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CN-122012634-A - Use of aldolase from Thermococcus and mutant thereof in diol synthesis

CN122012634ACN 122012634 ACN122012634 ACN 122012634ACN-122012634-A

Abstract

The invention relates to the field of biochemical engineering, and provides an aldolase from a thermochromatic bacterium and application of a mutant thereof in diol synthesis, and the aldolase mutant, a coding gene, a recombinant plasmid and a recombinant bacterium thereof. Wherein the amino acid sequence of the aldolase from the thermochromatic bacteria is shown as SEQ ID NO.1, and the mutant is obtained by mutating the aldolase. The aldolase and the mutant thereof have high catalytic efficiency, realize the efficient biosynthesis of the 1, 3-propanediol and the 1, 3-butanediol, and have good industrial application prospect.

Inventors

  • ZENG ANPING
  • LIU JIANMING
  • FENG JIACHENG
  • ZHU QICHENG

Assignees

  • 西湖大学

Dates

Publication Date
20260512
Application Date
20260317

Claims (10)

  1. 1. The application of the aldolase from the thermal coccus in the synthesis of 1, 3-propanediol and/or 1, 3-butanediol is characterized in that the amino acid sequence of the aldolase is shown as SEQ ID NO.1, methanol and ethanol are used as co-substrates when the 1, 3-propanediol is synthesized, ethanol is used as a substrate when the 1, 3-butanediol is synthesized, and a product is obtained through multienzyme catalytic reaction.
  2. 2. The application of claim 1, wherein the multienzyme catalytic reaction system comprises a coenzyme cycle enzyme and an auxiliary enzyme, wherein the coenzyme cycle enzyme is one or more selected from NADH dependent formate dehydrogenase, NAD (P) H dependent formate dehydrogenase, 1, 3-propanediol dehydrogenase and aldehyde ketone reductase, the auxiliary enzyme is one or more selected from catalase and alcohol oxidase, and the reaction condition is that the pH is 5.0-9.0 and the temperature is 20-60 ℃.
  3. 3. An aldolase mutant obtained by mutating the aldolase from a thermochromatic bacterium of claim 1, wherein the mutation site is one or more selected from the group consisting of 23, 64, 172, 213, and 214.
  4. 4. The aldolase mutant according to claim 3 wherein the mutation site is one or more of 64, 213 or 214 th site in the direction of mutation from 64 th site to cysteine, 213 th site to aspartic acid and 214 th site to aspartic acid.
  5. 5. The aldolase mutant according to claim 4 which is a triple mutant, wherein the mutation site is 64, 213 or 214, the 64 th mutation is cysteine, the 213 th mutation is aspartic acid, the 214 th mutation is aspartic acid, and the amino acid sequence is shown in SEQ ID NO. 8.
  6. 6. A gene encoding the aldolase mutant according to any one of claims 3 to 5.
  7. 7. A recombinant plasmid or recombinant bacterium comprising the coding gene of claim 6.
  8. 8. An aldolase mutant according to any one of claims 3 to 5 or an application of the coding gene according to claim 6 in synthesis of 1, 3-propanediol and/or 1, 3-butanediol, characterized in that methanol and ethanol are used as co-substrates in synthesis of 1, 3-propanediol, ethanol is used as a substrate in synthesis of 1, 3-butanediol, and a product is obtained by multienzyme catalytic reaction.
  9. 9. The use according to claim 8, wherein when the coding gene is used as an application object, the method further comprises the steps of constructing recombinant bacteria containing the coding gene, and performing the multienzyme catalytic reaction by using wet bacteria obtained by fermenting and culturing the recombinant bacteria or pure enzymes obtained by crushing and purifying the bacteria as catalysts.
  10. 10. The application of the multi-enzyme catalytic reaction system according to claim 8, wherein the multi-enzyme catalytic reaction system comprises coenzyme circulating enzyme and auxiliary enzyme, the coenzyme circulating enzyme is selected from one or more of NADH dependent formate dehydrogenase, NAD (P) H dependent formate dehydrogenase, 1, 3-propanediol dehydrogenase and aldehyde ketone reductase, the auxiliary enzyme is selected from one or more of catalase and alcohol oxidase, and the reaction condition is that the pH is 5.0-9.0 and the temperature is 20-60 ℃.

Description

Use of aldolase from Thermococcus and mutant thereof in diol synthesis Technical Field The invention relates to the field of biochemical engineering, in particular to an aldolase from thermococcus (Thermococcus onnurineus) and application of a mutant thereof in synthesis of diol (1, 3-propanediol and 1, 3-butanediol). Background 1, 3-Propanediol (1, 3-PDO) and 1, 3-butanediol (1, 3-BDO) are used as key C3 and C4 linear diol platform chemicals, and have irreplaceable application values in the fields of chemical industry, materials and daily chemicals. Among them, 1, 3-propanediol is a core raw material for producing polytrimethylene terephthalate (PTT), PTT fiber promotes the continuous increase of global 1,3-PDO market demand due to the excellent performance in the fields of high-end textiles, carpets and the like, the demand of China in 2025 years reaches 6.5 ten thousand tons, the market scale is about 2.64 hundred million dollars, and the market scale is expected to further increase to 5.18 hundred million dollars in 2031. 1, 3-butanediol is used as a multifunctional green solvent and a fine chemical intermediate, is widely used for synthesizing cosmetic moisturizers, fragrances and biodegradable polyesters, and has estimated 2030 to reach about 3.5 hundred million dollars in the global market scale and huge market potential. At present, the industrial production of 1, 3-propanediol is gradually changed to a biological method route, but the existing biological synthesis technology still has obvious defects, and is difficult to meet the large-scale application requirements, wherein the main flow technology takes glucose or glycerol as a substrate, the glucose raw material depends on grain resources, the grain safety problem of competing with people exists, the raw material cost is obviously influenced by grain price fluctuation, and the glycerol substrate route is subjected to the problems of large glycerol market price fluctuation and insufficient supply stability, so that the production economy is difficult to guarantee. Meanwhile, the existing microorganism fermentation method (such as producing 1, 3-propanediol by using klebsiella) also has the problems of complex metabolic pathway, multiple byproducts, high separation and purification difficulty of target products and the like, and further increases the production cost. The biosynthesis technology of 1, 3-butanediol is still immature, the existing path also depends on high-cost substrates or complex fermentation systems, and is difficult to be compatible with the production technology of 1, 3-propanediol, and the high-efficiency synthesis of multiple products on the same platform cannot be realized. The aldolase catalyzed carbon-carbon bond formation reaction provides a potential green biocatalysis path for synthesizing chiral diol from cheap substrates (such as formaldehyde, acetaldehyde, pyruvic acid and the like) in one step with high selectivity, has mild reaction conditions and high atom economy, completely meets the requirement of sustainable development, and is an ideal direction for solving the defects of the existing biological method. However, the application of the currently reported natural aldolase in the industrialized synthesis of 1, 3-propanediol and 1, 3-butanediol still has significant limitations that most of the natural aldolase has low catalytic activity on non-natural substrates such as formaldehyde, acetaldehyde and the like, is difficult to meet the yield requirement of industrial production, has problems of insufficient regio/stereoselectivity and obvious product inhibition effect of partial enzyme, and leads to limited purity and yield of target products, and in addition, the currently reported aldolase synthesis route takes formaldehyde and acetaldehyde as substrates, and has the problems of raw material safety and the like. These limitations together limit its scale-up applications. Although researchers have tried to screen aldolases of different sources or mutate them, it has not been found that a high-activity natural aldolases capable of efficiently synthesizing 1, 3-propanediol by a one-pot method using methanol, ethanol and the like as starting substrates and simultaneously having the synthesis capability of 1, 3-butanediol can not meet the requirements of industrial production on yield and cost far from the catalytic efficiency of the existing enzyme preparation. Accordingly, the high-activity natural aldolase resource is excavated, the catalytic performance is further optimized through directional mutation, an efficient and economical biocatalysis process is constructed, the green large-scale production of the 1, 3-propanediol and the 1, 3-butanediol is realized, and the key technical problem to be solved in the field is urgent. Disclosure of Invention The invention aims to solve the technical problem of providing an aldolase from thermochromatic bacteria and an application of a mutant thereof in synthesis of diol (