WO2014121724A1 - 5-氨基乙酰丙酸高产菌株及其制备方法和应用 - Google Patents

5-氨基乙酰丙酸高产菌株及其制备方法和应用 Download PDF

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WO2014121724A1
WO2014121724A1 PCT/CN2014/071712 CN2014071712W WO2014121724A1 WO 2014121724 A1 WO2014121724 A1 WO 2014121724A1 CN 2014071712 W CN2014071712 W CN 2014071712W WO 2014121724 A1 WO2014121724 A1 WO 2014121724A1
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strain
aminolevulinic acid
activity
ala
synthesis
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French (fr)
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郑平
陈久洲
蒲伟
孙际宾
吴新阳
马延和
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中国科学院天津工业生物技术研究所
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Priority to DE112014000710.2T priority Critical patent/DE112014000710B4/de
Priority to JP2015556388A priority patent/JP6341936B2/ja
Priority to KR1020157024407A priority patent/KR101814888B1/ko
Priority to US14/766,020 priority patent/US10975400B2/en
Publication of WO2014121724A1 publication Critical patent/WO2014121724A1/zh

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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • C—CHEMISTRY; METALLURGY
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01038—Malate dehydrogenase (oxaloacetate-decarboxylating) (1.1.1.38)
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    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01037—5-Aminolevulinate synthase (2.3.1.37)
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    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01031—Phosphoenolpyruvate carboxylase (4.1.1.31)
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    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01049—Phosphoenolpyruvate carboxykinase (ATP) (4.1.1.49)

Definitions

  • the invention relates to the field of genetic engineering and microbial fermentation technology.
  • the present invention relates to a high-yield strain of 5-aminoacetylpropionic acid, a preparation method and application thereof. Background technique
  • 5-aminolevulinic acid is a precursor of the synthesis of tetrapyrrole compounds such as heme, chlorophyll and vitamin B12, and tetrapyrrole compounds are important components of cytochromes, hemoglobin and chloroplast proteins. , plays an important role in life activities. Due to its degradable, non-toxic and residue-free nature, ALA has broad application prospects in the fields of medicine, agriculture, animal husbandry and household chemicals. It is an important high value-added bio-based chemical. As a new generation of photodynamic drugs, ALA can be used for cancer treatment, tumor diagnosis and treatment of skin diseases. As a plant growth regulator, ALA can greatly promote the growth of flowers, crops and vegetables, and improve the quality of crops, fruits and vegetables. Feed additive ALA enhances animal metabolism and immunity. In recent years, ALA has also been used as a major additive in the development of cosmetics and health foods, and related products have been on the market.
  • ALA is mainly prepared by chemical synthesis methods, and has many disadvantages such as many reaction steps, low conversion rate, high production cost, high energy consumption, use of toxic raw materials in the preparation process, serious environmental pollution, and high price.
  • the ex-factory price of ALA hydrochloride in the Chinese market is about 1.7-1.9 million yuan/kg, while the reagent grade price is as high as 2,000 yuan/gram. High manufacturing costs have become a bottleneck limiting the application of ALA.
  • the reaction synthesizes ALA, known as the C5 pathway.
  • Xie et al Xie L, Hall D, Eiteman MA, Altman E, Appl Microbiol Biotechnol, 2003, 63(3): 267-273
  • wild type E. coli MG1655 expressing a spheroidal Agrobacterium ALA synthetase, optimized for fermentation conditions,
  • the yield of ALA reached 5.2 g/L.
  • Lin Jianping (CN200710068168.6, CN201210013562.0) expressed the ALA synthase gene of Rhodobacter sphaeroides in Escherichia coli Rosetta 2 (DE3). After optimization by fermentation process, the ALA yield reached 6.6 g/L, further optimized after 15 The yield in the L fermenter reached 9.4 g/L, which is the highest yield reported in the literature. All of the above studies have used a relatively simple four-carbon pathway, mainly by expressing exogenous ALA synthase in E. coli and adding substrate succinic acid to abundant LB medium. It is achieved with glycine and inhibitors of downstream metabolic pathways.
  • Kang et al. (Kang Z, Wang Y, Gu P, Wang Q, Qi Q, Metab Eng, 2011, 13(5): 492-498) utilized the optimized engineered C5 pathway and ALA transport protein expression in E.
  • the present invention provides a method for constructing a 5-aminolevulinic acid producing strain, which comprises: enhancing the activity of an enzyme involved in promoting oxaloacetate synthesis in the 5-aminolevulinic acid producing strain or introducing an exogenous source Sexual enzymes that promote the synthesis of oxaloacetate, and/or
  • the activity of the enzyme involved in the downstream metabolic pathway of succinyl-CoA in the 5-aminolevulinic acid producing strain is attenuated.
  • the related enzyme that promotes oxaloacetate synthesis is phosphoenolpyruvate carboxylase or pyruvate carboxylase
  • the succinyl-CoA downstream metabolic pathway-related enzyme is succinyl-CoA Synthetase or succinate dehydrogenase.
  • the activity of the enzyme for promoting oxaloacetate synthesis in the enhanced 5-aminolevulinic acid producing strain can be achieved by one or a combination of the following methods: Enhancing the phosphoenolpyruvate carboxylase Activity, and / or enhance the activity of pyruvate carboxylase.
  • the activity of enhancing phosphoenolpyruvate carboxylase or pyruvate carboxylase can be achieved by one or a combination of the following methods: expressing a homologous or heterologous phosphoenol a gene encoding a pyruvate carboxylase or pyruvate carboxylase, and/or increasing the copy number of said coding gene in said strain, and/or modifying a promoter of said coding gene to enhance transcriptional initiation speed, and/or Or modified to carry the stated The translational regulatory region of the messenger RNA encoding the gene enhances translational strength.
  • said attenuating comprises deleting said succinyl-CoA downstream metabolic pathway-associated enzyme.
  • the succinyl-CoA downstream metabolic pathway-associated enzyme is succinyl-CoA synthetase or succinate dehydrogenase.
  • the attenuating the downstream metabolic pathway-related enzyme of succinyl-CoA in the 5-aminolevulinic acid producing strain can be achieved by one or a combination of the following methods: partial or total knockout of succinyl-CoA synthesis Enzyme or succinate dehydrogenase encoding gene, gene mutation inactivation, gene promoter or translational regulatory region changes its transcription or translational weakening, changing gene sequence to weaken mRNA stability or enzyme structure instability.
  • the method further comprises enhancing the 5-aminolevulinic acid synthesis pathway in the 5-aminolevulinic acid producing strain or introducing an exogenous 5-aminolevulinic acid synthesis pathway.
  • enhancing the 5-aminolevulinic acid synthesis pathway in the 5-aminolevulinic acid producing strain means enhancing 5-aminolevulinic acid synthase in the 5-aminolevulinic acid producing strain Activity or introduction of exogenous 5-aminolevulinic acid synthase.
  • the method comprises enhancing the activity of 5-aminolevulinic acid synthase or introducing an exogenous 5-aminolevulinic acid synthase in the 5-aminolevulinic acid producing strain, Enhance the activity of phosphoenolpyruvate carboxylase and knock out succinyl dehydrogenase.
  • the method further comprises measuring the yield of 5-aminolevulinic acid of the resulting strain.
  • the 5-aminolevulinic acid producing strain obtained by the method can produce 5-aminolevulinic acid at a high level without adding exogenous succinic acid.
  • the 5-aminolevulinic acid producing strain obtained by the method can produce 5-aminolevulinic acid at a high level under aerobic conditions without adding exogenous succinic acid.
  • the strain itself has a 5-aminolevulinic acid synthesis ability.
  • the method of the invention further comprises attenuating the activity of phosphoenolpyruvate carboxylase and/or malic enzyme.
  • the activity of attenuating phosphoenolpyruvate carboxylase and/or malic enzyme is achieved by knocking out the genes of phosphoenolpyruvate carboxylase and/or malic enzyme.
  • the present invention provides a method for constructing a 5-aminolevulinic acid producing strain, the method comprising: attenuating a phosphoenolpyruvate carboxylase in the 5-aminolevulinic acid producing strain And/or malic enzyme activity; and enhancing the 5-aminolevulinic acid synthesis pathway in the 5-aminolevulinic acid producing strain or introducing an exogenous 5-aminolevulinic acid synthesis pathway.
  • the present invention provides a 5-aminolevulinic acid producing strain, wherein the activity of an enzyme involved in promoting oxaloacetate synthesis is enhanced or comprises an exogenous enzyme which promotes synthesis of oxaloacetate, and / or
  • the related enzyme that promotes oxaloacetate synthesis is phosphoenolpyruvate carboxylase or pyruvate carboxylase
  • the succinyl-CoA downstream metabolic pathway-related enzyme is succinyl-CoA Synthetase or succinate dehydrogenase.
  • the 5-aminolevulinic acid synthesis pathway in the strain is also enhanced.
  • the strain has enhanced 5-aminolevulinic acid synthase activity or comprises an exogenous 5-aminolevulinic acid synthase.
  • the activity of 5-aminolevulinic acid synthase in the strain is enhanced or comprises exogenous 5-aminolevulinic acid synthase, phosphoenolpyruvate carboxylase activity or Exogenous phosphoenolpyruvate carboxylase is included and the succinate dehydrogenase is knocked out.
  • the strain is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodopseudomonas palustris, etc. .
  • the activity of phosphoenolpyruvate carboxylase and/or malic enzyme is reduced in the strain.
  • the gene knockout of phosphoenolpyruvate carboxylase and/or malic enzyme in the strain is a preferred embodiment, the gene knockout of phosphoenolpyruvate carboxylase and/or malic enzyme in the strain.
  • the present invention provides a 5-aminolevulinic acid producing strain in which the activity of phosphoenolpyruvate carboxylase and/or malic enzyme is decreased; and 5-amino in the strain The levulinic acid synthesis pathway is enhanced or contains an exogenous 5-aminolevulinic acid synthesis pathway.
  • the present invention provides an Escherichia coli producing 5-aminolevulinic acid, the strain being selected from the group consisting of CGMCC No. 6588 as a deposit number deposited in a Chinese microbial strain The strain of the General Microbiology Center of the Depository Management Committee or the strain deposited with CGMCC No. 6589 as the deposit number in the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • the strain produces 5-aminolevulinic acid without the exogenous addition of succinic acid.
  • the strain produces a yield of 5-aminolevulinic acid greater than 7 g/L. In another preferred embodiment, the strain produces glucose conversion of 5-aminolevulinic acid. The rate is higher than 0.35 (molar ratio), preferably higher than 0.45 (molar ratio), and most preferably higher than 0.5 (molar ratio).
  • the 5-aminolevulinic acid producing strain produces 5-aminolevulinic acid at a high level without the addition of exogenous succinic acid.
  • the 5-aminolevulinic acid producing strain produces 5-aminolevulinic acid at a high level without adding exogenous succinic acid under aerobic conditions.
  • the present invention provides a method of producing 5-aminolevulinic acid, the method comprising:
  • the yield of 5-aminolevulinic acid obtained by the process is greater than 7 g/L.
  • the method can achieve high yield of 5-aminolevulinic acid without additional addition of succinic acid.
  • the present invention also provides a method of producing 5-aminolevulinic acid, the method comprising:
  • the succinyl-CoA downstream metabolic pathway-associated enzyme is succinyl-CoA synthetase or succinate dehydrogenase.
  • the present invention provides the use of the strain of the second or third aspect of the present invention, which is for producing 5-aminolevulinic acid and/or producing 5-aminolevulinic acid as a precursor downstream product .
  • the downstream product is a heme or vitamin which is a precursor of ALA.
  • Figure 1 shows a schematic of the technical solution of the invention for increasing ALA production.
  • Figure 2 shows the genetic map of the expression vector used in the present invention.
  • Figure 3 shows a schematic of a further technical solution of the invention for increasing ALA production.
  • Figure 4 shows a schematic representation of the construction of the pZWAl and pZWA2 recombinant vectors. detailed description
  • exogenous refers to a system that contains substances that did not originally exist. For example, by introducing a gene encoding an enzyme which is not originally present in the strain into a strain by transformation or the like, thereby expressing the enzyme in the strain, the enzyme is "exogenous" to the strain.
  • enhancement refers to the increase, increase, increase or increase of the activity of a protein, such as an enzyme.
  • activity of the enhancing enzyme can be enhanced by expressing the endogenous or heterologous encoding gene of the enzyme, and/or increasing the copy number of the encoding gene, and/or modifying the promoter of the encoding gene. Transcriptional initiation rate, and/or modification of the translational regulatory region of messenger RNA carrying the encoded gene to enhance translational strength, and/or modification of the coding gene itself to enhance mRNA stability, protein stability, release of protein feedback inhibition, etc. to realise.
  • the term "attenuated” as used herein refers to the reduction, attenuation, reduction or complete elimination of certain proteins, such as the activity of an enzyme.
  • the activity of the attenuating enzyme may be caused by a partial or total knockdown of the coding gene, a mutation inactivation or partial inactivation, a change in a gene promoter or a translational regulatory region, such that the transcription or translation is weakened, and the gene sequence is altered. It is achieved by a method such as attenuating its mRNA stability or an unstable enzyme structure or a combination thereof.
  • related enzymes promoting oxaloacetate synthesis refers to an enzyme which is related to the synthesis of oxaloacetate, but which promotes, enhances, increases, etc., the amount of oxaloacetate synthesized.
  • related enzymes that promote oxaloacetate synthesis in the present invention include, but are not limited to, phosphoenolpyruvate carboxylase C or pyruvate carboxylase C.
  • the present invention may utilize various enzymes that promote oxaloacetate synthesis, as long as the enzyme is capable of promoting, enhancing, or Increase the amount of oxaloacetate synthesized.
  • the phosphoenolpyruvate carboxylase used in the present invention is of Escherichia coli origin, and the pyruvate carboxylase is of Rhizobium origin.
  • succinyl-CoA downstream metabolic pathway-associated enzyme refers to an enzyme which synthesizes other substances by using succinyl-CoA as a substrate, thereby consuming succinyl-CoA.
  • the succinyl-CoA downstream metabolic pathway-related enzymes include, but are not limited to, succinyl-CoA synthetase or succinate dehydrogenase.
  • 5-aminolevulinic acid synthesis pathway refers to a specific pathway for the production of 5-aminolevulinic acid in microorganisms, including various enzymes such as 5-aminolevulinic acid synthase, glutamyl-tRNA Synthetase, glutamyl-tRNA reductase or glutamate-1-semialdehyde aminotransferase, and the like.
  • enhanced 5-aminolevulinic acid synthesis pathway refers to a related enzyme involved in the 5-aminolevulinic acid synthesis pathway, such as 5-aminolevulinic acid synthase, glutamyl-tRNA synthetase.
  • the activity of glutamyl-tRNA reductase or glutamate-1-semialdehyde aminotransferase is enhanced.
  • the enzyme is derived from Rhodopseudomonas palustris 5- Aminolevulinic acid synthase.
  • the present invention provides a 5-aminolevulinic acid producing strain in which an activity of a related enzyme which promotes oxaloacetate synthesis is enhanced or contains an exogenous enzyme which promotes oxaloacetate synthesis, and/or The activity of enzymes related to downstream metabolic pathways of succinyl-CoA is diminished.
  • the related enzyme that promotes oxaloacetate synthesis includes, but is not limited to: phosphoenolpyruvate carboxylase or pyruvate carboxylase; the succinyl-CoA downstream metabolic pathway-related enzyme includes However, it is not limited to: succinyl-CoA synthetase or succinate dehydrogenase.
  • the 5-aminolevulinic acid synthesis pathway in the strain is also enhanced or contains an exogenous 5-aminolevulinic acid synthesis pathway. Therefore, in a specific embodiment, the 5-aminolevulinic acid synthesis pathway in the strain of the present invention is enhanced or contains an exogenous 5-aminolevulinic acid synthesis pathway, an activity of an enzyme involved in promoting oxaloacetate synthesis, or an exogenous source. The activity of the enzyme involved in the synthesis of oxaloacetate, and/or the activity of the enzyme involved in the downstream metabolic pathway of succinyl-CoA is diminished.
  • the activity of 5-aminolevulinic acid synthase in the strain of the invention is enhanced or comprises exogenous 5-aminolevulinic acid synthase, phosphoenolpyruvate carboxylase activity enhanced or included Derived pyruvate carboxylase and knocked out succinate dehydrogenase.
  • the activity of phosphoenolpyruvate carboxylase and/or malic enzyme is diminished in the strain of the invention.
  • the gene knockout of phosphoenolpyruvate carboxylase and/or malic enzyme in the strain of the invention is known.
  • Those skilled in the art are aware that many strains can be used to produce 5-aminolevulinic acid. Although these strains are different, their synthetic systems and pathways for the synthesis of 5-aminolevulinic acid are similar.
  • the strain of the present invention may be any strain that can be used to produce 5-aminolevulinic acid, including but not limited to: Escherichia coli C3 ⁇ 4 C /z e n' C /»'ac 0 / ), Cory neb acterium glutamicum, Rhodobacter sphaeroides ⁇ 3 ⁇ 4 j
  • the present invention provides Escherichia coli C&c/zen'c/z coli) deposited with CGMCC No. 6588 as a deposit number in the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • the present invention provides Escherichia coli C&c/zen'c/z ⁇ co/) deposited under the accession number CGMCC No. 6589 in the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • the strain of the present invention can produce 5-aminolevulinic acid without exogenous addition of the precursor succinic acid, and the yield of 5-aminolevulinic acid is higher than 7 g/L. Further, 5-aminolevulinic acid is produced by the strain of the present invention, and the glucose conversion rate is higher than 0.35 (molar ratio), preferably higher than 0.45 (molar ratio), and most preferably higher than 0.5 (molar ratio). In a preferred embodiment, the strain of the present invention can produce 5-aminolevulinic acid at a high level.
  • the strain of the present invention can produce 5-aminolevulinic acid at a high level under aerobic conditions, thereby eliminating the need to modify equipment such as fermenters of the prior art, and facilitating the enlargement at the industrial level. . Therefore, the strain of the present invention can produce 5-aminolevulinic acid at a lower cost and conveniently.
  • strains of the present invention can be used not only to produce 5-aminolevulinic acid, but also to produce various downstream products with 5-aminolevulinic acid as a precursor.
  • the downstream product is a heme or vitamin B 12 with ALA as a precursor.
  • the present invention also provides a method for constructing a 5-aminolevulinic acid producing strain, the method comprising: enhancing the activity of the related enzyme which promotes oxaloacetate synthesis in the 5-aminolevulinic acid producing strain or introducing exogenous An enzyme that promotes the synthesis of oxaloacetate, and/or attenuates the enzyme associated with the downstream metabolic pathway of succinyl-CoA in the 5-aminolevulinic acid producing strain.
  • the related enzyme that promotes oxaloacetate synthesis is phosphoenolpyruvate carboxylase or pyruvate carboxylase
  • the succinyl-CoA downstream metabolic pathway-associated enzyme is succinyl-CoA Synthetase or succinate dehydrogenase.
  • the activity of the enzyme for promoting oxaloacetate synthesis in the enhanced 5-aminolevulinic acid producing strain can be achieved by one or a combination of the following methods: Enhancing the phosphoenolpyruvate carboxylase Activity, and / or enhance the activity of pyruvate carboxylase.
  • the activity of enhancing phosphoenolpyruvate carboxylase or pyruvate carboxylase can be achieved by one or a combination of the following methods: expressing a heterologous phosphoenolpyruvate carboxylate Encoding the gene for the enzyme or pyruvate carboxylase, and/or increasing the copy number of the gene encoded in the strain, and/or modifying the promoter of the encoding gene to enhance transcriptional initiation, and/or modification carrying The translational regulatory region of the messenger RNA encoding the gene enhances translational strength.
  • the attenuating comprises deleting the succinyl-CoA downstream metabolic pathway-associated enzyme.
  • the succinyl-CoA downstream metabolic pathway-associated enzyme is succinyl-CoA synthetase or succinate dehydrogenase.
  • the attenuating the succinyl-CoA downstream metabolic pathway-associated enzyme in the 5-aminolevulinic acid producing strain can be achieved by one or a combination of the following methods: partial or total knockout of succinyl-CoA synthesis Enzyme or succinate dehydrogenase encoding gene, gene mutation inactivation or partial inactivation, gene promoter or translational regulatory region changes its transcription or translation weakening, changing gene sequence to weaken mRNA stability or enzyme structure instability method.
  • the method further comprises enhancing the 5-aminolevulinic acid synthesis pathway or introducing an exogenous 5-aminolevulinic acid synthesis pathway in the 5-aminolevulinic acid producing strain. Therefore, in a specific embodiment, the method comprises: enhancing a 5-aminolevulinic acid synthesis pathway in the 5-aminolevulinic acid production strain or introducing an exogenous 5-aminolevulinic acid synthesis pathway, enhancing the promotion grass
  • the activity of the enzyme involved in the synthesis of acylacetate either contains an exogenous enzyme that promotes the synthesis of oxaloacetate, and/or attenuates the activity of an enzyme involved in the downstream metabolic pathway of succinyl-CoA.
  • the method comprises: enhancing the activity of 5-aminolevulinic acid synthase in the 5-aminolevulinic acid producing strain or introducing exogenous 5-aminolevulinic acid synthase, enhancing phosphoric acid Enol pyruvate carboxylase and knock out succinyl dehydrogenase.
  • the method of the present invention further comprises attenuating the 5-aminolevulinic acid producing bacteria The activity of phosphoenolpyruvate carboxylase and/or malic enzyme in the strain.
  • the activity of attenuating phosphoenolpyruvate carboxylase and/or malic enzyme is achieved by knocking out the genes of phosphoenolpyruvate carboxylase and/or malic enzyme.
  • the method further comprises determining the 5-aminolevulinic acid production of the resulting strain.
  • the 5-aminolevulinic acid producing strain obtained by the method can produce 5-aminolevulinic acid at a high level under aerobic conditions without adding exogenous succinic acid.
  • the present invention enhances the activity of an enzyme involved in promoting oxaloacetate synthesis in an initial strain or introduces an exogenous enzyme that promotes the synthesis of oxaloacetate. And/or attenuating the activity of the enzyme involved in the downstream metabolic pathway of succinyl-CoA in the strain, and/or attenuating the activity of phosphoenolpyruvate carboxylase and/or malic enzyme to increase the production of the strain 5 - The ability of aminolevulinic acid.
  • the method for increasing the yield of 5-aminolevulinic acid or the strain thus obtained should fall within the protection scope of the present invention.
  • the scope of protection of the present invention is not limited to the specific methods employed in the examples and the strains obtained.
  • the present invention further provides a method for producing 5-aminolevulinic acid, the method comprising: 1) cultivating the strain of the present invention, thereby obtaining 5-aminolevulinic acid; 2) 5-aminolevulinic acid is obtained from the fermentation culture system of 1).
  • the yield of 5-aminolevulinic acid obtained by the process is greater than 7 g/L.
  • the method does not add succinic acid and/or uses only glucose as a carbon source.
  • succinyl-CoA downstream metabolic pathway-related enzyme inhibitor to a culture system of a 5-aminolevulinic acid producing strain can also increase 5-aminoacetyl.
  • the production of propionic acid is also increase.
  • the succinyl-CoA downstream metabolic pathway-associated enzyme is succinyl-CoA synthetase or succinate dehydrogenase.
  • the strain of the present invention increases the conversion of sugar and increases the synthesis of succinyl-CoA, which is one of the substrates necessary for the synthesis of ALA, thereby increasing the yield of ALA;
  • strain of the present invention for the preparation of ALA does not require the exogenous addition of the precursor succinic acid, which frees the production process from dependence on the substrate succinic acid addition and the use of expensive media, such as LB, which greatly reduces production costs.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. Suggested article Pieces. Materials and Methods
  • the DNA polymerase used in the examples of the present invention was purchased from Fastpfu of Beijing Quanjin Company; the restriction enzymes and DNA ligase were purchased from Fermentas;
  • Yeast powder and peptone were purchased from the British company Oxoid; glycine and IPTG were purchased from Promega; 5-ALA and p-dimethylaminobenzaldehyde were purchased from Sigma; agar powder and antibiotics were purchased from Beijing Suobao; glucose, glacial acetic acid Perchloric acid, trichloroacetic acid, acetylacetone, chloroform and other commonly used chemical reagents are purchased from Chinese medicine.
  • the plasmid extraction kit and the agarose gel electrophoresis recovery kit were purchased from Shanghai Shenggong, and the relevant operations were carried out in strict accordance with the instructions;
  • DH5a competent cells were purchased from Beijing Quanjin Company.
  • yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, 2% agar powder was added to the solid medium.
  • the antibiotic concentrations were: ampicillin 100 g/mL, kanamycin 30 g/mL.
  • ALA assay 200 ⁇ L of diluted fermentation broth was added to 100 ⁇ L of pH 4.6 sodium acetate buffer, then 5 ⁇ L of acetylacetone was added and incubated for 15 min in a 100 ° C water bath. After cooling to room temperature, an equal volume of Ehrlish's reagent (42 mL) was added. The glacial acetic acid, 8 mL of 70% perchloric acid, and 1 g of dimethylaminobenzaldehyde were mixed, and the absorbance at 553 nm was measured after 10 min of color development.
  • the glucose analysis method was carried out by using the SBA-40D biosensor analyzer manufactured by Shandong Academy of Sciences.
  • Example 1 Construction of a succinyl-CoA A synthase deletion mutant and a succinate dehydrogenase deletion mutant
  • Escherichia coli gene knockout is performed by the classical Red recombination method, and the specific literature is as follows: For the knockout of the succinyl-CoA synthetase structural gene cO) gene, firstly according to the genome sequence and auxiliary of E. coli MG1655 published by NCBI The sequence design of the vector pKD13 primer cC-1 and the specific sequence are shown in the primer sequence table, and the KK resistance gene fragment carrying the homologous arm upstream and downstream of the cC gene was amplified by using pKD13 as a template.
  • the PCR amplification parameters were 94 °C for 2 min; 94 °C for 20 s, 64 °C for 20 s, 72 °C for 1 min, 30 cycles; 72 °C for 5 min.
  • the PCR product was recovered and electroporated into MG1655/pKD46 strain. Competent cell preparation and transformation process was referred to J. Sambrook et al. The transformants were verified by PCR and primers.
  • the size of the wild type strain was 2267 bp, and the size of the cO) gene deletion strain was 1558 bp.
  • the cC deletion mutant was selected according to the band size and named ZPEcAl.
  • primers and sdhAB-2 were designed based on the genomic sequence of E. coli MG1655 published by NCBI and the sequence of the helper vector pKD13, and amplified by pKD 13 as a template.
  • the PCR amplification parameters were 94 °C for 2 min; 94 °C for 20 s, 64 °C for 20 s, 72 V for 1 min, 30 cycles; 72 °C for 5 min.
  • the PCR product was recovered and electroporated into MG1655/pKD46 strain. The process of preparation and transformation of competent cells was referred to J.
  • the PCR amplification parameters were 94 °C for 2 min; 94 °C for 20 s, 60 V for 20 s, 72 V for 1.5 min, 30 cycles; 72 °C extension 5 Min.
  • the c gene fragment was recovered and treated with H dlll, and the plasmid pZGA24 carrying ALA synthase was also constructed (pZGA24 construction see reference: Guo Xiaofei et al., 5-aminoacetyl acetylation by recombinant Escherichia coli deleted by 5-aminolevulinic acid dehydratase Acid, Journal of Tianjin University of Science and Technology, 2012, 27(4): 1-6 ) Also treated with this enzyme, the vector and the fragment are recovered and then ligated with T4 ligase to transform DH5 (competent cells, coated with LB plate containing Amp, The positive clones were picked and extracted and verified by enzyme digestion. The correct recombinant plasmid was named as
  • the primers/ ⁇ c-1 and py C -2 were designed according to the sequence of the promoter BBB-J23 105 in BioBrick and the genomic sequence of Rhizobium CFN42 published by NCBi, and the constitutive start-up was carried out by PCR amplification of the Rhizobium CFN42 genome as a template.
  • the gene fragment of the subsequence, the PCR amplification parameters were 94 °C for 2 min; 94 °C for 20 s, 60 °C for 20 s, 72 °C for 2 min, 30 cycles; 72 °C for 5 min.
  • the target fragment was recovered and treated with phosphorylase, and the pWSK29 vector was treated with restriction endonuclease PvuII and then treated with dephosphorylation enzyme, and the obtained vector fragment and the phosphorylated c gene fragment were ligated with T4 ligase.
  • DH5 competent cells were transformed, LB plates containing Amp were plated, positive clones were picked and plasmids were verified, and the correct recombinant plasmid was designated as pSLS33.
  • Primers/ ⁇ cF and / ⁇ cR were designed according to the sequence of pSLS33 above. The specific sequence is shown in the primer sequence table.
  • the pyc gene fragment with constitutive promoter was amplified by pSLS33 as template.
  • the PCR amplification parameter was 94 °C for 2 min. 94 °C 20 s, 60V 20 s, 72V 2 min, 30 cycles; 72 °C extension for 5 min.
  • Primers pA-1 and pA-2 were designed according to the sequence of pZGA24 vector, and pTrc-/ze vector containing ALA synthase gene was amplified by reverse PCR using pZGA24 as template.
  • the PCR amplification parameters were 94 °C for 2 min; 94 ° C 20 s, 60 °C 20 s, 72 °C 3 min, 30 cycles; 72 °C extension for 5 min.
  • the / ⁇ c gene fragment was recovered and digested with the endonuclease Smal and ligated to the reverse-amplified and purified pZGA24 vector with T4 ligase, and the ligated product was transformed into DH5a competent cells, and the LB plate containing Amp was coated and picked. Positive gram The plasmid was extracted and verified by enzyme digestion. The correct recombinant plasmid was sequenced as pZPA7.
  • sucCD- GTTTAACGTGTCTTATCAGGCCT SEQ ID NO: 3
  • sucCD CGAAAATCATCGCGATAAGCACA SEQ ID NO: 4
  • GCTGCTTCG SEQ ID NO: 6
  • Example 4 Construction of recombinant strain and comparison of ALA yield
  • the recombinant plasmids pZGA24, ⁇ and ⁇ 7 constructed above were transformed into wild-type E. coli MG1655 and sucCD deletion mutant ZPEcAl and sdhAB deletion mutant ZPEcA2, and coated with Amp-resistant LB plates. After overnight culture, the positive clones were extracted and verified. Recombinant strains MG1655/pZGA24, ZPEcAl/pZGA24, ZPEcA2/pZGA24, MG 1655/pZPA6, ZPEcAl/pZPA6, ZPEcA2/pZPA6, MG1655/pZPA7, ZPEcAl/pZPA7 and ZPEcA2/pZPA7 were obtained, respectively.
  • the above recombinant single colonies were inoculated separately with 5 mL of LB liquid medium containing 100 g/mL ampicillin, and cultured at 37 ° C, 220 rpm for 12 h.
  • the 250 mL flask containing 50 mL of fermentation medium was transferred at an initial OD of 0.05, and cultured at 37 °C, 220 rpm for 2.5 h, and finally added with IPTG at a final concentration of 25 ⁇ M.
  • the fermentation broth was collected for 19 h after induction, and ALA was detected. concentration.
  • the fermentation medium is M9 medium supplemented with a small amount of yeast powder.
  • the main components are: Na 2 HP0 4 ' 12H 2 0 12.8 g/L, KH 2 P0 4 3.0 g/L, NaCl 0.5 g/L, NH 4 C1 1.0 g/L, MgS0 4 2 mM, CaCl 2 O. lmM, glucose 10 g/L, yeast powder 2 g/L, glycine 4 g/L, ampicillin concentration 100 g/mL.
  • ALA detection and glucose analysis methods are described in the "Materials and Methods" section.
  • the results of ALA production of each recombinant strain are shown in Table 2.
  • the yield of ALA in control strain MG1655/pZGA24 was only 1.06 g/L, and the yield of ALA in ZPEcAl/pZGA24 and ZPEcA2/pZGA24 strains with sucCD or sdhAB deletion was 1.33 g/L, respectively. 1.45 g/L, which was 25% and 37% higher than the original strain, respectively, indicating that partial or total loss of succinyl-CoA synthetase or succinate dehydrogenase activity in E. coli can increase ALA production.
  • the yields of ALA in ZPEcAl/pZPA6, ZPEcA2/pZPA6, ZPEcAl/pZPA7 and ZPEcA2/pZPA7 strains with SU cCD or sdhAB deletion and overexpression of ppc or pyc reached 2.43 g/L, 3.08 g/L, 2.12 g/L and 2.66 g/L, both higher than the corresponding control strains, indicating that sucCD or sdhAB deletion has a certain overlapping effect with ppc or ⁇ c overexpression, which can promote the biosynthesis of ALA.
  • ZPEcA2/pZPA6 strain ALA with sdhAB deletion and ppc expression had the highest yield, 2.91 times of control strain MG1655/pZGA24, and glucose conversion rate reached the highest 0.47 (molar ratio), which was 2.57 times that of the original strain.
  • the yield of ALA can reach 7 g/L or more, which has reached a good level in China.
  • the above experimental results indicate that the expression of succinyl-CoA synthetase or succinate dehydrogenase is partially or completely deleted based on the expression of exogenous ALA synthase in E. coli, and the expression of related enzymes promoting oxaloacetate synthesis can be enhanced. Greatly increase the output of ALA.
  • the fermentation methods of the recombinant flasks and the ALA and glucose detection methods are the same as above.
  • the results are shown in Table 3. It can be seen from the table that the yield and changes of the recombinant strains are consistent with the corresponding MG1655 recombinant strains. The highest yield is ⁇ A deletion and expression c
  • the JW0713/pZPA7 strain had an ALA yield of 3.21 g/L and a glucose conversion rate of 0.56 (molar ratio), indicating that the method provided by the present invention is equally applicable to other strains of Escherichia coli.
  • the experimental results show that the ALA yield of the recombinant bacteria reaches 3.08 g/L and the yield per unit of ALA reaches 0.76 g/L/OD by using the method provided by the present invention in a shake flask fermentation medium containing glucose as the main carbon source. , 2.91 times and 2.59 times of the starting strain, respectively, the conversion rate of glucose reached 0.47 (mol/mol), which was 2.57 times that of the starting strain.
  • the recombinant bacteria constructed by the method provided by the present invention and the method for producing ALA thereof are free from the dependence on succinic acid addition and the use of expensive LB medium, and have good industrial application prospect and economic value.
  • Example 6 Construction of a recombinant strain with enhanced phosphoenolpyruvate carboxylase and malic enzyme activity and comparison of ALA yield
  • the inventors constructed a recombinant strain of phosphoenolpyruvate carboxylase (encoding gene/and malic enzyme (coding gene ae) with an activity similar to that described in Examples 1-5 and tested their ALA. Yield.
  • Example 2 a gene similar to that of Example 2 was used to clone the gene encoding plc of phosphoenolpyruvate carboxykinase of Escherichia coli MG1655 using the primers shown in Table 4 below, and ligated to the pZGA24 vector, and the obtained recombinant plasmid was named pZPA12. .
  • the primers maeB-F and maeB-R shown in Table 4 below were designed according to the genome sequence of Escherichia coli MG1655 published by NCBI, and the apple acid dehydrogenase encoding gene maeB was amplified by PCR using the E. coli MG 1655 genome as a template.
  • the target fragment was phosphorylated with T4 polynucleotide kinase and ligated to the pZGA24 vector fragment obtained by reverse amplification.
  • the correct recombinant vector was identified as pZPA14 by transformation, restriction enzyme digestion and sequencing.
  • the underlined part is the enzyme cleavage site.
  • the recombinant plasmids pZPA12 and pZPA14 constructed above were transformed into wild-type Escherichia coli MG 1655 by a method similar to that of Example 4 to obtain recombinant strains MG1655/pZPA12 and MG1655/pZPA14, respectively. Then, the ALA production of each recombinant strain was examined by a method similar to that of Example 4, and the results are shown in Table 6 below.
  • the ALA yields of the engineered strains MG1655/pZPA12 and MG1655/pZPA14 expressing PCK and MaeB were 46% and 17% lower than the control strain MG 1655/pZGA24, respectively.
  • the enhanced activity of the phosphoenolpyruvate carboxylase kinase pck did not produce a technical effect of increasing the yield of the strain ALA.
  • Example 7 Effect of knockout of pck or maeB gene on ALA production
  • Example 9 the inventors further studied the effect of the ae gene deletion on ALA synthesis.
  • Primer / ze ⁇ -F was designed according to the sequence of the ATCC 17001 ALA synthase encoding gene published by NCBI (R/zo6fo/weM6fo oM£w palustris) (GenBank: JQ048720.1) (primer sequence:
  • the pZGA24 vector (for the construction of pZGA24, see Reference: Guo Xiaofei et al., Journal of Tianjin University of Science and Technology, 2012, 27(4): 1 -6) was used as a template to obtain the target gene/ze ⁇ by PCR.
  • the PCR amplification system was as follows: PCR amplification The parameters were as follows: 94 °C for 10 min, 94 °C for 20 s, 65 °C for 30 s, 72 V for 40 s, 30 cycles, and 72 °C for 5 min.
  • the target fragment was digested with coRI and S al, and then the obtained fragment was ligated to the same digested plasmid pEC-XK99E by DNA ligase, and the ligation product was transformed into DH5a competent cells, and Kanamycin was coated.
  • the resistant plates were cultured overnight, and the positive clones were picked for colony PCR verification.
  • the correct transformants were verified by sequencing, and the correct recombinant vector was named pZWAl (Fig. 4).
  • PCR amplification parameters were: 94 °C for 10 min, 94 °C for 20 s, 65 °C for 30 s, 72 °C for 2 min, Cycle 30 times, extend at 72 ° C for 5 min.
  • the target fragment was treated with S al and double digestion, and then the obtained fragment was ligated with the same digested pZWAl vector under the action of DNA ligase, and the ligation product was transformed into DH5 (competent cells, coated kanamycin antibiotic) The plates were cultured overnight, and positive clones were picked for colony PCR verification. The correct transformants were verified by sequencing, and the correct recombinant vector was named pZWA2 (Fig. 4).
  • the above recombinant vectors pZWAl and pZWA2 and the control empty vector pEC-XK99E were transformed into Corynebacterium glutamicum ATCC 13032, respectively, to obtain recombinant strains ATCC 13032/pEC-XK99E, ATCC 13032/pZWAl and ATCC 13032/pZWA2.
  • the above recombinant single colonies were inoculated with 10 mL of LB liquid medium containing 25 g/mL kanamycin and 24 g/L glucose, and cultured at 30 ° C, 200 rpm for 12 h. According to the initial OD of 0.3, a 500 1 ⁇ triangle flask containing 50 mL of fermentation medium was transferred, 30 ° ⁇ , 200 ⁇ 111 was cultured for 3 11 and then IPTG was added at a final concentration of 100 ⁇ . The fermentation broth was collected after 32 hours of induction culture. The concentration of ALA was measured.
  • Shake flask fermentation medium The formula is: Glu 50 g/L, (NH 4 ) 2 SO 4 10 g/L, MnSO 4 1 g/L, K 2 HPO 4 1.5 g/L, MgSO 4 0.6 g/L, corn syrup lg/L, Glycine 4 g / L, MOPS 31.395 g / L, adjust the pH to 7.0. The final concentration of kanamycin was 25 g/mL. ALA detection and glucose analysis methods are described in the "Materials and Methods" section.
  • the results of shake flask fermentation are shown in Table 8.
  • the ALA yield of the ATCC 13032/pZWAl strain expressing exogenous ALA synthase alone was 1.3 1 g/L, and the molar conversion of glucose was 0.052.
  • the ALA yield and glucose molar conversion rate of strain ATCC 13032/pZWA2 reached 1.95 g/L and 0.077, respectively, which were 48% higher than ATCC 13032/pZWAl strain, and the effect was obvious. Therefore, the method for modifying the ALA producing strain provided by the present invention is also applicable to microorganisms commonly used in the fermentation industry such as Corynebacterium glutamicum.
  • the present inventors Based on the synthetic route of 5-aminolevulinic acid, the present inventors aimed at the recombinant microorganism producing ALA, and rationally designed and modified the glucose metabolism pathway of the host bacteria, and the results showed that the activity of the enzyme involved in promoting the synthesis of oxaloacetate can significantly increase glucose. Conversion rate and ALA yield. Specifically, the present invention enhances the recovery of oxaloacetate by expressing phosphoenolpyruvate carboxylase or pyruvate carboxylase, and greatly increases the yield of ALA and the conversion rate of glucose.
  • the present inventors have also found that the activity of reducing the downstream metabolic pathway-related enzymes of succinyl-CoA, SP, succinyl-CoA synthetase and succinate dehydrogenase can also be improved to exert similar technical effects.
  • the biological common sense tells us that multi-subunit enzymes need close coordination of subunits to complete the complete biological function. Any subunit sequence or structural changes and changes in expression levels may affect the overall activity of the enzyme.
  • Both succinyl-CoA synthetase and succinate dehydrogenase are multi-subunit enzymes.
  • Succinyl-CoA synthetase includes two subunits, which are encoded by sucC and sucD, respectively.
  • Succinate dehydrogenase contains four subunits, respectively. Encoded by ⁇ / ⁇ , sdhB, and sdhD. By the examples 4 and 5, the inventors confirmed that deletion of the cC and sucD, sucC, sdhA and sdhB, sdhA genes caused the inactivation of the corresponding enzyme, which in turn facilitated ALA synthesis. According to biological common sense, other genetic modifications capable of causing the weakening of the activity of the two enzymes may be inhibited or reduced by other methods such as addition of an inhibitor of exogenous succinyl-CoA synthetase or succinate dehydrogenase. The activity will be beneficial to the biosynthesis of ALA.
  • the modification method of the present invention is also applicable to microorganisms commonly used in fermentation industries such as Corynebacterium glutamicum, and has a fairly good universality.
  • the strains and methods provided by the present invention can significantly increase the yield of ALA and the conversion rate of glucose, and are free from the dependence on succinic acid addition and the use of expensive complex medium, such as LB medium, in the conventional synthesis of ALA. Production costs have good industrial prospects.
  • the inventors also initially expected to enhance the activity of phosphoenolpyruvate carboxylase to increase the amount of precursors required for ALA synthesis, thereby increasing the yield of ALA.
  • specific experiments have demonstrated that enhancing the activity of phosphoenolpyruvate carboxylase does not have the expected technical effect.
  • the inventors have also found that increasing the activity of malic enzyme does not result in an increase in ALA production.
  • the inventors unexpectedly found that attenuating the activity of phosphoenolpyruvate carboxylase and malic enzyme could significantly increase the yield of ALA.
  • the deletion of phosphoenolpyruvate carboxylase can significantly increase ALA production and glucose conversion, and the loss of malic enzyme is also beneficial to increase glucose conversion.

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Abstract

一种构建ALA生产菌株的方法，该方法增强5-氨基乙酰丙酸生产菌株中促进草酰乙酸合成的相关酶的活性或导入外源性促进草酰乙酸合成的相关酶，例如磷酸烯醇式丙酮酸羧化酶或丙酮酸羧化酶，和/或减弱所述菌株中琥珀酰辅酶A下游代谢途径相关酶，例如琥珀酰辅酶A合成酶或琥珀酸脱氢酶的活性，和/或减弱磷酸烯醇式丙酮酸羧化激酶和/或苹果酸酶的活性。利用所述方法构建的ALA高产菌株和利用所述菌株制备ALA的方法。

Description

5-氨基乙酰丙酸高产菌株及其制备方法和应用
技术领域
本发明涉及基因工程和微生物发酵技术领域。具体地说， 本发明涉及 5-氨基乙 酰丙酸的高产菌株及其制备方法和应用。 背景技术
5-氨基乙酰丙酸 （5-aminolevulinic acid, ALA)是生物体合成血红素、 叶绿素、 维生素 B12等四吡咯化合物的前体， 四吡咯化合物则是细胞色素、 血红蛋白、 叶 绿体蛋白等的重要组成部分， 在生命活动中发挥着重要作用。 因具有可降解和无 毒无残留等特点， ALA在医药、 农业、 畜牧业、 日用化学品等领域应用前景广阔， 是一种重要的高附加值生物基化学品。 ALA作为新一代光动力学药物可用于癌症 治疗、肿瘤诊断和皮肤病的治疗等；作为植物生长调节剂 ALA可以大幅促进花卉、 作物和蔬菜的生长， 提高作物、 果品、 蔬菜的品质； 作为动物饲料添加剂 ALA可 以增强动物的新陈代谢和免疫力。 近年来， ALA还作为主要添加成分用于化妆品 以及保健食品的开发， 相关产品已上市销售。
然而， 目前 ALA主要通过化学合成方法制备， 存在反应步骤多、 转化率低、 生产成本高、 能耗物耗高、 制备过程中使用有毒原料、 环境污染严重和价格居高 不下等缺点。 目前中国市场上， ALA盐酸盐的出厂价格大约为 1.7-1.9万元 /公斤， 而试剂级售价高达 2000元 /克。 制造成本居高不下， 已经成为限制 ALA推广应用 的瓶颈因素。
随着社会和科学技术的发展， 以高效清洁的生物发酵法代替化学合成法生产 ALA成为人们研究的重点。 目前利用微生物生产 ALA的方法主要有两类： 一是 对光合细菌进行诱变育种， 选育高产 ALA的突变株并在特定培养基上培养， 积累 较高浓度的 ALA, 但该方法发酵周期较长， 条件控制复杂， 底物和抑制剂的添加 也增加了生产成本。 二是利用代谢工程技术改造微生物的代谢途径， 通过强化 ALA的合成实现 ALA的过量积累。 微生物合成 ALA的途径主要有两条， 一条是 在 ALA合成酶的作用下以琥珀酰 CoA和甘氨酸为前体合成 ALA, 称为 C4途径， 另一条是以谷氨酰 tRNA为前体通过两步反应合成 ALA,称为 C5途径。 Xie等 (Xie L, Hall D， Eiteman MA, Altman E， Appl Microbiol Biotechnol, 2003, 63(3): 267-273) 利用表达球形红细菌 ALA合成酶的野生型大肠杆菌 MG1655 ,经过发酵条件优化， ALA的产量达到 5.2 g/L。 林建平等 (CN200710068168.6, CN201210013562.0)将球 形红细菌的 ALA合成酶基因在大肠杆菌 Rosetta 2 (DE3)中表达， 经过发酵工艺优 化后 ALA产量达到 6.6 g/L, 进一步优化后在 15 L发酵罐中产量达到了 9.4 g/L, 为目前文献报道的最高产量。 以上研究都选用了比较简单的四碳途径， 主要是通 过在大肠杆菌中表达外源 ALA合成酶， 并在丰富的 LB培养基中添加底物琥珀酸 和甘氨酸以及下游代谢途径的抑制剂实现。虽然上述方法的 ALA产量已经达到较 高的水平， 但 LB 培养基的使用以及底物和抑制剂的添加不但造成发酵工艺控制 复杂， 而且导致生产成本增加， 限制了大规模工业化应用。 Shin等 (Shin JA， Kwon YD, Kwon OH, Lee HS， Kim P, J Microbiol Biotechnol, 2007, 17(9): 1579-1584)在表 达类球红细菌 ALA合成酶的大肠杆菌中共表达大肠杆菌自身的 NADP依赖型的 苹果酸酶，其在厌氧条件下，在不添加琥珀酸的丰富培养基中可以提高 ALA产量， 但就 ALA 的提高程度而言， 其总体产量依然很低， 并不能达到生产要求。 Kang 等 (Kang Z, Wang Y， Gu P, Wang Q， Qi Q， Metab Eng, 2011, 13(5): 492-498)在大肠 杆菌中利用优化改造的 C5途径及 ALA运输蛋白的表达， 5 L发酵罐上 ALA的产 量达到 4.13 g/L，并实现了利用以葡萄糖为主要碳源的合成培养基发酵生产 ALA, 但发酵周期较长， 且葡萄糖转化率比较低， 只有 0.168 g/g (摩尔比约为 0.23 ) 。 虽然上述研究对 ALA合成底物的胞内供应进行了部分尝试， 但总体效果不好， 因 此， 目前为止对于 ALA合成的底物供应普遍采用的还是直接外源添加的方式， 尤 其是现在 ALA合成中最常用的 C4合成途径， 尚未见到有利用 C4途径的重组工 程菌在以廉价的葡萄糖为主要碳源的培养基中发酵生产 ALA的报道。
综上所述， 本领域急需开发高效、 低成本、 低污染的 ALA制备方法。 发明内容
本发明的目的在于提供一种构建高水平的 5-氨基乙酰丙酸生产菌株的方法以 及相应获得的菌株。 在第一方面， 本发明提供 5-氨基乙酰丙酸生产菌株的构建方法， 所述方法： 增强所述 5-氨基乙酰丙酸生产菌株中促进草酰乙酸合成的相关酶的活性或导 入外源性的促进草酰乙酸合成的相关酶， 和 /或
减弱所述 5-氨基乙酰丙酸生产菌株中琥珀酰辅酶 A下游代谢途径相关酶的活 性。
在具体的实施方式中， 所述促进草酰乙酸合成的相关酶是磷酸烯醇式丙酮酸 羧化酶或丙酮酸羧化酶， 所述琥珀酰辅酶 A下游代谢途径相关酶是琥珀酰辅酶 A 合成酶或琥珀酸脱氢酶。
在优选的实施方式中， 所述增强 5-氨基乙酰丙酸生产菌株中促进草酰乙酸合 成的相关酶的活性可通过以下方法之一或组合实现： 增强磷酸烯醇式丙酮酸羧化 酶的活性， 和 /或增强丙酮酸羧化酶的活性。
在另一优选的实施方式中， 所述增强磷酸烯醇式丙酮酸羧化酶或丙酮酸羧化 酶的活性， 可通过以下方法之一或组合实现： 表达同源或异源磷酸烯醇式丙酮酸 羧化酶或丙酮酸羧化酶的编码基因， 和 /或增加所述菌株中所述编码基因的拷贝 数，和 /或改造所述编码基因的启动子以增强转录启动速度， 和 /或修改携带有所述 编码基因的信使 RNA的翻译调控区以增强翻译强度。
在优选的实施方式中， 所述减弱包括将所述琥珀酰辅酶 A下游代谢途径相关 酶缺失。
在另一优选的实施方式中， 所述琥珀酰辅酶 A下游代谢途径相关酶是琥珀酰 辅酶 A合成酶或琥珀酸脱氢酶。
在优选的实施方式中， 所述减弱所述 5-氨基乙酰丙酸生产菌株中琥珀酰辅酶 A下游代谢途径相关酶可通过以下方法之一或组合实现： 部分或全部敲除琥珀酰 辅酶 A合成酶或琥珀酸脱氢酶的编码基因、 基因突变失活、 基因启动子或翻译调 控区改变令其转录或翻译弱化、改变基因序列使其 mRNA稳定性减弱或酶结构不 稳定等。
在另一优选的实施方式中， 所述方法还包括增强所述 5-氨基乙酰丙酸生产菌 株中的 5-氨基乙酰丙酸合成途径或导入外源性 5-氨基乙酰丙酸合成途径。
在优选的实施方式中， 增强所述 5-氨基乙酰丙酸生产菌株中的 5-氨基乙酰丙 酸合成途径是指增强所述 5-氨基乙酰丙酸生产菌株中 5-氨基乙酰丙酸合成酶的活 性或导入外源性 5-氨基乙酰丙酸合成酶。
在另一具体的实施方式中， 所述方法包括在所述 5-氨基乙酰丙酸生产菌株中 增强 5-氨基乙酰丙酸合成酶的活性或导入外源性 5-氨基乙酰丙酸合成酶、 增强磷 酸烯醇式丙酮酸羧化酶的活性并敲除琥珀酰脱氢酶。
在另一优选的实施方式中， 所述方法还包括测定所得菌株的 5-氨基乙酰丙酸 产量。
在另一优选的实施方式中， 所述方法获得的 5-氨基乙酰丙酸生产菌株可以无 需添加外源琥珀酸而高水平地产生 5-氨基乙酰丙酸。
在另一优选的实施方式中， 所述方法获得的 5-氨基乙酰丙酸生产菌株可以在 好氧条件下， 无需添加外源琥珀酸而高水平地产生 5-氨基乙酰丙酸。
在另一具体的实施方式中， 所述菌株本身具有 5-氨基乙酰丙酸合成能力。 在另一具体的实施方式中， 本发明的方法还包括减弱磷酸烯醇式丙酮酸羧化 激酶和 /或苹果酸酶的活性。
在优选的实施方式中，所述减弱磷酸烯醇式丙酮酸羧化激酶和 /或苹果酸酶的 活性通过敲除磷酸烯醇式丙酮酸羧化激酶和 /或苹果酸酶的基因得以实现。
在另一方面， 本发明提供了一种 5-氨基乙酰丙酸生产菌株的构建方法， 所述 方法包括：减弱所述 5-氨基乙酰丙酸生产菌株中的磷酸烯醇式丙酮酸羧化激酶和 / 或苹果酸酶的活性； 并且增强所述 5-氨基乙酰丙酸生产菌株中的 5-氨基乙酰丙酸 合成途径或引入外源 5-氨基乙酰丙酸合成途径。 在第二方面， 本发明提供一种 5-氨基乙酰丙酸生产菌株， 所述菌株中促进草 酰乙酸合成的相关酶的活性增强或包含外源性的促进草酰乙酸合成的相关酶， 和 / 或
所述琥珀酰辅酶 A下游代谢途径相关酶的活性减弱。
在具体的实施方式中， 所述促进草酰乙酸合成的相关酶是磷酸烯醇式丙酮酸 羧化酶或丙酮酸羧化酶， 所述琥珀酰辅酶 A下游代谢途径相关酶是琥珀酰辅酶 A 合成酶或琥珀酸脱氢酶。
在另一具体的实施方式中，所述菌株中 5-氨基乙酰丙酸合成途径也得到增强。 在优选的实施方式中， 所述菌株的 5-氨基乙酰丙酸合成酶活性增强或包含外源性 的 5-氨基乙酰丙酸合成酶。
在另一具体的实施方式中， 所述菌株中 5-氨基乙酰丙酸合成酶的活性增强或 包含外源性 5-氨基乙酰丙酸合成酶、 磷酸烯醇式丙酮酸羧化酶活性增强或包含外 源性的磷酸烯醇式丙酮酸羧化酶并敲除琥珀酸脱氢酶。
在另一具体的实施方式中， 所述菌株选自大肠杆菌 (Escherichia coli), 谷氨 酸棒杆菌 {Corynebacterium glutamicum)^ 球形红细菌 (Rhodobacter sphaeroides)、 沼泽红叚单胞菌 (Rhodopseudomonas palustris、等。
在另一具体的实施方式中，所述菌株中磷酸烯醇式丙酮酸羧化激酶和 /或苹果 酸酶的活性减弱。
在优选的实施方式中，所述菌株中磷酸烯醇式丙酮酸羧化激酶和 /或苹果酸酶 的基因敲除。
在另一方面， 本发明提供一种 5-氨基乙酰丙酸生产菌株， 所述菌株中磷酸烯 醇式丙酮酸羧化激酶和 /或苹果酸酶的活性减弱； 并且所述菌株中 5-氨基乙酰丙酸 合成途径增强或含有外源 5-氨基乙酰丙酸合成途径。 在第三方面， 本发明提供一种产生 5-氨基乙酰丙酸的大肠埃希氏菌 {Escherichia coli) , 所述菌株选自下组： 以 CGMCC No.6588为保藏号保藏于中国 微生物菌种保藏管理委员会普通微生物中心的菌株或以 CGMCC No.6589为保藏 号保藏于中国微生物菌种保藏管理委员会普通微生物中心的菌株。
在具体的实施方式中， 所述菌株无需外源性添加琥珀酸即可产生 5-氨基乙酰 丙酸。
在另一优选的实施方式中， 所述菌株产生 5-氨基乙酰丙酸的产量高于 7 g/L o 在另一优选的实施方式中， 所述菌株产生 5-氨基乙酰丙酸的葡萄糖转化率高 于 0.35 (摩尔比）， 优选高于 0.45 (摩尔比）， 最优选高于 0.5 (摩尔比）。
在另一优选的实施方式中， 所述 5-氨基乙酰丙酸生产菌株无需添加外源琥珀 酸而高水平地产生 5-氨基乙酰丙酸。
在另一优选的实施方式中， 所述 5-氨基乙酰丙酸生产菌株在好氧条件下， 无 需添加外源琥珀酸而高水平地产生 5-氨基乙酰丙酸。 在第四方面， 本发明提供一种产生 5-氨基乙酰丙酸的方法， 所述方法包括：
1) 培养本发明第二或第三方面所述的菌株， 从而得到 5-氨基乙酰丙酸； 和
2) 从 1)的发酵培养体系中获得 5-氨基乙酰丙酸。
在优选的实施方式中， 所述方法获得的 5-氨基乙酰丙酸的产量高于 7 g/L。 在另一优选的实施方式中， 所述方法可以在不额外添加琥珀酸的情况下也能 实现 5-氨基乙酰丙酸的高产。 在第五方面，本发明还提供一种产生 5-氨基乙酰丙酸的方法，所述方法包括：
1) 在含有琥珀酰辅酶 A下游代谢途径相关酶抑制剂的培养基中培养 5-氨基 乙酰丙酸的生产菌株， 从而得到 5-氨基乙酰丙酸； 和
2) 从 1)的培养体系中获得 5-氨基乙酰丙酸。
在优选的实施方式中， 所述琥珀酰辅酶 A下游代谢途径相关酶是琥珀酰辅酶 A合成酶或琥珀酸脱氢酶。 在第六方面， 本发明提供本发明第二或第三方面所述菌株的用途， 所述菌株 用于产生 5-氨基乙酰丙酸和 /或产生以 5-氨基乙酰丙酸为前体下游产物。
在优选的实施方式中， 所述下游产物是以 ALA 为前体的血红素或维生素
B 12。 应理解， 在本发明范围内中， 本发明的上述各技术特征和在下文 (如实施例) 中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。 限于篇幅， 在此不再一一赘述。 附图说明
图 1显示了提高 ALA产量的本发明技术方案的示意图。
图 2显示了本发明所用表达载体的遗传图谱。
图 3显示了提高 ALA产量的本发明进一步的技术方案的示意图。
图 4显示了 pZWAl和 pZWA2重组载体的构建示意图。 具体实施方式
发明人经过广泛而深入的研究， 出乎意料地发现增强 5-氨基乙酰丙酸生产菌 株中促进草酰乙酸合成的相关酶的活性可以极大提高所述生产菌株的 5-氨基乙酰 丙酸的产量； 发明人还发现减弱所述生产菌株中琥珀酰辅酶 A下游代谢途径相关 酶也能够提高所得生产菌株的 5-氨基乙酰丙酸的产量； 进一步地， 发明人发现减 弱磷酸烯醇式丙酮酸羧化激酶和苹果酸酶的活性能够显著提高 5-氨基乙酰丙酸的 产量； 而将上述手段中的任意两种或三种结合起来能够进一步提高所得生产菌株 的 5-氨基乙酰丙酸的产量。 在此基础上完成了本发明。 术语定义
本文所用的术语 "外源性" 是指某体系中包含了原来不存在的物质。 例如， 通过转化等方式在某菌株中引入该菌株中原本不存在的酶的编码基因， 从而在该 菌株中表达该酶， 则该酶对于该菌株是 "外源性" 的。
本文所用的术语 "增强" 是指增加、 提高、 增大或升高某种蛋白， 例如酶的 活性。鉴于本发明的教导和现有技术，本领域技术人员也不难理解本文所用的"增 强" 还应包括通过表达酶的异源编码基因而增强其活性。 在具体的实施方式中， 增强酶的活性可以通过表达酶的内源或异源性编码基因， 和 /或增加所述编码基因 的拷贝数，和 /或改造所述编码基因的启动子以增强转录启动速度，和 /或修改携带 有所述编码基因的信使 RNA的翻译调控区以增强翻译强度， 和 /或修改编码基因 本身以增强 mRNA稳定性、蛋白质稳定性、解除蛋白质的反馈抑制等方法来实现。
类似地， 本文所用的术语 "减弱" 是指降低、 削弱、 减小或完全消除某种蛋 白， 例如酶的活性。 在具体的实施方式中， 减弱酶的活性可以通过部分或全部敲 除酶的编码基因、 基因突变失活或部分失活、 基因启动子或翻译调控区改变令其 转录或翻译弱化、改变基因序列使其 mRNA稳定性减弱或酶结构不稳定等方法或 其组合来实现。
本文所用的术语 "促进草酰乙酸合成的相关酶" 是指与草酰乙酸的合成相关， 但对于草酰乙酸的合成量起到促进、 提高、 增加等正面作用的酶。 在具体的实施 方式中， 本发明中促进草酰乙酸合成的相关酶包括但不限于： 磷酸烯醇式丙酮酸羧 化酶 C或丙酮酸羧化酶^ C。 此外， 鉴于本发明的教导和现有技术， 本领域普通 技术人员不难理解， 本发明可利用各种来源的促进草酰乙酸合成的相关酶， 只要所 述酶能在菌株中促进、 提高或增加草酰乙酸的合成量。 在具体的实施方式中， 本 发明所用的磷酸烯醇式丙酮酸羧化酶是大肠杆菌来源的， 而丙酮酸羧化酶是根瘤 菌来源的。
本文所用的术语 "琥珀酰辅酶 A下游代谢途径相关酶"是指利用琥珀酰辅酶 A作为底物合成其它物质， 从而消耗琥珀酰辅酶 A的酶。 在具体的实施方式中， 所述琥珀酰辅酶 A下游代谢途径相关酶包括但不限于：琥珀酰辅酶 A合成酶或琥 珀酸脱氢酶。
本文所用的术语" 5-氨基乙酰丙酸合成途径"是指微生物中产生 5-氨基乙酰丙酸 的具体途径， 其中包括各种酶， 例如 5-氨基乙酰丙酸合成酶、 谷氨酰 -tRNA合成酶、 谷氨酰 -tRNA还原酶或谷氨酸 -1-半醛氨基转移酶，等等。类似地，本文所用的术语" 5- 氨基乙酰丙酸合成途径增强"是指涉及 5-氨基乙酰丙酸合成途径的相关酶， 例如 5- 氨基乙酰丙酸合成酶、谷氨酰 -tRNA合成酶、谷氨酰 -tRNA还原酶或谷氨酸 -1-半醛氨 基转移酶的活性增强。 在优选的实施方式中， 所述酶是来源于沼泽红假单胞菌的 5- 氨基乙酰丙酸合成酶。 本发明的 5-氨基乙酰丙酸生产菌株
本发明提供了一种 5-氨基乙酰丙酸生产菌株， 所述菌株中促进草酰乙酸合成的 相关酶的活性增强或包含外源性的促进草酰乙酸合成的相关酶， 和 /或所述琥珀酰辅 酶 A下游代谢途径相关酶的活性减弱。 在优选的实施方式中， 所述促进草酰乙酸合 成的相关酶包括但不限于： 磷酸烯醇式丙酮酸羧化酶或丙酮酸羧化酶； 所述琥珀酰 辅酶 A下游代谢途径相关酶包括但不限于：琥珀酰辅酶 A合成酶或琥珀酸脱氢酶。
在其它实施方式中， 所述菌株中 5-氨基乙酰丙酸合成途径也得到增强或含有外 源 5-氨基乙酰丙酸合成途径。 因此， 在具体的实施方式中， 本发明菌株中 5-氨基乙酰 丙酸合成途径增强或含有外源 5-氨基乙酰丙酸合成途径、促进草酰乙酸合成的相关酶 的活性增强或包含外源性的促进草酰乙酸合成的相关酶，和 /或所述琥珀酰辅酶 A下 游代谢途径相关酶的活性减弱。 在优选的实施方式中， 本发明菌株中 5-氨基乙酰 丙酸合成酶的活性增强或包含外源性 5-氨基乙酰丙酸合成酶、 磷酸烯醇式丙酮酸羧 化酶活性增强或包含外源性的丙酮酸羧化酶并敲除琥珀酸脱氢酶。
在另一具体的实施方式中， 本发明的菌株中磷酸烯醇式丙酮酸羧化激酶和 / 或苹果酸酶的活性减弱。
在优选的实施方式中，本发明的菌株中磷酸烯醇式丙酮酸羧化激酶和 /或苹果 酸酶的基因敲除。 本领域技术人员知道许多菌株可以用于产生 5-氨基乙酰丙酸。 这些菌株虽然 不同， 但它们合成 5-氨基乙酰丙酸的合成体系、 途径却是类似的。 因此， 本领域 普通技术人员鉴于本发明的教导和现有技术可以明白， 本发明的菌株可以是任何 可用于产生 5-氨基乙酰丙酸的菌株， 包括但不限于： 大肠杆菌 C¾C/zen'C/»'a c0/ )、 谷氨酸棒杆菌 (Cory neb acterium glutamicum)、 球形红细菌 (Rhodobacter sphaeroides)^ ¾ j|¾ -^- ffi ¾ {Rhodopseudomonas palustris、等。
在具体的实施方式中， 本发明提供以 CGMCC No.6588为保藏号保藏于中国 微生物菌种保藏管理委员会普通微生物中心的大肠埃希氏菌 C&c/zen'c/z coli)。 在 另一具体的实施方式中， 本发明提供以 CGMCC No.6589为保藏号保藏于中国微 生物菌种保藏管理委员会普通微生物中心的大肠埃希氏菌 C&c/zen'c/z^ co/ )。
本发明菌株无需外源性添加前体琥珀酸即可产生 5-氨基乙酰丙酸， 5-氨基乙 酰丙酸的产量高于 7 g/L。 此外， 利用本发明的菌株产生 5-氨基乙酰丙酸， 葡萄糖转 化率高于 0.35 (摩尔比)， 优选高于 0.45 (摩尔比)， 最优选高于 0.5 (摩尔比)。 在优选 的实施方式中， 本发明的菌株可以高水平地生产 5-氨基乙酰丙酸。在另一优选的实施 方式中， 本发明的菌株可以在好氧条件下高水平地生产 5-氨基乙酰丙酸， 从而无需对 现有技术水平的发酵罐等设备进行改造， 便于在工业化水平放大。 因此， 本发明的菌株可以更低的成本、 方便地制备 5-氨基乙酰丙酸。
本领域技术人员还应明白， 本发明的菌株不仅可用于产生 5-氨基乙酰丙酸， 还 能产生以 5-氨基乙酰丙酸为前体的各种下游产物。 在具体的实施方式中， 所述下游 产物是以 ALA为前体的血红素或维生素 B 12。 本发明还提供一种 5-氨基乙酰丙酸生产菌株的构建方法， 所述方法包括： 增强 所述 5-氨基乙酰丙酸生产菌株中促进草酰乙酸合成的相关酶的活性或导入外源性的 促进草酰乙酸合成的相关酶， 和 /或减弱所述 5-氨基乙酰丙酸生产菌株中琥珀酰辅酶 A 下游代谢途径相关酶。 在优选的实施方式中， 所述促进草酰乙酸合成的相关酶是 磷酸烯醇式丙酮酸羧化酶或丙酮酸羧化酶， 所述琥珀酰辅酶 A下游代谢途径相关 酶是琥珀酰辅酶 A合成酶或琥珀酸脱氢酶。
在优选的实施方式中， 所述增强 5-氨基乙酰丙酸生产菌株中促进草酰乙酸合成 的相关酶的活性可通过以下方法之一或组合实现： 增强磷酸烯醇式丙酮酸羧化酶 的活性， 和 /或增强丙酮酸羧化酶的活性。
在进一步优选的实施方式中， 所述增强磷酸烯醇式丙酮酸羧化酶或丙酮酸羧 化酶的活性， 可通过以下方法之一或组合实现： 表达异源磷酸烯醇式丙酮酸羧化 酶或丙酮酸羧化酶的编码基因， 和 /或增加所述菌株中所述编码基因的拷贝数， 和 /或改造所述编码基因的启动子以增强转录启动速度，和 /或修改携带有所述编码基 因的信使 RNA的翻译调控区以增强翻译强度。
在另一优选的实施方式中，所述减弱包括将所述琥珀酰辅酶 A下游代谢途径相 关酶缺失。 在进一步优选的实施方式中， 所述琥珀酰辅酶 A下游代谢途径相关酶 是琥珀酰辅酶 A合成酶或琥珀酸脱氢酶。
在优选的实施方式中，所述减弱所述 5-氨基乙酰丙酸生产菌株中琥珀酰辅酶 A 下游代谢途径相关酶可通过以下方法之一或组合实现： 部分或全部敲除琥珀酰辅 酶 A合成酶或琥珀酸脱氢酶的编码基因、 基因突变失活或部分失活、 基因启动子 或翻译调控区改变令其转录或翻译弱化、改变基因序列使其 mRNA稳定性减弱或 酶结构不稳定等方法。
在另一优选的实施方式中， 所述方法还包括增强所述 5-氨基乙酰丙酸生产菌株 中 5-氨基乙酰丙酸合成途径或引入外源 5-氨基乙酰丙酸合成途径。因此，在具体的实 施方式中， 所述方法包括： 增强所述 5-氨基乙酰丙酸生产菌株中 5-氨基乙酰丙酸合 成途径或引入外源 5-氨基乙酰丙酸合成途径、增强促进草酰乙酸合成的相关酶的活性 或包含外源性的促进草酰乙酸合成的相关酶，和 /或减弱所述琥珀酰辅酶 A下游代谢 途径相关酶的活性。 在优选的实施方式中， 所述方法包括： 增强所述 5-氨基乙酰 丙酸生产菌株中 5-氨基乙酰丙酸合成酶的活性或导入外源性 5-氨基乙酰丙酸合成酶、 增强磷酸烯醇式丙酮酸羧化酶并敲除琥珀酰脱氢酶。
在另一具体的实施方式中， 本发明的方法还包括减弱 5-氨基乙酰丙酸生产菌 株中磷酸烯醇式丙酮酸羧化激酶和 /或苹果酸酶的活性。
在优选的实施方式中，所述减弱磷酸烯醇式丙酮酸羧化激酶和 /或苹果酸酶的 活性通过敲除磷酸烯醇式丙酮酸羧化激酶和 /或苹果酸酶的基因得以实现。
在进一步的优选实施方式中， 所述方法还包括测定所得菌株的 5-氨基乙酰丙 酸产量。
在另一优选实施方式中， 所述方法获得的 5-氨基乙酰丙酸生产菌株可以在好氧 条件下， 无需添加外源琥珀酸而高水平地产生 5-氨基乙酰丙酸。
鉴于本发明的教导和现有技术，本领域技术人员还应明白本发明是通过增强起始 菌株中促进草酰乙酸合成的相关酶的活性或导入外源性的促进草酰乙酸合成的相关 酶， 和 /或减弱起始该菌株中琥珀酰辅酶 A下游代谢途径相关酶的活性， 和 /或减弱 磷酸烯醇式丙酮酸羧化激酶和 /或苹果酸酶的活性来提高所述菌株产生 5-氨基乙 酰丙酸的能力。 因此， 只要通过以上手段或它们中任意二者或三者的组合来构建或 改造菌株，进而提高 5-氨基乙酰丙酸产量的方法或由此得到的菌株均应落在本发明的 保护范围内， 本发明的保护范围并不限于实施例中采用的具体方法和得到的菌株。 在本发明方法以及获得的菌株的基础上， 本发明进一步提供了产生 5-氨基乙 酰丙酸的方法， 所述方法包括： 1) 培养本发明的菌株， 从而得到 5-氨基乙酰丙酸； 和 2) 从 1)的发酵培养体系中获得 5-氨基乙酰丙酸。 在优选的实施方式中， 所述方法 获得的 5-氨基乙酰丙酸的产量高于 7 g/L。 在另一优选的实施方式中， 所述方法不额 外添加琥珀酸和 /或仅仅利用葡萄糖作为碳源。
鉴于本发明的提示和教导， 本领域普通技术人员还可知道， 在 5-氨基乙酰丙 酸的生产菌株的培养体系中添加琥珀酰辅酶 A下游代谢途径相关酶抑制剂也可以 提高 5-氨基乙酰丙酸的产量。 在具体的实施方式中， 所述琥珀酰辅酶 A下游代谢 途径相关酶是琥珀酰辅酶 A合成酶或琥珀酸脱氢酶。 本发明的优点：
1. 本发明的菌株提高糖的转化率， 增加了 ALA合成必需底物之一琥珀酰辅 酶 A的合成， 从而提高了 ALA的产量；
2. 利用本发明菌株制备 ALA无需外源性添加前体琥珀酸， 使得生产过程摆 脱了对底物琥珀酸添加的依赖和昂贵培养基， 例如 LB 的使用， 大大降低了生产 成本。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件如 Sambrook 等人， 分子克隆： 实验室手册 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条 件。 材料与方法
本发明实施例所用的 DNA聚合酶购自北京全式金公司的 Fastpfu; 限制性内 切酶及 DNA连接酶等均购自 Fermentas公司；
酵母粉和蛋白胨购自英国 Oxoid公司产品； 甘氨酸和 IPTG购自 Promega公 司； 5-ALA和对二甲氨基苯甲醛等购自 Sigma公司； 琼脂粉和抗生素购自北京索 来宝； 葡萄糖、 冰醋酸、 高氯酸、 三氯乙酸、 乙酰丙酮、 氯仿以及其他常用化学 试剂均购自国药。
质粒提取试剂盒和琼脂糖凝胶电泳回收试剂盒均购自上海生工， 相关操作均 严格按照说明书执行；
质粒构建测序验证由华大基因完成；
DH5a感受态细胞购自北京全式金公司。
LB培养基成分： 酵母粉 5 g/L， 蛋白胨 10 g/L， NaCl 10 g/L, 固体培养基中 添加 2%的琼脂粉。
抗生素浓度为： 氨苄青霉素 100 g/mL， 卡那霉素 30 g/mL。
ALA的检测方法： 200 μL稀释的发酵液加入 100 μL pH 4.6乙酸钠缓冲液， 然后加入 5 μL 乙酰丙酮， 100°C水浴温育 15 min, 冷却至室温后加入等体积的 Ehrlish's试剂 (42 mL冰醋酸， 8 mL 70%高氯酸， 1 g二甲氨基苯甲醛)混匀， 显色 10 min后测 553 nm波长下的吸光度。
葡萄糖分析方法采用山东科学院生产的 SBA-40D生物传感分析仪进行检测。 实施例 1. 琥珀酰辅酶 A合成酶缺失突变株和琥珀酸脱氢酶缺失突变株的构 建
大肠杆菌基因敲除采用经典的 Red重组方法， 参考相关文献进行， 具体操作 如下： 对于琥珀酰辅酶 A合成酶结构基因 cO)基因的敲除， 首先根据 NCBI公 布的大肠杆菌 MG1655的基因组序列及辅助载体 pKD13的序列设计引物 cC -l 和 具体序列见引物序列表， 以 pKD13为模板扩增得到带有 cC 基因 上下游同源臂的 Kan抗性基因片段。 PCR扩增参数为 94°C 2 min; 94 °C 20 s, 64 °C 20 s , 72 °C 1 min, 循环 30 次； 72°C延伸 5 min。 PCR 产物胶回收后电转化 MG1655/pKD46 菌株， 感受态细胞制备和转化过程参考 J.萨姆布鲁克 (Sambrook) 等编写的 《分子克隆实验指南》 。 转化子利用 和 引物进行 PCR 验证， 野生型菌株片段大小为 2267 bp , 而 cO)基因缺失的菌株中目的条带大 小为 1558 bp , 根据条带大小挑选 cC 缺失突变株并命名为 ZPEcAl。
对于 sdhAB的敲除， 首先根据 NCBI公布的大肠杆菌 MG1655的基因组序列 及辅助载体 pKD13的序列设计引物^ 和 sdhAB-2，以 pKD 13为模板扩增得 到带有 sdhAB基因上下游同源臂的 Kan抗性基因片段。 PCR扩增参数为 94°C 2 min; 94 °C 20 s， 64 °C 20 s， 72 V 1 min, 循环 30次； 72°C延伸 5 min。 PCR产物 胶回收后电转化 MG1655/pKD46菌株， 感受态细胞制备和转化过程参考 J.萨姆布 鲁克 (Sambrook)等编写的《分子克隆实验指南》 。 转化子利用^ 和^ 引物进行 PCR验证， 野生型菌株片段大小为 2553 bp , 而 sucCD基因缺失的菌株 中目的条带大小为 1408 bp , 根据条带大小挑选 sdhAB 缺失突变株并命名为 ZPEcA2。 实施例 2. 磷酸烯醇式丙酮酸羧化酶 ppc和 ALA合成酶共表达质粒的构建 根据 NCBI公布的大肠杆菌 MG1655的基因组序列设计引物/^ c-F和 ppc-R， 以大肠杆菌 MG1655基因组为模板 PCR扩增得到带有自身启动子的 pPc基因片 段， PCR扩增参数为 94°C 2 min; 94 °C 20 s， 60V 20 s， 72V 1.5 min, 循环 30 次； 72 °C延伸 5 min。 c基因片段回收后用 H dlll处理， 同时将携带有 ALA合 成酶的质粒 pZGA24 ( pZGA24构建参见参考文献： 郭小飞等， 利用 5-氨基乙酰 丙酸脱水酶缺失的重组大肠杆菌合成 5-氨基乙酰丙酸， 天津科技大学学报， 2012， 27(4)： 1-6 ) 也用该酶处理， 载体和片段回收后用 T4连接酶连接， 转化 DH5( 感 受态细胞， 涂布含有 Amp的 LB平板， 挑取阳性克隆提取质粒并进行酶切验证， 测序正确的重组质粒命名为 pZPA6。 实施例 3. 丙酮酸羧化酶 pyc和 ALA合成酶共表达质粒的构建
根据 BioBrick中启动子 BBa— J23 105的序列和 NCBi公布的根瘤菌 CFN42的 基因组序列设计引物 /^c- l和 pyC-2， 以根瘤菌 CFN42基因组为模板 PCR扩增得 到带有组成型启动子序列的 基因片段， PCR扩增参数为 94 °C 2 min; 94°C 20 s , 60°C 20 s , 72°C 2 min, 循环 30次； 72°C延伸 5 min。 目的片段回收后用磷酸化酶 处理， 同时将 pWSK29载体用限制性内切酶 PvuII处理后再用去磷酸化酶处理， 将得到的载体片段与磷酸化的^ c基因片段用 T4连接酶连接，转化 DH5( 感受态 细胞， 涂布含有 Amp的 LB平板， 挑取阳性克隆提取质粒并进行酶切验证， 测序 正确的重组质粒命名为 pSLS33。
根据上述 pSLS33的序列设计引物/^ c-F和/^ c-R， 具体序列见引物序列表， 以 pSLS33为模版扩增得到带有组成型启动子的 pyc基因片段， PCR扩增参数为 94 °C 2 min; 94 °C 20 s， 60V 20 s， 72V 2 min, 循环 30次； 72°C延伸 5 min。 同 时根据 pZGA24载体的序列设计引物 pA- 1和 pA-2， 以 pZGA24为模板反向 PCR 扩增含有 ALA合成酶基因的 pTrc-/ze 载体， PCR扩增参数为 94 °C 2 min; 94 °C 20 s， 60 °C 20 s， 72 °C 3 min, 循环 30次； 72°C延伸 5 min。 /^c基因片段回收后 用核酸内切酶 Smal消化处理并用 T4连接酶连接到反向扩增并纯化的 pZGA24载 体上， 连接产物转化 DH5a感受态细胞， 涂布含有 Amp的 LB平板， 挑取阳性克 隆提取质粒并进行酶切验证， 测序正确的重组质粒命名为 pZPA7
表 1 引物序列表
物名称 引物序列
sucCD-l
CCGTCGACC(SEQ ID NO: 1)
sue CD -2 CGGCGA GGGCTA TTTC
CTGCTTCG(SEQ ID NO: 2)
sucCD- GTTTAACGTGTCTTATCAGGCCT(SEQ ID NO: 3)
sucCD CGAAAATCATCGCGATAAGCACA(SEQ ID NO: 4)
sdhAB-l CTGGTGGTTTA CGTGA TTTA
CCGTCGACC(SEQ ID NO: 5)
sdhAB-2 A CGGTTTA CGCA TTA CGI
GCTGCTTCG(SEQ ID NO: 6)
sdhAB- TTATGGATTCGTTGTGGTGTGGGGT(SEQ ID NO: 7)
sdhAB-4 TGCGCGTCTTATCAGGCCTA(SEQ ID NO: 8)
ppc-¥ CCGCAAGCTTTATCCGACCTACACCTTTGGT(SEQ ID NO: 9)
ppc-R CCGCAAGC1TGGACTTCTGTGGAATGCATAGT(SEQ ID NO: 10)
pyc- l
ATGCCCATATCCAAGATACTC(SEQ ID NO: 1 1)
pyc-2 AACAGCCTGACTTTACACAATCGG(SEQ ID NO: 12)
pyc-¥ GATACCCGGGTTTACGGCTAGCTCAGTCCTAGG(SEQ ID NO: 13)
pyc-R CAAGCCCGGGAACAGCCTGACTTTACACAATCGG(SEQ ID NO: 14)
pA- 1 GCGGATGAGAGAAGATTTTCAG(SEQ ID NO: 15)
pA-2 CAAAACAGCCAAGCTTTCAGT(SEQ ID NO: 16)
注： 斜体加粗部分为同源臂序列， 下划线部分为酶切位点 实施例 4. 重组菌株的构建及 ALA产量的比较
分别将上述构建的重组质粒 pZGA24、 ρΖΡΑό和 ρΖΡΑ7转化野生型大肠杆菌 MG1655及 sucCD缺失突变株 ZPEcAl和 sdhAB缺失突变株 ZPEcA2， 涂布 Amp 抗性 LB 平板， 过夜培养后挑取阳性克隆提取质粒验证， 分别获得重组菌株 MG1655/pZGA24、 ZPEcAl /pZGA24、 ZPEcA2/pZGA24、 MG 1655/pZPA6、 ZPEcAl/pZPA6 、 ZPEcA2/pZPA6 、 MG1655/pZPA7 、 ZPEcAl /pZPA7 禾口 ZPEcA2/pZPA7。
将上述重组菌单菌落分别接种 5 mL含有 100 g/mL氨苄青霉素的 LB液体培 养基， 37°C， 220 rpm培养 12 h。 按照初始 OD为 0.05转接装有 50 mL发酵培养 基的 250 mL三角瓶， 37 °C， 220 rpm培养 2.5 h后加入终浓度为 25 μΜ的 IPTG， 诱导培养 19 h后收集发酵液， 检测 ALA的浓度。 其中发酵培养基为添加了少量 酵母粉的 M9培养基， 主要成分为： Na2HP04' 12H20 12.8 g/L， KH2P04 3.0 g/L， NaCl 0.5 g/L , NH4C1 1.0 g/L, MgS04 2 mM， CaCl2 O. lmM, 葡萄糖 10 g/L， 酵母 粉 2 g/L， 甘氨酸 4 g/L， 氨苄青霉素浓度为 100 g/mL。 ALA的检测和葡萄糖分 析方法如 "材料与方法" 部分所述。
各重组菌 ALA产量结果见表 2， 对照菌株 MG1655/pZGA24中 ALA的产量 仅为 1.06 g/L, sucCD或 sdhAB缺失的 ZPEcAl/pZGA24和 ZPEcA2/pZGA24菌株 中 ALA的产量分别为 1.33 g/L和 1.45 g/L , 分别比出发菌株提高了 25%和 37%， 表明在大肠杆菌中部分或全部缺失琥珀酰辅酶 A合成酶或琥珀酸脱氢酶的活性能 够提高 ALA的产量。表达/?/ ^的 MG 1655/pZPA6菌株和表达/ 的 MG1655/pZPA7 菌株中 ALA的产量分别达到 2.52 g/L和 2 g/L, 分别是出发菌株的 2.38倍和 1.89 倍，结果表明增强促进草酰乙酸合成的相关酶能够显著提高 ALA的产量。而 SUcCD 或 sdhAB 缺失且 ppc 或 pyc 过量表达的 ZPEcAl/pZPA6、 ZPEcA2/pZPA6 , ZPEcAl/pZPA7和 ZPEcA2/pZPA7菌株中 ALA的产量分别达到 2.43 g/L, 3.08 g/L, 2.12 g/L和 2.66 g/L, 均高于相应的对照菌株， 表明 sucCD或 sdhAB缺失与 ppc 或^ c过量表达有一定的叠加效果， 能够促进 ALA的生物合成。 其中， sdhAB缺 失且表达 ppc 的 ZPEcA2/pZPA6 菌株 ALA 的产量最高， 是对照菌株 MG1655/pZGA24的 2.91倍， 葡萄糖转化率也达到最高的 0.47 (摩尔比）， 是出发 菌株的 2.57倍。 而利用本发明的菌株进行发酵罐发酵时， ALA的产量可以达到 7 g/L以上，达到了国内的较好水平。上述实验结果表明在大肠杆菌中表达外源 ALA 合成酶的基础上， 部分或全部缺失琥珀酰辅酶 A合成酶或琥珀酸脱氢酶的活性， 同时增强促进草酰乙酸合成的相关酶的表达可以大大提高 ALA的产量。
上述重组菌株 MG1655/pZPA6和 ZPEcA2/pZPA6菌株已于 2012年 9月 19日 保藏于中国微生物菌种保藏管理委员会普通微生物中心， 北京市朝阳区北辰西路 1号院 3号， 保藏号分别为： CGMCC No.6588和 CGMCC No.6589。
表 2 不同菌株摇瓶发酵数据
菌株 ALA(g/L) ALA/ODgoo 葡萄糖摩尔转化率
MG 1655/pZGA24 1.06 0.30 0.18
ZPEcA l/pZGA24 1.33 0.35 0.23
ZPEcA2/pZGA24 1.45 0.41 0.24
MG 1655/pZPA6 2.52 0.56 0.39
ZPEcA l/pZPA6 2.43 0.65 0.37
ZPEcA2/pZPA6 3.08 0.76 0.47
MG 1655/pZPA7 2.00 0.47 0.35
ZPEcA l/pZPA7 2.12 0.40 0.36
ZPEcA2/pZPA7 2.66 0.48 0.41 实施例 5. 在 BW25113菌株中重复验证
重组菌株的构建： 分别将上述质粒 pZGA24、 pZPA6和 pZPA7转化大肠杆菌 BW25 1 13(作为对照的菌株 BW251 13来源于 CGSC (美国耶鲁大学大肠杆菌保藏中 心))及相应的 cC缺失突变株 JW0717 (缺失了琥珀酰辅酶 A合成酶 β亚基 ( cC) 的 JW0717 菌株和 sdhA 缺失突变株 JW0713 (JW0717 和 JW0713 均来自 Keio Collection 大肠杆菌单基因缺失菌株库， National BioResource Project E. coli, Microbial Genetics Laboratory, National Institute of Genetics 1 1 1 1 Yata, Mishima, Shizuoka, 41 1 -8540 Japan) , 涂布 Amp抗性 LB平板， 过夜培养后挑取阳性克隆提 取质粒验证， 分别获得重组菌株 BW251 13/pZGA24、 JW0717/pZGA24、 JW0713/pZGA24 、 BW251 13/pZPA6 、 JW0717/pZPA6 、 JW0713/pZPA6 、 BW25 1 13/pZPA7、 JW0717/pZPA7和 JW0713/pZPA7。
各重组菌摇瓶发酵及 ALA和葡萄糖检测方法同上， 结果见表 3， 从表中可以 看出各重组菌与相应的 MG1655重组菌产量及变化基本一致，产量最高的是 ^ A 缺失且表达 c的 JW0713/pZPA7菌株， ALA产量达到 3.21 g/L , 葡萄糖转化率 更是达到 0.56 (摩尔比)， 表明本发明提供的方法同样适用于大肠杆菌其他菌株。
表 3. 不同菌株摇瓶发酵数据
菌株 ALA (g/L) ALA/ODgoo 葡萄糖摩尔转化率
BW251 13/pZGA24 1.59 0.39 0.27
JW0717/pZGA24 1.21 0.32 0.23
JW0713/pZGA24 2.23 0.49 0.38
BW251 13/pZPA6 3. 1 1 0.55 0.43
JW0717/pZPA6 1.88 0.53 0.32
JW0713/pZPA6 3.08 0.74 0.52
BW251 13/pZPA7 2.61 0.58 0.44
JW0717/pZPA7 2.25 0.50 0.39
JW0713/pZPA7 3.21 0.72 0.56
实验结果表明， 利用本发明提供的方法， 在以葡萄糖为主要碳源的培养基中 摇瓶发酵， 重组菌中 ALA产量达到 3.08 g/L , 单位菌体 ALA 的产量达到 0.76 g/L/OD，分别为出发菌株的 2.91倍和 2.59倍，葡萄糖的转化率达到 0.47 (mol/mol) , 是出发菌株的 2.57倍。利用本发明提供的方法构建的重组菌以及利用其产生 ALA 的方法摆脱了对琥珀酸添加的依赖和昂贵 LB 培养基的使用， 具有很好工业应用 前景和经济价值。 实施例 6. 磷酸烯醇式丙酮酸羧化激酶和苹果酸酶活性增强的重组菌株的构 建及 ALA产量的比较
发明人采用与实施例 1-5所述类似的方法构建了磷酸烯醇式丙酮酸羧化激酶 (编码基因/ 和苹果酸酶 （编码基因 ae ) 的活性增强的重组菌株并检测了 它们的 ALA产量。
首先， 采用与实施例 2 类似的方法， 利用下表 4 所示引物克隆大肠杆菌 MG1655的磷酸烯醇式丙酮酸羧激酶的编码基因 pck， 并连接到 pZGA24载体上， 获得的重组质粒命名为 pZPA12。
其次， 根据 NCBI公布的大肠杆菌 MG1655的基因组序列设计下表 4所示引 物 maeB-F和 maeB-R， 以大肠杆菌 MG 1655基因组为模板， 作 PCR扩增得到苹 果酸脱氢酶编码基因 maeB。 目的片段用 T4多核苷酸激酶进行磷酸化处理后与上 述反向扩增得到的 pZGA24载体片段进行连接。 经转化、 酶切及测序验证正确的 重组载体命名为 pZPA14。
表 4 引物序列表二 引物名称 引物序列
pck-V CCGCAAGCTTGCGTGGTGAATCGATACTTT(SEQ ID NO: 17)
pck-R CCGCAAGCTTTGCCTCCCGTTTTGCTTTCT(SEQ ID NO: 18)
maeB-K CAATGGATCCCTAAACTGCTTACCCTGAAT(SEQ ID NO: 20)
注： 下划线部分为酶切位点
采用与实施例 4类似的方法，分别将上述构建的重组质粒 pZPA12和 pZPA14 转化野生型大肠杆菌 MG 1655， 获得重组菌株 MG1655/pZPA12 和 MG1655/pZPA14。 然后利用与实施例 4类似的方法， 检测各重组菌 ALA产量， 结果见下表 6。
表 6. 摇瓶发酵数据
菌株 ALA (g/L) OD600 葡萄糖摩尔转化率
MG1655/pZGA24 1.06 0.30 0.18
MG1655/pZPA12 0.57 4.20 0.10
MG1655/pZPA14 0.88 3.90 0.16
由上表可见， 表达 PCK 和 MaeB 的工程菌株 MG1655/pZPA12 和 MG1655/pZPA14 的 ALA产量分别比对照菌株 MG 1655/pZGA24 下降了 46%和 17%。 磷酸烯醇式丙酮酸羧化激酶 pck的活性增强并没有产生提高菌株 ALA产量 的技术效果。 实施例 7. 敲除 pck或 maeB基因对 ALA产量的影响
鉴于实施例 9的结果， 本发明人进一步研究 或 ae 基因缺失对 ALA合 成的影响。
将 ALAS 和 PPC 的共表达载体 pZPA6 分别导入来源于 Kelio Collection
( National BioResource Project E. coli, Microbial Genetics Laboratory, National Institute of Genetics 1 1 1 1 Yata, Mishima, Shizuoka, 41 1 -8540 Japan) 的单基因缺失 的菌株 JW3366 (^ 基因缺失菌株)和 JW2447 ( ae 基因缺失菌株）中， 经验证得 到正确的工程菌株 JW3366/pZPA6 和 JW2447/pZPA6。 以实施例 5 获得的 BW25 1 13/pZPA6为对照， 在初始添加 15 g/L葡萄糖和 4 g/L甘氨酸的发酵培养基 中发酵验证上述重组菌株产 ALA的能力。 发酵过程中在培养 12 h之后补加 5 g/L 葡萄糖和 2 g/L甘氨酸。 发酵结果如下表 7所示， 发酵 25 h之后 JW3366/pZPA6 的 ALA产量达到了 4.84 g/L， 同时糖转化率达到了 0.51 mol/mol， 分别比对照菌 株 BW25 1 13/pZPA6提高了 29%和 20.8% ; 而 JW2447/pZPA6的 ALA产量略微下 降， 但转化率提高 7.8%。
该结果表明 pck或 maeB基因的缺失有助于 ALA积累， 尤其是敲除 基因 更有利于 ALA积累和糖转化率的提高。
表 7. 或 maeB基因缺失对 ALA积累的影响
菌株 ALA (g/L) ΟΡβοο 葡萄糖摩尔转化率
BW251 13/pZPA6 3.75 8.37 0.42 JW3366/pZPA6 4.84 8.71 0.50
JW2447/pZPA6 3.60 7.9 0.45 实施例 8. 谷氨酸棒杆菌表达载体构建及发酵验证
根据 NCBI公布的沼泽红假单胞菌 (R/zo6fo/weM6fo oM£w palustris) ATCC 17001 ALA合成酶编码基因的序列 （GenBank: JQ048720.1) , 设计引物/ ze ^-F (引物序 列 ：
SEQ ID NO: 21) 和 fwmA-R ( 引 物 序 列 ： TATACCCCGGGTCAGGCCGCCTTGGCGAGAC； SEQ ID NO: 22)。 以 pZGA24 载体 （pZGA24构建参见参考文献： 郭小飞等， 天津科技大学学报， 2012, 27(4) : 1 -6)为模板通过 PCR扩增得到目的基因 / ze ^ , PCR扩增体系如下： PCR扩增参 数为： 94 °C 10 min, 94 °C 20 s , 65 °C 30 s , 72V 40 s， 循环 30次， 72°C延伸 5 min。 目的片段用 coRI和 S al双酶切处理， 然后在 DNA连接酶作用下将获得 片段与同样酶切处理的质粒 pEC-XK99E连接，并将连接产物转化到 DH5a感受态 细胞， 涂布卡那霉素抗性平板过夜培养， 挑阳性克隆进行菌落 PCR验证， 验证正 确的转化子进行测序验证， 将正确的重组载体命名为 pZWAl (图 4)。
其次， 根据 NCBI公布的谷氨酸棒杆菌 (Coryfiebacterium glutamicum) ATCC 13032的磷酸烯醇式丙酮酸羧化酶编码基因 ppc的序列 （GenBank: BA000036.3) , 设 计 引 物 Cg，c-F ID NO: 23)和 Cg-/^c-R: GCTCTAGACTAGCCGGAGTTGCGCAGCGCAGT； SEQ ID NO: 24。 以 ATCC 13032基因组为模板通过 PCR扩增得到目的基因， PCR扩 增体系如下： PCR扩增参数为： 94 °C 10 min, 94 °C 20 s , 65 °C 30 s , 72 °C 2 min, 循环 30次， 72°C延伸 5 min。 目的片段用 S al和 双酶切处理， 然后在 DNA 连接酶作用下将获得片段与同样酶切处理的 pZWAl 载体连接， 并将连接产物转 化到 DH5( 感受态细胞， 涂布卡那霉素抗性平板过夜培养， 挑阳性克隆进行菌落 PCR验证，验证正确的转化子进行测序验证，将正确的重组载体命名为 pZWA2 (图 4)。
将上述重组载体 pZWAl和 pZWA2及对照空载体 pEC-XK99E分别转化谷氨 酸棒杆菌 ATCC 13032 , 获得重组菌株 ATCC 13032/pEC-XK99E、 ATCC 13032/ pZWAl和 ATCC 13032/pZWA2。
将上述重组菌单菌落分别接种 10 mL含有 25 g/mL卡那霉素和 24 g/L葡萄 糖的 LB液体培养基， 30°C， 200 rpm培养 12 h。 按照初始 OD为 0.3转接装有 50 mL发酵培养基的 500 1^三角瓶，30 °〇，200卬111培养3 11后加入终浓度为 100 μΜ 的 IPTG , 诱导培养 32 h后收集发酵液， 检测 ALA的浓度。 其中摇瓶发酵培养基 配方为： Glu 50 g/L , (NH4)2SO4 10 g/L, MnSO4 1 g/L, K2HPO4 1.5 g/L , MgSO4 0.6 g/L , 玉米浆 l g/L， 甘氨酸 4 g/L， MOPS 31.395 g/L, 调 pH至 7.0。 卡那霉素终 浓度为 25 g/mL。 ALA的检测和葡萄糖分析方法如 "材料与方法" 部分所述。
摇瓶发酵结果见表 8，单独表达外源 ALA合成酶的 ATCC 13032/pZWAl菌株 中 ALA产量为 1.3 1 g/L,葡萄糖摩尔转化率为 0.052。而菌株 ATCC 13032/pZWA2 中 ALA 产量和葡萄糖摩尔转化率分别达到 1.95 g/L 和 0.077， 均比 ATCC 13032/pZWAl菌株提高了 48%， 效果明显。 因此， 本发明提供的 ALA生产 菌株的改造方法同样适用于谷氨酸棒杆菌等发酵工业常用微生物。
表 8 谷氨酸棒杆菌不同菌株摇瓶发酵数据
菌株 A1A (g/L) OD600 葡萄糖摩尔转化率
ATCC13032/pEC-XK99E 0.02 18.05 0.0008
ATCC13032/pZWAl 1.31 20.35 0.052
ATCC13032/pZWA2 1.95 23.18 0.077 讨论
本发明人基于 5-氨基乙酰丙酸的合成途径， 以产 ALA的重组微生物为目标， 通过理性设计改造宿主菌的糖代谢途径，结果表明促进草酰乙酸合成的相关酶的活 性能够显著提高葡萄糖的转化率和 ALA的产量。具体地说， 本发明通过表达磷酸 烯醇式丙酮酸羧化酶或丙酮酸羧化酶增强草酰乙酸的回补，大幅提高了 ALA的产 量和葡萄糖的转化率。
本发明人还发现降低琥珀酰辅酶 A下游代谢途径相关酶， SP , 琥珀酰辅酶 A 合成酶和琥珀酸脱氢酶的活性也能提高起到类似的技术效果。 而生物学常识告诉 我们， 多亚基酶需要各亚基密切配合才能完成完整的生物学功能， 任何一个亚基 序列或结构变化、 表达水平变化都可能影响酶的整体活性。 琥珀酰辅酶 A合成酶 和琥珀酸脱氢酶均是多亚基酶，琥珀酰辅酶 A合成酶包括两个亚基，分别由 sucC、 sucD编码， 琥珀酸脱氢酶含有四个亚基， 分别由 ^ /^、 sdhB、 和 sdhD编码。 通过实施例 4禾卩 5， 发明人证实了 cC和 sucD、 sucC、 sdhA和 sdhB、 sdhA基因 缺失造成相应酶失活， 进而有利于 ALA合成。 根据生物学常识可以推理， 其他能 够引起这两个酶活性弱化的遗传修饰或者通过其它方法， 例如添加外源性琥珀酰 辅酶 A 合成酶或琥珀酸脱氢酶的抑制剂来抑制或降低二者的活性， 都会有利于 ALA的生物合成。
而将以上两种改造策略整合更是极大提高了葡萄糖的转化率和 ALA的产量， 与仅表达 ALA合成酶的对照菌株相比， 改造后的菌株 ALA的产量和葡萄糖的转 化率分别提高了 1.91倍和 1.57倍， 而利用本发明的菌株进行发酵罐发酵时， ALA 的产量可以达到 7 g/L以上， 达到了较好水平。 此外， 本发明的改造方法还适用 于谷氨酸棒杆菌等发酵工业常用的微生物， 具有相当好的普遍性。 因此， 本发明提供的菌株和方法能够显著提高 ALA 的产量和葡萄糖的转化 率， 摆脱了以往合成 ALA 时对琥珀酸添加的依赖和昂贵复杂培养基的使用， 如 LB培养基， 大幅度降低了生产成本， 具有很好的工业化前景。
发明人最初还预计增强磷酸烯醇式丙酮酸羧化激酶的活性也可以起到增加草 酰乙酸合成， 从而增加 ALA合成所需的前体量， 进而提高 ALA的产量的作用。 然而， 具体实验证明增强磷酸烯醇式丙酮酸羧化激酶的活性并未起到预计的技术 效果。 同样， 发明人还发现增强苹果酸酶的活性也不能实现 ALA产量的提高。 发 明人通过进一步研究， 出乎意料地发现减弱磷酸烯醇式丙酮酸羧化激酶和苹果酸 酶的活性反而能够显著提高 ALA的产量。具体地说， 磷酸烯醇式丙酮酸羧化激酶 的缺失可以大幅提高 ALA的产量和葡萄糖转化率，而苹果酸酶的缺失也有利于提 高葡萄糖的转化率。
在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所限定的范围。

Claims

权 利 要 求
I . 5-氨基乙酰丙酸生产菌株的构建方法， 其特征在于， 所述方法包括： 增强所述 5-氨基乙酰丙酸生产菌株中促进草酰乙酸合成的相关酶的活性或导 入外源性的促进草酰乙酸合成的相关酶， 和 /或
减弱所述 5-氨基乙酰丙酸生产菌株中琥珀酰辅酶 A下游代谢途径相关酶的活 性。
2. 如权利要求 1所述的构建方法， 其特征在于， 所述促进草酰乙酸合成的相 关酶是磷酸烯醇式丙酮酸羧化酶或丙酮酸羧化酶， 所述琥珀酰辅酶 A下游代谢途 径相关酶是琥珀酰辅酶 A合成酶或琥珀酸脱氢酶。
3. 如权利要求 1 所述的构建方法， 其特征在于， 所述方法还包括增强所述 5-氨基乙酰丙酸生产菌株中的 5-氨基乙酰丙酸合成途径或引入外源 5-氨基乙酰丙 酸合成途径。
4. 如权利要求 1或 3所述的构建方法， 其特征在于， 所述方法还包括减弱磷 酸烯醇式丙酮酸羧化激酶和 /或苹果酸酶的活性。
5. 5-氨基乙酰丙酸生产菌株的构建方法， 其特征在于， 所述方法包括： 减弱所述 5-氨基乙酰丙酸生产菌株中的磷酸烯醇式丙酮酸羧化激酶和 /或苹 果酸酶的活性； 和
增强所述 5-氨基乙酰丙酸生产菌株中的 5-氨基乙酰丙酸合成途径或引入外源 5-氨基乙酰丙酸合成途径。
6. 一种 5-氨基乙酰丙酸生产菌株， 其特征在于， 所述菌株中促进草酰乙酸合 成的相关酶的活性增强或包含外源性的促进草酰乙酸合成的相关酶， 和 /或
所述琥珀酰辅酶 A下游代谢途径相关酶的活性减弱。
7. 如权利要求 6所述的菌株， 其特征在于， 所述促进草酰乙酸合成的相关酶 是磷酸烯醇式丙酮酸羧化酶或丙酮酸羧化酶， 所述琥珀酰辅酶 A下游代谢途径相 关酶是琥珀酰辅酶 A合成酶或琥珀酸脱氢酶。
8. 如权利要求 7所述的菌株， 其特征在于， 所述菌株中 5-氨基乙酰丙酸合成 途径增强或含有外源 5-氨基乙酰丙酸合成途径。
9. 如权利要求 6-8中任一项所述的菌株， 其特征在于， 所述菌株选自大肠杆 ¾ (Escherichia coif)、 # f ¾ (Corynebacterium glutamicum)、 球形红细菌
(Rhodobacter sphaeroides)、 沼 j|¾ -^- ffi ¾ Rhodopseudomonas palustris)等。
10. 如权利要求 6-8 中任一项所述的菌株， 其特征在于， 所述菌株中磷酸烯 醇式丙酮酸羧化激酶和 /或苹果酸酶的活性减弱。
I I . 一种 5-氨基乙酰丙酸生产菌株， 其特征在于， 所述菌株中磷酸烯醇式丙 酮酸羧化激酶和 /或苹果酸酶的活性减弱；和所述菌株中 5-氨基乙酰丙酸合成途径 增强或含有外源 5-氨基乙酰丙酸合成途径。
12. —种产生 5-氨基乙酰丙酸的大肠杆菌菌株， 所述菌株选自下组： 以 CGMCC No.6588 为保藏号保藏于中国微生物菌种保藏管理委员会普通微生物中 心的菌株或以 CGMCC No.6589为保藏号保藏于中国微生物菌种保藏管理委员会 普通微生物中心的菌株。
13. 一种产生 5-氨基乙酰丙酸的方法， 其特征在于， 所述方法包括：
1) 培养权利要求 6-12中任一项所述的菌株， 从而得到 5-氨基乙酰丙酸； 和
2) 从 1)的发酵培养体系中获得 5-氨基乙酰丙酸。
14. 权利要求 6-12中任一项所述菌株的用途， 其特征在于， 所述菌株用于产 生 5-氨基乙酰丙酸和 /或产生以 5-氨基乙酰丙酸为前体的下游产物。
PCT/CN2014/071712 2013-02-07 2014-01-28 5-氨基乙酰丙酸高产菌株及其制备方法和应用 WO2014121724A1 (zh)

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