟，共30个循环；72℃5分钟。PCR反应结束后，将PCR扩增产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到上游和下游同源片段。
使用限制性内切酶EcoRI和BamHI酶切上游同源片段，使用限制性内切酶BamHI和PstI酶切下游同源片段，使用限制性内切酶EcoRI和PstI酶切自杀质粒pKR6K(Wang et al.,J.Biol.Chem.2014,289:6080-6090)，将酶切产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到线性化的质粒pKR6K和带有粘性末端的上游和下游同源臂片段。
使用T4连接酶(NEB公司)将带有粘性末端的上游同源片段、下游同源片段和线性化的质粒pKR6K连接，得到可部分缺失budB基因的自杀质粒pKR6K-ΔbudB。
(4)budB基因部分缺失的产酸克雷伯氏菌的构建
将pKR6K-ΔbudB转化至大肠杆菌S17-1(λpir)中，得到供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔbudB)。将供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔbudB)与受体菌budA基因缺陷的菌株进行双亲本杂交，使pKR6K-ΔbudB上的budB基因上游同源片段和下游同源片段与budA基因缺陷的菌株的基因组发生同源重组，从而budA基因缺陷的菌株的budB基因缺失722bp，达到使budB基因缺陷的目的。具体方法与步骤(2)相同，只是PCR验证时，使用引物budB-1：5’-ACGCGAATTCGTGGATAATCAACATCAACCGCGCC-3’(如SEQ ID NO:12 所示)和引物budB-4：5’-ACGCCTGCAGATTTGACTGAGATGAAGCTGGCCCA-3’(如SEQ ID NO:15所示)进行PCR验证。获得的budA基因和budB基因缺陷的菌株，命名为产酸克雷伯氏菌PDL-1。
实施例3：产酸克雷伯氏菌PDL-1中醛脱氢酶基因(adhE)的缺陷
(1)用于在产酸克雷伯氏菌PDL-1中部分缺失adhE基因的载体的构建
根据adhE基因序列(如SEQ ID NO:3所示)设计引物，PCR扩增adhE基因的上游和下游同源片段。以产酸克雷伯氏菌PDL-1的基因组DNA为模板，使用引物adhE-1：5’-ACATGATTACGAATTCATGGCTGTTACTAATGTCGC-3’(如SEQ ID NO:16所示)和引物adhE-2：5’-TGCTGTCTGTTGGCGTTACGGGTCTTCAGG-3’(如SEQ ID NO:17所示)进行PCR扩增，得到上游同源片段；使用引物adhE-3：5’-CGTAACGCCAACAGACAGCATTCAGCCAGT-3’(如SEQ ID NO:18所示)和引物adhE-4：5’-TACCGAGCTCGAATTCTTAAGCGGATTTTTTCGCTT-3’(如SEQ ID NO:19所示)进行PCR扩增，得到下游同源片段。PCR扩增条件为：95℃5分钟；95℃30秒，60℃30秒，72℃1分钟，共30个循环；72℃5分钟。PCR反应结束后，将PCR扩增产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到上游和下游同源片段。
使用限制性内切酶EcoRI酶切自杀质粒pKR6K(Wang et al.,J.Biol.Chem.2014,289:6080-6090)，将酶切产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到线性化的质粒pKR6K。
使用无缝克隆和组装试剂盒(pEASY-Uni Seamless Cloning and Assembly Kit，北京全式金生物技术有限公司)，将上游同源片段、下游同源片段和线性化的质粒pKR6K连接，得到可部分缺失adhE基因的自杀质粒pKR6K-ΔadhE。
(2)adhE基因部分缺失的产酸克雷伯氏菌的构建
将pKR6K-ΔadhE转化至大肠杆菌S17-1(λpir)中，得到供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔadhE)。将供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔadhE)与受体菌产酸克雷伯氏菌PDL-1进行双亲本杂交，使pKR6K-ΔadhE上的adhE基因上游同源片段和下游同源片段与产酸克雷伯氏菌PDL-1的基因组发生同源重组，从而使产酸克雷伯氏菌PDL-1的adhE基因缺失1876bp，达到使adhE基因缺陷的目的。具体方法与实施例2相同，只是PCR验证时，使用引物adhE-1：5’-ACATGATTACGAATTCATGGCTGTTACTAATGTCGC-3’(如SEQ ID NO:16所示)和引物adhE-4：5’-TACCGAGCTCGAATTCTTAAGCGGATTTTTTCGCTT-3’(如SEQ ID NO:19 所示)进行PCR验证。获得的adhE基因缺陷的菌株，命名为产酸克雷伯氏菌PDL-2。
实施例4：产酸克雷伯氏菌PDL-2中乙酸激酶和乙酰磷酸转移酶基因(ackA-pta)的缺陷
(1)用于在产酸克雷伯氏菌PDL-2中部分缺失ackA-pta基因的载体的构建
根据ackA-pta基因序列(如SEQ ID NO:4所示)设计引物，PCR扩增ackA-pta基因的上游和下游同源片段。以产酸克雷伯氏菌PDL-2的基因组DNA为模板，使用引物ackA-pta-1：5’-ACATGATTACGAATTCATGTCGAGTAAGTTAGTACT-3’(如SEQ ID NO:20所示)和引物ackA-pta-2：5’-CACGCGCGGTCCTCAGCGATACCGATCAGG-3’(如SEQ ID NO:21所示)进行PCR扩增，得到上游同源片段；使用引物ackA-pta-3：5’-ATCGCTGAGGACCGCGCGTGGCCATGCTCT-3’(如SEQ ID NO:22所示)和引物ackA-pta-4：5’-TACCGAGCTCGAATTCTTATGCTTGCTGCTGGGACG-3’(如SEQ ID NO:23所示)进行PCR扩增，得到下游同源片段。PCR扩增条件为：95℃5分钟；95℃30秒，60℃30秒，72℃1分钟，共30个循环；72℃5分钟。PCR反应结束后，将PCR扩增产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到上游和下游同源片段。
使用限制性内切酶EcoRI酶切自杀质粒pKR6K(Wang et al.,J.Biol.Chem.2014,289:6080-6090)，将酶切产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到线性化的质粒pKR6K。
使用无缝克隆和组装试剂盒(pEASY-Uni Seamless Cloning and Assembly Kit，北京全式金生物技术有限公司)，将上游同源片段、下游同源片段和线性化的质粒pKR6K连接，得到可部分缺失ackA-pta基因的自杀质粒pKR6K-ΔackA-pta。
(2)ackA-pta基因部分缺失的产酸克雷伯氏菌的构建
将pKR6K-ΔackA-pta转化至大肠杆菌S17-1(λpir)中，得到供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔackA-pta)。将供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔackA-pta)与受体菌产酸克雷伯氏菌PDL-2进行双亲本杂交，使pKR6K-ΔackA-pta上的ackA-pta基因上游同源片段和下游同源片段与产酸克雷伯氏菌PDL-2的基因组发生同源重组，从而使产酸克雷伯氏菌PDL-2的ackA-pta基因缺失2749bp，达到使ackA-pta基因缺陷的目的。具体方法与实 施例2相同，只是PCR验证时，使用引物ackA-pta-1：5’-ACATGATTACGAATTCATGTCGAGTAAGTTAGTACT-3’(如SEQ ID NO:20所示)和引物ackA-pta-4：5’-TACCGAGCTCGAATTCTTATGCTTGCTGCTGGGACG-3’(如SEQ ID NO:23所示)进行PCR验证。获得的ackA-pta基因缺陷的菌株，命名为产酸克雷伯氏菌PDL-3。
实施例5：产酸克雷伯氏菌PDL-3中丙酮酸氧化酶基因(poxB)的缺陷
(1)用于在产酸克雷伯氏菌PDL-3中部分缺失poxB基因的载体的构建
根据poxB基因序列(如SEQ ID NO:5所示)设计引物，PCR扩增poxB基因的上游和下游同源片段。以产酸克雷伯氏菌PDL-3的基因组DNA为模板，使用引物poxB-1：5’-ACATGATTACGAATTCATGAAACAGACCGTGGCGGC-3’(如SEQ ID NO:24所示)和引物poxB-2：5’-AAAATCCCCCGGGTTGAGACCAGTTCACAG-3’(如SEQ ID NO:25所示)进行PCR扩增，得到上游同源片段；使用引物poxB-3：5’-GTCTCAACCCGGGGGATTTTCTCTCGCTGG-3’(如SEQ ID NO:26所示)和引物poxB-4：5’-TACCGAGCTCGAATTCTTACCTTAGCCAGTTAGTTT-3’(如SEQ ID NO:27所示)进行PCR扩增，得到下游同源片段。PCR扩增条件为：95℃5分钟；95℃30秒，60℃30秒，72℃1分钟，共30个循环；72℃5分钟。PCR反应结束后，将PCR扩增产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到上游和下游同源片段。
使用限制性内切酶EcoRI酶切自杀质粒pKR6K(Wang et al.,J.Biol.Chem.2014,289:6080-6090)，将酶切产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到线性化的质粒pKR6K。
使用无缝克隆和组装试剂盒(pEASY-Uni Seamless Cloning and Assembly Kit，北京全式金生物技术有限公司)，将上游同源片段、下游同源片段和线性化的质粒pKR6K连接，得到可部分缺失poxB基因的自杀质粒pKR6K-ΔpoxB。
(2)poxB基因部分缺失的产酸克雷伯氏菌的构建
将pKR6K-ΔpoxB转化至大肠杆菌S17-1(λpir)中，得到供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔpoxB)。将供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔpoxB)与受体菌产酸克雷伯氏菌PDL-3进行双亲本杂交，使pKR6K-ΔpoxB上的poxB基因上游同源片段和下游同源片段与产酸克雷伯氏菌PDL-3的基因组发生同源重组，从而使产酸克雷伯氏菌PDL-3的poxB基因缺失919bp，达到使poxB基因缺陷的目的。具体方法与实施例2相同，只是PCR验证时，使用引物poxB-1： 5’-ACATGATTACGAATTCATGAAACAGACCGTGGCGGC-3’(如SEQ ID NO:24所示)和引物poxB-4：5’-TACCGAGCTCGAATTCTTACCTTAGCCAGTTAGTTT-3’(如SEQ ID NO:27所示)进行PCR验证。获得的poxB基因缺陷的菌株，命名为产酸克雷伯氏菌PDL-4。
实施例6：产酸克雷伯氏菌PDL-4中富马酸还原酶基因(frdA)的缺陷
(1)用于在产酸克雷伯氏菌PDL-4中部分缺失frdA基因的载体的构建
根据frdA基因序列(如SEQ ID NO:6所示)设计引物，PCR扩增frdA基因的上游和下游同源片段。以产酸克雷伯氏菌PDL-4的基因组DNA为模板，使用引物frdA-1：5’-ACATGATTACGAATTCGTGCAAACTTTTCAAGCCGA-3’(如SEQ ID NO:28所示)和引物frdA-2：5’-GTAGATGCCGAGCCGGTTTTATCGGCAGCG-3’(如SEQ ID NO:29所示)进行PCR扩增，得到上游同源片段；使用引物frdA-3：5’-AAAACCGGCTCGGCATCTACCGTACGCCGG-3’(如SEQ ID NO:30所示)和引物frdA-4：5’-TACCGAGCTCGAATTCTCAGCCATTCGTCGTCTCCT-3’(如SEQ ID NO:31所示)进行PCR扩增，得到下游同源片段。PCR扩增条件为：95℃5分钟；95℃30秒，60℃30秒，72℃1分钟，共30个循环；72℃5分钟。PCR反应结束后，将PCR扩增产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到上游和下游同源片段。
使用限制性内切酶EcoRI酶切自杀质粒pKR6K(Wang et al.,J.Biol.Chem.2014,289:6080-6090)，将酶切产物进行1.0％琼脂糖凝胶电泳，回收并纯化，得到线性化的质粒pKR6K。
使用无缝克隆和组装试剂盒(pEASY-Uni Seamless Cloning and Assembly Kit，北京全式金生物技术有限公司)，将上游同源片段、下游同源片段和线性化的质粒pKR6K连接，得到可部分缺失frdA基因的自杀质粒pKR6K-ΔfrdA。
(2)frdA基因部分缺失的产酸克雷伯氏菌的构建
将pKR6K-ΔfrdA转化至大肠杆菌S17-1(λpir)中，得到供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔfrdA)。将供体菌大肠杆菌S17-1(λpir)(pKR6K-ΔfrdA)与受体菌产酸克雷伯氏菌PDL-4进行双亲本杂交，使pKR6K-ΔfrdA上的frdA基因上游同源片段和下游同源片段与产酸克雷伯氏菌PDL-4的基因组发生同源重组，从而使产酸克雷伯氏菌PDL-4的frdA基因缺失991bp，达到使frdA基因缺陷的目的。具体方法与实施例2相同，只是PCR验证时，使用引物frdA-1：5’-ACATGATTACGAATTCGTGCAAACTTTTCAAGCCGA-3’(如SEQ ID NO:28所示)和引物frdA-4： 5’-TACCGAGCTCGAATTCTCAGCCATTCGTCGTCTCCT-3’(如SEQ ID NO:31所示)进行PCR验证。获得的frdA基因缺陷的菌株，命名为产酸克雷伯氏菌PDL-5。
实施例2-6给出了构建budA、budB、adhE、ackA-pta、poxB和frdA酶失活菌株的方法，即通过基因同源重组的方式来实现基因缺陷从而导致其所编码的酶失活。然而导致酶失活的方式并不限于基因同意重组，还可以是：小RNA干扰，点突变，加入相关酶的抑制剂等等。
实施例7：使用产酸克雷伯氏菌PDL-5CCTCC M 2016185分批发酵生产1,3-PD和D-LAC
(1)菌株选择：选用产酸克雷伯氏菌PDL-5CCTCC M 2016185；
(2)种子培养：选用步骤(1)的菌株，在无菌条件下接种至甘油培养基中，培养温度为30℃，摇床震荡转速为200rpm，培养时间为15小时，制得种子培养液；
(3)发酵：将步骤(2)中制得的种子培养液接种至装有甘油培养基的发酵罐中，接种量为5％(v/v)，发酵温度为30℃，通气量为0.5vvm，搅拌转速为200rpm，发酵过程中，使用25％(w/v)氢氧化钠水溶液作为中和剂调节发酵液pH为7.0，发酵方式为分批发酵，当甘油培养基中的甘油耗尽时，停止发酵。
其中，所述甘油培养基的配方为：酵母粉2g/L、K2HPO4·3H2O 5g/L、KH2PO4 1g/L、NH4Cl 2g/L、MgSO4·7H2O 0.1g/L、FeCl3·6H2O 10mg/L、CoCl2·6H2O 10mg/L和甘油60g/L；121℃灭菌20分钟。
发酵14小时后，甘油培养基中的甘油耗尽，停止发酵，检测发酵液中的产物组成和浓度。发酵的主要产物为1,3-PD和D-LAC，1,3-PD浓度为23.1g/L，摩尔转化率为46.6％；D-LAC浓度为27.2g/L，摩尔转化率为46.3％。副产物仅能检测到少量的乙酸和琥珀酸，乙酸浓度为0.4g/L，摩尔转化率为1.0％；琥珀酸浓度为0.6g/L，摩尔转化率为0.8％。发酵液中检测不到2,3-丁二醇、乙醇和甲酸。
实施例8：使用产酸克雷伯氏菌PDL-5CCTCC M 2016185分批补料发酵生产1,3-PD和D-LAC
(1)菌株选择：选用产酸克雷伯氏菌PDL-5CCTCC M 2016185；
(2)种子培养：选用步骤(1)的菌株，在无菌条件下接种至甘油培养基中，培养温度为37℃，摇床震荡转速为150rpm，培养时间为12小时，制得种子培养液；
(3)发酵：将步骤(2)中制得的种子培养液接种至装有甘油培养基的发 酵罐中，接种量为2.5％(v/v)，发酵温度为37℃，通气量为1vvm，搅拌转速为250rpm，发酵过程中，使用25％(w/v)的氢氧化钙和水的混合乳液作为中和剂调节发酵液pH为6.5，发酵方式为分批补料发酵，当甘油培养基中的甘油耗尽时，通过向发酵罐中补加700g/L的甘油溶液，控制发酵液中甘油浓度为5-30g/L，当发酵液中1,3-PD或D-LAC浓度不再升高时，停止发酵。
其中，所述甘油培养基的配方为：酵母粉5g/L、K2HPO4·3H2O 10g/L、KH2PO4 2g/L、NH4Cl 1g/L、MgSO4·7H2O 0.1g/L、FeCl3·6H2O 20mg/L、CoCl2·6H2O 15mg/L和甘油20g/L；121℃灭菌20分钟。
发酵30小时后，发酵液中1,3-PD和D-LAC浓度不再升高时，停止发酵，检测发酵液中的产物组成和浓度。发酵的主要产物为1,3-PD和D-LAC，1,3-PD浓度为76.2g/L，摩尔转化率为42.6％；D-LAC浓度为111.9g/L，摩尔转化率为52.8％。副产物仅能检测到少量的乙酸和琥珀酸，乙酸浓度为2.3g/L，摩尔转化率为1.6％；琥珀酸浓度为4.1g/L，摩尔转化率为1.5％。发酵液中检测不到2,3-丁二醇、乙醇和甲酸。
实施例7和8只是本发明在应用方法上的两个较佳实施方式，其所限定的数值在合理范围内的变化也可以实现同一目的，因此不能以实施例7和8中给出的数值对本发明进行限制。虽然实施例7和8是对产酸克雷伯氏菌PDL-5的发酵产物进行检测，证明了产酸克雷伯氏菌PDL-5可以生产高摩尔转化率的1,3-PD和D-LAC，但是并不能仅仅理解为本发明所公开的产酸克雷伯氏菌进行基因改造的技术方案只适用于产酸克雷伯氏菌PDL-5，而应该理解为只要是通过本发明所公开的对产酸克雷伯氏菌进行基因改造的技术方案所得到的人工菌都具有同时提高1,3-PD和D-LAC的摩尔转化率和减少副产物的能力。此外，实施例1-8是以产酸克雷伯氏菌PDL-0为野生菌进行基因改造的，若通过其他与产酸克雷伯氏菌PDL-0具有相同代谢通路的菌进行同样的基因改造，应当理解为也能同时提高1,3-PD和D-LAC的摩尔转化率和减少副产物。
使用产酸克雷伯氏菌PDL-5CCTCC M 2016185，以甘油为底物，进行分批补料发酵，得到的发酵液中目标产物1,3-PD和D-LAC浓度高，副产物种类少且浓度低，此外，产酸克雷伯氏菌PDL-5CCTCC M 2016185易于离心和过滤，这些优势都有利于1,3-PD和D-LAC的高效生物法生产，同时有利于产物的提取，说明本发明的产酸克雷伯氏菌PDL-5CCTCC M 2016185，具有重要的实际应用价值。
实施例9：发酵中和剂的选择
采用了生产1,3-PD常用的中和剂NaOH来作为中和剂调节pH，分批补料 发酵PDL-5菌株30h后，1,3-PD产量为41.1g/L，D-LAC为71.5g/L。细胞在620nm处的光密度(OD)可以达到5.3。本文中出现的细胞OD均指在细胞在620nm处的光密度。在换用了其他常用的中和剂，例如KOH，NH3H2O后，1,3-PD及D-LAC的产量也没有明显提升，1,3-PD的产量分别为34.7g/L及34.8g/L；D-Lac的产量分别为61.6g/L及73.5g/L；细胞OD分别为5.2及6.1。
采用Ca(OH)2作为联产1,3-PD及D-LAC的中和剂，其分批补料发酵PDL-5菌株30h的数据显示，1,3-PD及D-LAC的产量分别为76.2g/L、111.9g/L，细胞OD达到了12.5，比用NaOH作为中和剂分别提高了85.4％、56.5％及135.8％，如图2所示。因此，选用Ca(OH)2作为最终的发酵中和剂。
分析使用Ca(OH)2作为发酵中和剂更有利于联产1,3-PD及D-LAC的原因是：一般的克雷伯氏菌在代谢甘油合成1,3-PD时，主要的副产物一般是2,3-丁二醇，乙醇等有机醇，合成的酸并不多，少量的NaOH等中和剂就可以满足生产需要。而本发明所涉及的菌合成较多的乳酸，如果用NaOH或者氨水去调试pH，会生成大量的钠盐或者氨盐，这两种盐容易解离，造成细胞渗透压过大，严重抑制细胞的生长及代谢。而乳酸钙不易解离，对细胞渗透压影响小，所以可以让细胞合成更高产量的乳酸。
实施例10：发酵过程中溶氧量的优化
由于合成1,3-PD的关键酶甘油脱水酶是氧敏感的酶，溶氧过高会抑制1,3-PD的合成。因此，通入的空气对其生产是有影响的。在代谢甘油合成1,3-PD的其他报道中也有体现。通常发酵甘油合成1,3-PD的最佳发酵条件为微好氧发酵。而LAC的生物法合成中，通常采用厌氧发酵。而在最近报道的利用克雷伯氏菌发酵甘油合成D-LAC的生产过程中，作者发现，在微好氧的发酵条件下，随着通入空气量的增加，D-LAC的合成也随之增加。由此，发明人在联产两种物质的实验中，也要十分重视溶氧量对细胞中碳代谢流的影响。因此，发明人通过不同的通气速率来达到不同的溶氧量，从而观察并选取最优的条件来进行联产发酵。
发明人在实验中采用了厌氧条件及微好氧条件来进行联产发酵。具体的实现方法体现在通气速率的改变。通气速率分别为0,0.5vvm，1.0vvm，2.0vvm。搅拌速度都为250rpm。发酵数据见表2。
表2：通气速率对发酵产物的影响
Figure PCTCN2017093235-appb-000002
Figure PCTCN2017093235-appb-000003
由表2和图3(根表2绘制的柱状图)中的数据，选取1,3-PD产量最高的条件，即通气速率为1.0vvm的条件为最终分批发酵条件。
实施例11：产酸克雷伯氏菌PDL-0产1,3-PD和D-LAC分析
使用产酸克雷伯氏菌PDL-0，以甘油为底物，进行分批补料发酵，得到的发酵液中目标产物1,3-PD和D-LAC浓度高，以6M的NaOH作为发酵中和剂时，产量分别为35.0g/L及44.9g/L，摩尔量接近1：1。副产物种类为乙酸、琥珀酸、甲酸等有机酸及2,3-丁二醇、乙醇等有机醇。各副产物浓度相对较低。野生型PDL-0在以25％(w/v)的氢氧化钙和水的混合乳液作为中和剂调节发酵液pH时，最终的1,3-PD及D-LAC的产量分别为50.7g/L和64.2g/L，摩尔转化率分别是36.5％和39.0％，摩尔量仍然接近1:1。该菌天然的D-乳酸脱氢酶的酶学特性为基因工程改造此菌提供了便利。
此外，该菌易于离心和过滤，这些优势都有利于在其基因工程改造后实现1,3-PD和D-LAC的高效生物法生产，同时有利于产物的提取，具有重要的实际应用价值。
实施例12：产酸克雷伯氏菌PDL-1产1,3-PD和D-LAC分析
发酵方法：
(1)菌株选择：选用产酸克雷伯氏菌PDL-1；
(2)种子培养：选用步骤(1)的菌株，在无菌条件下接种至甘油培养基中，培养温度为37℃，摇床震荡转速为150rpm，培养时间为12小时，制得种子培养液；
(3)发酵：将步骤(2)中制得的种子培养液接种至装有甘油培养基的发酵罐中，接种量为2.5％(v/v)，发酵温度为37℃，通气量为1vvm，搅拌转速为250rpm，发酵过程中，使用6M的氢氧化钠作为中和剂调节发酵液pH为6.5，发酵方式为分批补料发酵，当甘油培养基中的甘油耗尽时，通过向发酵罐中补加700g/L的甘油溶液，控制发酵液中甘油浓度为5-30g/L，当发酵液中1,3-PD或D-LAC浓度不再升高时，停止发酵。
其中，所述甘油培养基的配方为：酵母粉5g/L、K2HPO4·3H2O 10g/L、KH2PO4 2g/L、NH4Cl 1g/L、MgSO4·7H2O 0.1g/L、FeCl3·6H2O 20mg/L、CoCl2·6H2O 15mg/L和甘油20g/L；121℃灭菌20分钟。
发酵30小时后，发酵液中1,3-PD和D-LAC浓度不再升高时，停止发酵，检测发酵液中的产物组成和浓度。
PDL-1的发酵主要产物为1,3-PD和D-LAC，1,3-PD浓度为32.1g/L，D-LAC浓度为60.4g/L。副产物能检测到乙醇、乙酸、甲酸和琥珀酸，乙醇浓度为1.7g/L，乙酸浓度为0.9g/L，甲酸浓度为2.2g/L，琥珀酸浓度为1.5g/L。与野生型相比，发酵液中检测不到2,3-丁二醇的合成，D-LAC的产量从48.2g/L增加到60.1g/L，摩尔转化率从野生型的39.1％增长到52.7％。
实施例13：产酸克雷伯氏菌PDL-2产1,3-PD和D-LAC分析
发酵方法同实施例12中的发酵方法，但选用菌株是产酸克雷伯氏菌PDL-2。发酵结果显示，主要产物为1,3-PD和D-LAC，1,3-PD浓度为43.5g/L，D-LAC浓度为59.3g/L。副产物能检测到乙酸、甲酸和琥珀酸，乙酸浓度为3.9g/L，甲酸浓度为1.1g/L，琥珀酸浓度为1.5g/L。与PDL-1相比，发酵液中检测不到乙醇的合成，D-LAC产量基本没变，1,3-PD的产量从32.1g/L增加到43.5g/L，摩尔转化率从PDL-1的33.2％增长到40.0％。
实施例14：产酸克雷伯氏菌PDL-3产1,3-PD和D-LAC分析
发酵方法同实施例12中的发酵方法，但选用菌株是产酸克雷伯氏菌PDL-3。发酵结果显示，主要产物为1,3-PD和D-LAC，1,3-PD浓度为44.5g/L，D-LAC浓度为71.9g/L。副产物能检测到少量乙酸、甲酸和琥珀酸，乙酸浓度为1.5g/L，甲酸浓度为0.3g/L，琥珀酸浓度为1.5g/L。与PDL-2相比，敲除乙酸一条途径抑制了部分乙酸的合成，1,3-PD产量基本没变，D-LAC的产量从59.3g/L增加到71.9g/L，摩尔转化率从PDL-2的46.0％增长到54.0％。敲除乙酸的一条途径后，乙酸的摩尔转化率从PDL-2的4.5％降到1.6％。
实施例15：副产物甲酸的去除
甲酸在微好氧及厌氧发酵中是微生物中非常重要的代谢途径。由丙酮酸-甲酸裂解酶催化，丙酮酸裂解生成甲酸和乙酰辅酶A。在野生型中检测到了甲酸的合成。因此，在敲除丁二醇途径及乙醇途径后，发明人敲除了编码丙酮酸甲酸裂解酶的基因pflB。发现细胞生长受到了明显抑制，细胞OD从未敲除的7.50降到了敲除后的4.85，1,3-PD的产量也从之前的43.4g/L降到了37.9g/L。这就说明了甲酸途径对细胞代谢非常重要，敲除该途径会影响细胞生长，原因可能是细胞内无法提供足够的乙酰辅酶A来进行TCA循环，从而无法为细胞生长提供足够的ATP。在本发明中构建的PDL-5基础上，发明人敲除了pflB基因，发现在Ca(OH)2作为中和剂的条件下，细胞OD从之前的12.5降到了7.0，1,3-PD的产量从74.5g/L降到了40.5 g/L。由此进一步说明了甲酸途径对细胞代谢的重要性。该途径是不应该失活的。
由于甲酸可以被甲酸脱氢酶及氢化酶代谢为H2和CO2，因此，只要发酵条件控制在一定条件下，中间代谢中合成的甲酸可以被分解掉，从而既可以实现不影响菌体生长，同时在最后又不积累甲酸。这在工程菌构建的过程中，可以在PDL-5的发酵过程中体现。
在优化了发酵条件后，大量的代谢流流向了1,3-PD及LAC，中间少量的甲酸在发酵结束的时候被代谢掉，因此，得到了不积累甲酸的敲除株PDL-5。菌株PDL-4中也有体现，最终积累的甲酸是比菌株PDL-0～3的代谢过程中积累的少。甲酸的消除是个很复杂的过程。调节发酵条件非常重要，因为不同的溶氧可以造成丙酮酸裂解途径的增强或者减弱，从而增加或者减少甲酸的合成。
实施例16：外源引入新的途径，从而实现联产1,3-PD及D-LAC
使用基因工程改造的大肠杆菌K12分批发酵生产1,3-PD和D-LAC
(1)菌株选择：选用大肠杆菌K12；
(2)基因工程改造，在K12中引入外源1,3-PD合成途径
选用产酸克雷伯氏菌中的1,3-PD合成途径中的甘油脱水酶编码基因dhaB及1,3-PD氧化还原酶编码基因dhaT，进行PCR克隆后连接到质粒DNA pet-Duet上，并转化到大肠杆菌K12中。该菌确定为K12-dhaBdhaT。
(3)基因工程改造，在K12-dhaBdhaT中引入外源D-LAC合成途径
选用来自于凝集芽孢杆菌2-6中编码D-乳酸脱氢酶的基因dldhBc，进行适用于大肠杆菌表达的密码子优化，并将其利用穿梭质粒置换到大肠杆菌K12基因组中的D-乳酸脱氢酶位置，从而实现dldhBc在大肠杆菌基因组上的组成型表达。该菌确定为K12-dhaBdhaTdldhBc。
(4)种子培养：选用步骤(3)的菌株，在无菌条件下接种至甘油培养基中，培养温度为37℃，摇床震荡转速为200rpm，培养时间为15小时，制得种子培养液；
(5)发酵：将步骤(4)中制得的种子培养液接种至装有甘油培养基的发酵罐中，接种量为5％(v/v)，发酵温度为37℃，通气量为0.5vvm，搅拌转速为200rpm，发酵过程中，使用氢氧化钙水溶液作为中和剂调节发酵液pH为7.0，发酵方式为分批发酵，当甘油培养基中的甘油耗尽时，停止发酵。
其中，所述甘油培养基的配方为：10mM硫酸铵,50mM MOPS/KOH缓冲液，pH 7.5,5mM磷酸钾缓冲液，pH 7.5,2mM氯化镁，0.7mM氯化钙，50uM氯化锰，1uM氯化锌，1.72uM硫酸铜，2.53uM氯化钴，2.4uM钼酸钠，2uM盐酸硫胺素、0.8ug/mL维生素B12以及50ng/ul氨苄霉素。根据需要，碳源选用甘油60g/L；121℃灭菌20分钟。
发酵3小时培养基中菌的OD为1.0，加IPTG诱导1,3-PD的合成。35小时后，甘油培养基中的甘油耗尽，停止发酵，检测发酵液中的产物组成和浓度。发酵的主要产物为1,3-PD和D-LAC，1,3-PD浓度为14.5g/L，D-LAC浓度为17.2g/L。副产物可以检测到乙醇、甲酸、乙酸和琥珀酸，乙醇浓度为2.1g/L，甲酸浓度为0.6g/L，乙酸浓度为4.4g/L，琥珀酸浓度为0.8g/L。
这里仅仅以大肠杆菌K12为例说明了可以向不具备1,3-PD合成途径和/或D-LAC合成途径的菌株中通过基因工程的手段引入外源1,3-PD合成途径和/或外源D-LAC合成途径。将本实施例给出的构思与本发明实施例1-8进行结合，可以进一步地获得联产高摩尔转化率的1,3-PD和D-LAC的菌株，这也是在本发明的保护范围内。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (65)

  1. 一种菌，其特征在于，所述菌具有联产1,3-丙二醇和D-乳酸的特性。
  2. 如权利要求1所述的菌，其特征在于，所述菌是由野生菌改造获得的人工菌。
  3. 如权利要求2所述的菌，其特征在于，所述野生菌具有如下代谢途径：
    1)甘油→1,3-丙二醇；和/或
    2)甘油→丙酮酸→D-乳酸；
    所述野生菌还具有如下代谢途径中一个或多个：
    3)丙酮酸→α-乙酰乳酸，α-乙酰乳酸合成酶是催化该代谢途径的酶；
    4)α-乙酰乳酸→乙偶姻，α-乙酰乳酸脱羧酶是催化该代谢途径的酶；
    5)丙酮酸→乙酸，丙酮酸氧化酶是催化该代谢途径的酶；
    6)乙酰辅酶A→乙酰磷酸，乙酰磷酸转移酶是催化该代谢途径的酶；
    7)乙酰磷酸→乙酸，乙酰激酶是催化该代谢途径的酶；
    8)乙酰辅酶A→乙醛，醛脱氢酶是催化该代谢途径的酶；
    9)富马酸→琥珀酸，富马酸还原酶是催化该代谢途径的酶；
    所述改造包括：阻断所述代谢途径中的3)－9)中的一个或多个。
  4. 如权利要求3所述的菌，其特征在于，所述野生菌为产酸克雷伯氏菌(Klebsiella oxytoca)。
  5. 如权利要求3所述的菌，其特征在于，所述野生菌为产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0于2016年4月8日保藏于中国典型培养物保藏中心，保藏登记号为CCTCC M 2016184。
  6. 如权利要求3所述的菌，其特征在于，所述改造包括：通过抑制或去除酶的活性来阻断所述代谢途径中的3)－9)中的一个或多个。
  7. 如权利要求6所述的菌，其特征在于，所述改造包括：通过改变酶的基因来抑制或去除酶的活性。
  8. 如权利要求7所述的菌，其特征在于，所述改造包括：通过基因重组的办法来改变酶的基因。
  9. 如权利要求8所述的菌，其特征在于，
    编码α-乙酰乳酸脱羧酶基因的序列如SEQ ID NO:1所示；
    编码α-乙酰乳酸合成酶基因的序列如SEQ ID NO:2所示；
    编码醛脱氢酶基因的序列如SEQ ID NO:3所示；
    编码乙酸激酶和乙酰磷酸转移酶基因的序列如SEQ ID NO:4所示；
    编码丙酮酸氧化酶基因的序列如SEQ ID NO:5所示；
    编码富马酸还原酶基因的序列如SEQ ID NO:6所示。
  10. 如权利要求4所述的菌，其特征在于，所述人工菌的budA、budB、adhE、ackA-pta、poxB和frdA中的一种基因或者多种基因缺陷。
  11. 如权利要求10所述的菌，其特征在于，所述人工菌的budA、budB基因缺陷。
  12. 如权利要求10所述的菌，其特征在于，所述人工菌的budA、budB和adhE基因缺陷。
  13. 如权利要求10所述的菌，其特征在于，所述人工菌的budA、budB、adhE和ackA-pta基因缺陷。
  14. 如权利要求10所述的菌，其特征在于，所述基因缺陷通过同源重组的方法产生。
  15. 如权利要求10所述的菌，其特征在于，所述人工菌的基因型包括ΔbudA ΔbudB ΔadhE ΔackA-pta ΔpoxB ΔfrdA。
  16. 如权利要求10所述的菌，其特征在于，所述人工菌的基因型是ΔbudA ΔbudB ΔadhE ΔackA-pta ΔpoxB ΔfrdA。
  17. 如权利要求4所述的菌，其特征在于，所述人工菌是由所述野生菌的budA、budB、adhE、ackA-pta、poxB和frdA基因缺陷获得；所述人工菌产1,3-PD 和D-LAC，且1,3-PD和D-LAC的总转化率超过90％。
  18. 如权利要求17所述的菌，其特征在于，budA、budB、adhE、ackA-pta、poxB和frdA基因缺陷的获得采用同源重组的方法，且各基因缺陷的获得不存在固定的先后顺序；所述同源重组的方法是指通过PCR扩增以上所述基因的上游同源片段和下游同源片段，并将所述上游同源片段和所述下游同源片段构建到自杀质粒中转化至大肠杆菌(Escherichia coli)中，得到供体菌；将所述供体菌与相应的受体菌进行双亲本杂交，使所述上游同源片段和所述下游同源片段与所述受体菌的基因组发生同源重组，从而获得以上所述基因缺陷的菌株，即所述人工菌。
  19. 如权利要求17所述的菌，其特征在于，所述野生菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0于2016年4月8日保藏于中国典型培养物保藏中心，保藏登记号为CCTCC M 2016184。
  20. 如权利要求10-19任一项所述的菌，其特征在于，所述budA的DNA序列如SEQ ID NO:1所示；和/或所述budB的DNA序列如SEQ ID NO:2所示；和/或所述adhE的DNA序列如SEQ ID NO:3所示；和/或所述ackA-pta的DNA序列如SEQ ID NO:4所示；和/或所述poxB的DNA序列如SEQ ID NO:5所示；和/或所述frdA的DNA序列如SEQ ID NO:6所示。
  21. 如权利要求2所述的菌，其特征在于，所述改造包括向所述野生菌中引入外源1,3-PD合成途径和/或D-LAC合成途径。
  22. 如权利要求21所述的菌，其特征在于，所述引入外源1,3-PD合成途径是将甘油脱水酶编码基因dhaB及1,3-PD氧化还原酶编码基因dhaT转化到所述野生菌中。
  23. 如权利要求21所述的菌，其特征在于，所述引入外源D-LAC合成途径是将D-乳酸脱氢酶的基因dldhBc进行适用于所述野生菌表达的密码子优化，并将其利用穿梭质粒置换到所述野生菌基因组中的D-乳酸脱氢酶位置，从而实现dldhBc在所述野生菌基因组上的组成型表达。
  24. 如权利要求23所述的菌，其特征在于，所述外源引入的D-乳酸脱氢 酶的基因dldhBc来自于凝集芽孢杆菌2-6。
  25. 如权利要求21所述的菌，其特征在于，所述改造还包括：通过基因重组的办法来改变budA、budB、adhE、ackA-pta、poxB和/或frdA基因。
  26. 如权利要求1所述的菌，其特征在于，所述菌为野生菌。
  27. 如权利要求26所述的菌，其特征在于，所述菌是产酸克雷伯氏菌(Klebsiella oxytoca)。
  28. 如权利要求1所述的菌，其特征在于，所述菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0于2016年4月8日保藏于中国典型培养物保藏中心，保藏登记号为CCTCC M 2016184。
  29. 如权利要求1所述的菌，其特征在于，所述菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5于2016年4月8日保藏于中国典型培养物保藏中心，保藏登记号为CCTCC M 2016185。
  30. 如权利要求1所述的菌，其特征在于，所述菌具有如下代谢途径：
    1)甘油→1,3-丙二醇；
    2)甘油→丙酮酸→D-乳酸。
  31. 如权利要求1所述的菌，其特征在于，联产得到的：1,3-丙二醇的摩尔转化率≥36.5％；D-乳酸的摩尔转化率≥39.0％。
  32. 如权利要求31所述的菌，其特征在于，联产得到的：D-乳酸的摩尔转化率≥52.7％。
  33. 如权利要求31所述的菌，其特征在于，联产得到的：1,3-丙二醇的摩尔转化率≥40％。
  34. 如权利要求31所述的菌，其特征在于，联产得到的：D-乳酸的摩尔转化率≥54.0％。
  35. 如权利要求31所述的菌，其特征在于，联产得到的：1,3-丙二醇的摩尔转化率≥42.6％；D-乳酸的摩尔转化率≥52.8％。
  36. 如权利要求1所述的菌，其特征在于，联产得到的：1,3-丙二醇和D-乳酸的总转化率超过90％。
  37. 如权利要求1所述的菌，其特征在于，联产得到的1,3-丙二醇和D-乳酸的质量比为1:0.1-10。
  38. 如权利要求37所述的菌，其特征在于，联产得到的1,3-丙二醇和D-乳酸的质量比为1:0.2-5。
  39. 如权利要求38所述的菌，其特征在于，联产得到的1,3-丙二醇和D-乳酸的质量比为1:0.5-2。
  40. 如权利要求1-19或21-39任一项所述的菌，其特征在于，所述菌来自于真菌中的曲霉属、酵母属、接合酵母属、毕赤酵母属、克鲁维酵母属、假丝酵母属、汉逊酵母属、德巴利酵母属、毛霉属、球拟酵母属或链霉菌属；或者来自细菌中的甲基菌属、沙门氏菌属、芽孢杆菌属、假单胞菌属、克雷伯氏菌、乳杆菌属、肠杆菌属、柠檬酸杆菌属、暗杆菌属、泥杆菌属或者梭菌属。
  41. 一种如权利要求1或3所述的菌的构建方法，其特征在于，包括以下步骤：
    步骤一、从土壤样品中筛选能生产1,3-丙二醇和乳酸的产酸克雷伯氏菌；
    步骤二、使得所述步骤一中得到的产酸克雷伯氏菌的budA、budB、adhE、ackA-pta、poxB和frdA中的一种基因或者多种基因缺陷，得到所述菌。
  42. 如权利要求41所述的菌的构建方法，其特征在于，所述步骤一中得到的产酸克雷伯氏菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0，所述产酸克雷伯氏菌(Klebsiella oxytoca)PDL-0于2016年4月8日保藏于中国典型培养物保藏中心，保藏登记号为CCTCC M 2016184。
  43. 如权利要求41所述的菌的构建方法，其特征在于，所述budA的DNA序列如SEQ ID NO:1所示；所述budB的DNA序列如SEQ ID NO:2所示；所述adhE的DNA序列如SEQ ID NO:3所示；所述ackA-pta的DNA序列如SEQ  ID NO:4所示；所述poxB的DNA序列如SEQ ID NO:5所示；所述frdA的DNA序列如SEQ ID NO:6所示。
  44. 如权利要求42所述的菌的构建方法，其特征在于，所述步骤二包括以下步骤：
    步骤2-1、使所述产酸克雷伯氏菌PDL-0 CCTCC M 2016184的α-乙酰乳酸脱羧酶基因和α-乙酰乳酸合成酶基因缺陷，得到α-乙酰乳酸脱羧酶和α-乙酰乳酸合成酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-1；
    步骤2-2、使所述产酸克雷伯氏菌PDL-1的醛脱氢酶基因缺陷，得到醛脱氢酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-2；
    步骤2-3、使所述产酸克雷伯氏菌PDL-2的乙酸激酶和乙酰磷酸转移酶基因缺陷，得到乙酸激酶和乙酰磷酸转移酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-3；
    步骤2-4、使所述产酸克雷伯氏菌PDL-3的丙酮酸氧化酶基因缺陷，得到丙酮酸氧化酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-4；
    步骤2-5、使所述产酸克雷伯氏菌PDL-4的富马酸还原酶基因缺陷，得到富马酸还原酶活性丢失的菌株，命名为产酸克雷伯氏菌PDL-5；
    所述α-乙酰乳酸脱羧酶基因序列如SEQ ID NO.1所示；所述α-乙酰乳酸合成酶基因序列如SEQ ID NO.2所示；所述醛脱氢酶基因序列如SEQ ID NO.3所示；所述乙酸激酶和乙酰磷酸转移酶基因序列如SEQ ID NO.4所示；所述丙酮酸氧化酶基因序列如SEQ ID NO.5所示；所述富马酸还原酶基因序列如SEQ ID NO.6所示。
  45. 如权利要求41所述的菌的构建方法，其特征在于，所述步骤二中，使得所述步骤一中得到的产酸克雷伯氏菌的α-乙酰乳酸脱羧酶基因、α-乙酰乳酸合成酶基因、醛脱氢酶基因、乙酸激酶和乙酰磷酸转移酶基因、丙酮酸氧化酶基因和富马酸还原酶基因缺陷是通过PCR扩增以上所述基因的上游同源片段和下游同源片段，并将所述上游同源片段和所述下游同源片段构建到自杀质粒上，并转化至大肠杆菌中，得到供体菌；将所述供体菌与相应的受体菌进行双亲本杂交，使所述上游同源片段和所述下游同源片段与所述受体菌的基因组发生同源重组，从而获得以上所述基因缺陷的菌株，即所述菌。
  46. 如权利要求45所述的菌的构建方法，其特征在于，所述自杀质粒包括自杀质粒pKR6K；所述大肠杆菌包括大肠杆菌(Escherichia coli)S17-1(λpir)；所述供体菌包括产酸克雷伯氏菌PDL-0 CCTCC M 2016184、产酸克雷伯氏菌 PDL-1、产酸克雷伯氏菌PDL-2和产酸克雷伯氏菌PDL-3；所述受体菌包括产酸克雷伯氏菌PDL-1、产酸克雷伯氏菌PDL-2和产酸克雷伯氏菌PDL-3和产酸克雷伯氏菌PDL-4。
  47. 一种如权利要求1所述的菌的构建方法，其特征在于，通过基因工程改造向菌株中引入外源1,3-PD合成途径和/或外源D-LAC合成途径。
  48. 如权利要求47所述的构建方法，其特征在于，所述引入外源1,3-PD合成途径指：将甘油脱水酶编码基因dhaB及1,3-PD氧化还原酶编码基因dhaT通过质粒转化到所述菌株中，得到菌株A，所述菌株A产1,3-PD。
  49. 如权利要求47所述的构建方法，其特征在于，所述引入外源1,3-PD合成途径指：选用产酸克雷伯氏菌中的1,3-PD合成途径中的甘油脱水酶编码基因dhaB及1,3-PD氧化还原酶编码基因dhaT，进行PCR克隆后连接到质粒DNApet-Duet上，并转化到所述菌株中，得到菌株A，所述菌株A产1,3-PD。
  50. 如权利要求47所述的构建方法，其特征在于，所述引入外源D-LAC合成途径指：将D-乳酸脱氢酶的基因dldhBc通过质粒转化到所述菌株中，得到菌株B，所述菌株B产D-LAC。
  51. 如权利要求47所述的构建方法，其特征在于，所述引入外源D-LAC合成途径指：选用来自于凝集芽孢杆菌2-6中编码D-乳酸脱氢酶的基因dldhBc，进行适用于所述菌株表达的密码子优化，并将其利用穿梭质粒置换到所述菌株的基因组中的D-乳酸脱氢酶位置，得到菌株B，所述菌株B产D-LAC。
  52. 如权利要求47-51任一项所述的构建方法，其特征在于，还包括如下操作：向引入外源1,3-PD合成途径和/或引入外源D-LAC合成途径的菌株，采用同源重组的方法产生budA、budB、adhE、ackA-pta、poxB和/或frdA基因缺陷的菌株。
  53. 一种如权利要求1或26所述的菌的筛选方法，其特征在于，包括如下操作：将土壤样品加入第一液体培养基中培养，再转入固体培养基；将所述固体培养基上长出的单菌落接种到第二液体培养基中培养；培养结束后检测所述第二液体培养基中1,3-丙二醇和D-乳酸的产量，挑取1,3-丙二醇和D-乳酸产量满足要求的菌株。
  54. 如权利要求53所述的筛选方法，其特征在于，所述第一液体培养基和第二液体培养基均是甘油液体培养基；所述固体培养基是甘油固体培养基。
  55. 如权利要求53所述的筛选方法，其特征在于，所述单菌落是指带有产酸透明圈的单菌落。
  56. 如权利要求1-19或21-39任一项所述的菌在生产1,3-丙二醇或D-乳酸或联产1,3-丙二醇和D-乳酸中的应用。
  57. 产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5 CCTCC M 2016185在生产1,3-丙二醇或D-乳酸或联产1,3-丙二醇和D-乳酸中的应用。
  58. 一种通过发酵如权利要求1-19或21-39任一项所述的菌联产1,3-丙二醇和D-乳酸的方法。
  59. 如权利要求58所述的联产1,3-丙二醇和D-乳酸的方法，其特征在于，所述菌是产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5 CCTCC M 2016185。
  60. 如权利要求58所述的联产1,3-丙二醇和D-乳酸的方法，其特征在于，发酵时通气量为0-2.0vvm。
  61. 如权利要求58所述的联产1,3-丙二醇和D-乳酸的方法，其特征在于，发酵时通气量为1.0vvm。
  62. 如权利要求58所述的联产1,3-丙二醇和D-乳酸的方法，其特征在于，发酵时采用氢氧化钙和水的混合乳液作为中和剂调节发酵液的pH。
  63. 如权利要求58所述的联产1,3-丙二醇和D-乳酸的方法，其特征在于，包括以下步骤：
    步骤(1)、菌株选择：选用产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5 CCTCC M 2016185；
    步骤(2)、种子培养；
    步骤(3)、发酵：发酵过程中，使用中和剂调节发酵液pH为5.5-7.5。
  64. 如权利要求58所述的联产1,3-丙二醇和D-乳酸的方法，其特征在于，包括以下步骤：
    步骤(1)、菌株选择：选用产酸克雷伯氏菌(Klebsiella oxytoca)PDL-5 CCTCC M 2016185；
    步骤(2)、种子培养：选用所述步骤(1)的菌株，在无菌条件下接种至甘油培养基中，培养温度为25-40℃，摇床震荡转速为100-300rpm，培养时间为6-24小时，制得种子培养液；
    步骤(3)、发酵：将所述步骤(2)中制得的种子培养液接种至装有甘油培养基的发酵罐中，接种量v/v为0.5-10％，发酵温度为25-40℃，通气量为0.3-2.0vvm，搅拌转速为50-400rpm，发酵过程中，使用中和剂调节发酵液pH为5.5-7.5，发酵方式为分批发酵或分批补料发酵，进行分批发酵时，当甘油培养基中的甘油耗尽时，停止发酵；进行分批补料发酵时，当甘油培养基中的甘油耗尽时，通过向发酵罐中补加400-800g/L的甘油溶液，控制发酵液中甘油浓度为5-40g/L，当发酵液中1,3-丙二醇或D-乳酸浓度不再升高时，停止发酵。
  65. 如权利要求64所述的联产1,3-丙二醇和D-乳酸的方法，其特征在于，所述步骤(2)中培养温度为30-37℃，摇床震荡转速为150-250rpm，培养时间为10-16小时；所述步骤(3)中接种量v/v为2-6％，发酵温度为30-37℃，通气量为0.7-1.5vvm，搅拌转速为150-300rpm，调节发酵液pH为6.0-7.0，发酵方式为分批补料发酵，补加的甘油溶液中甘油浓度为500-700g/L，控制发酵液中甘油浓度为10-30g/L；所述步骤(3)中的中和剂包括氢氧化钠水溶液、氢氧化钾水溶液、氨水水溶液，以及氢氧化钙和水的混合乳液中的任一一种或者多种。
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