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CN-122012541-A - D-lactate dehydrogenase, engineering strain containing same and construction and application thereof

CN122012541ACN 122012541 ACN122012541 ACN 122012541ACN-122012541-A

Abstract

The invention provides a D-lactate dehydrogenase, an engineering strain containing the same, and construction and application thereof. The present invention discloses a d-lactate dehydrogenase from the genus Thermodesulphus (Thermodesulfatatorindicus) having unique properties, which has very good thermophilicity and thermostability. By utilizing the d-lactic dehydrogenase and the genetic engineering modification method, the fermentation product of the modified bacillus licheniformis is redirected from natural 2, 3-butanediol to high-yield optical pure d-lactic acid, the optical purity of the produced d-lactic acid reaches 99.9%, the price of raw materials used for fermentation is low, and the fermentation state is between anaerobic and micro-aerobic. The method for producing d-lactic acid by high-temperature fermentation can save cost, improve production efficiency and has wide industrial application prospect.

Inventors

  • XU PING
  • LI CHAO
  • TAO FEI
  • TANG HONGZHI

Assignees

  • 上海交通大学

Dates

Publication Date
20260512
Application Date
20170125
Priority Date
20160127

Claims (10)

  1. 1. A nucleotide sequence, wherein the nucleotide sequence encodes a d-lactate dehydrogenase comprising a substitution, deletion, insertion, or addition of one or more amino acid residues, wherein the d-lactate dehydrogenase comprises an amino acid sequence having at least 80% and less than 100% homology with the amino acid sequence set forth in SEQ ID No. 1, and wherein the d-lactate dehydrogenase has d-lactate dehydrogenase activity.
  2. 2. The nucleotide sequence of claim 1, wherein the nucleotide sequence has one of the following nucleotide sequences: 1) A nucleotide sequence shown as SEQ ID No. 2; 2) A nucleotide sequence shown as SEQ ID No. 3; 3) A nucleotide sequence having a homology of 80% or more with the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 3; 4) A nucleotide sequence which hybridizes under high stringency conditions to the complement of the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 3.
  3. 3. A genetically engineered strain, wherein the genetically engineered strain has a d-lactate dehydrogenase, wherein the d-lactate dehydrogenase comprises a substitution, deletion, insertion, or addition of one or more amino acid residues, wherein the d-lactate dehydrogenase comprises an amino acid sequence having at least 80% and less than 100% homology to the amino acid sequence shown in SEQ ID No. 1, and wherein the d-lactate dehydrogenase has d-lactate dehydrogenase activity.
  4. 4. A genetically engineered strain according to claim 3, wherein the nucleotide sequence encoding the d-lactate dehydrogenase has one of the following nucleotide sequences: 1) A nucleotide sequence shown as SEQ ID No. 2; 2) A nucleotide sequence shown as SEQ ID No. 3; 3) A nucleotide sequence having a homology of 80% or more with the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 3; 4) A nucleotide sequence which hybridizes under high stringency conditions to the complement of the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 3.
  5. 5. The genetically engineered strain according to claim 3, wherein a pyruvate synthesis path of l-lactic acid is blocked and a pyruvate synthesis path of 2, 3-butanediol is blocked in the genetically engineered strain.
  6. 6. The genetically engineered strain according to claim 5, wherein the pathway for synthesizing l-lactic acid from pyruvic acid is blocked by inactivation or deletion of l-lactate dehydrogenase gene, and the pathway for synthesizing 2, 3-butanediol from pyruvic acid is blocked by inactivation or deletion of one or both of acetolactate synthase gene and acetolactate decarboxylase gene.
  7. 7. The genetically engineered strain of claim 5, wherein one or more of the pathways of pyruvic acid to formate, pyruvic acid to acetate, and pyruvic acid to ethanol in the genetically engineered strain is blocked.
  8. 8. The genetically engineered strain of claim 7, wherein the pathway for pyruvate synthesis of formate is blocked by inactivation or deletion of one or both of a pyruvate formate lyase gene and a pyruvate formate lyase activating enzyme gene, the pathway for pyruvate synthesis of acetate is blocked by inactivation or deletion of one or both of a pyruvate dehydrogenase gene and an acetate kinase, and the pathway for pyruvate synthesis of ethanol is blocked by inactivation or deletion of one or both of a pyruvate dehydrogenase gene and an alcohol dehydrogenase gene.
  9. 9. The genetically engineered strain of claim 5, wherein the original d-lactate dehydrogenase gene in the genetically engineered strain is inactivated or deleted.
  10. 10. The genetically engineered strain according to claim 5, wherein the starting strain of the genetically engineered strain is a thermophilic strain.

Description

D-lactate dehydrogenase, engineering strain containing same and construction and application thereof The application relates to a divisional application of the following application, namely, the application date is 25 days of 20171 month, the application number is 201710056240.7, and the application is named as D-lactate dehydrogenase, engineering strain containing the D-lactate dehydrogenase and construction and application of the D-lactate dehydrogenase. Technical Field The invention relates to the field of genetic engineering, in particular to D-lactic dehydrogenase, construction of bacillus genetic engineering bacteria containing the enzyme and application of the bacillus genetic engineering bacteria in production of D-lactic acid. Background Lactic acid (LACTIC ACID) is an important industrial raw material which can be applied to medicine, food, cosmetics, petrochemical industry and the like, and in recent years, lactic acid has also been used as a monomer for synthesizing high-strength biodegradable plastic polylactic acid (PLA). Conventionally, PLA is polymerized by high optical purity l-lactic acid, and the mechanical property, the thermal stability and the hydrolytic resistance of a product taking polylactic acid as a material can be obviously improved by using mixed l-lactic acid and d-lactic acid, so that the market demand of d-lactic acid is greatly stimulated. Although lactic acid can be synthesized from petroleum, the synthesized lactic acid is a mixture of two isomers, and is not suitable for the production of PLA. The high optical purity of l and d-lactic acid required for the production of PLA can only be produced by microbial fermentation, and many researchers have therefore studied the production of d-lactic acid by microbial fermentation. The high-temperature fermentation can minimize pollution risk, improve raw material conversion rate, and reduce heat supply cost. Up to now, high Wen Shengchan optically pure l-lactic acid has been widely studied, which makes a great contribution to the commercial production of l-lactic acid. Regarding d-lactic acid, it has been desired to realize its high-temperature fermentation production, but there are few reports on thermophilic production of d-lactic acid, and the concentration and production rate of d-lactic acid reported and the abundant medium components thereof are not suitable for industrial scale production. Therefore, there is a need to develop a low-cost, robust microbial platform for high-temperature and high-yield d-lactic acid. Nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide, NADH) -dependent d-lactate dehydrogenase (d-lactate dehydrogenase) is a key enzyme for the microbial synthesis of d-lactic acid. However, most naturally occurring d-lactate dehydrogenases are not thermostable, which is also one of the main bottlenecks in the high temperature production of d-lactic acid by microorganisms. The in vitro measurement of d-lactate dehydrogenase activity in Lactobacillus plantarum (Lactobacillus plantarum) has been reported to show that the enzyme has a very high activity at 42℃but is completely inactivated after incubation for 3 minutes at 50℃while 94% of the activity remains after the same procedure for the l-lactate dehydrogenase in the cell. Other in vivo experiments have also shown that d-lactate dehydrogenase is also susceptible to inactivation under high temperature conditions. In summary, the low activity, or poor thermostability, of the above reported strains of d-lactate dehydrogenase prevents them from producing d-lactate under high temperature conditions. In order to realize high-temperature and high-efficiency d-lactic acid production, it is an important task to find a d-lactic acid dehydrogenase with high thermal stability. Thermotolerant bacillus licheniformis (Bacillus licheniformis) ATCC 14580 is a facultative anaerobic, gram positive, endospores producing bacterium. It has many potential advantages as a microbial fermentation platform, 1) it can utilize various five-carbon sugars and six-carbon sugars, 2) it has a faster cell growth rate, thus shortening the fermentation period, 3) it can be genetically manipulated, 4) it is a recognized "generally recognized safe" strain by the food and drug administration, indicating that strain ATCC 14580 can act as an ideal platform strain. At present, the strain has proved to be useful for the high temperature fermentative production of 2, 3-butanediol. Accordingly, those skilled in the art have focused on developing a high-temperature high-yield engineering strain of d-lactic acid, and its preparation and application, starting from heat-resistant Bacillus licheniformis ATCC 14580. Disclosure of Invention Aiming at the defects that most of naturally occurring d-lactic dehydrogenase in the prior art has poor thermal stability, the existing thermophilic production cost for producing d-lactic acid is high, but the d-lactic acid concentration and the production rate