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Biological synthesis method of branched ketose

A branched-chain ketose and microbial technology, applied in the field of branched-chain ketose biosynthesis, can solve the problems of high price of dihydroxyacetone, harsh reaction conditions, increased production costs, etc.

Inactive Publication Date: 2020-04-21
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional method of synthesizing 4-hydroxymethylfurfural is mainly through chemical modification of the furan ring, but this method requires very harsh reaction conditions, and the solution used is also very expensive; studies have reported that 4-hydroxymethylfurfural can be easily obtained after branched chain ketose dehydration Methylfurfural, the reaction conditions are relatively simple, and has a high yield, so the development of a high-efficiency branched chain ketose synthesis method is of great significance for the preparation of 4-hydroxymethylfurfural
[0003] At present, branched chain ketose is mainly synthesized by chemically condensing dihydroxyacetone (US8455668B2), yet this method needs a strong base as a catalyst, which is easy to pollute the environment; the catalytic process produces a small amount of by-product straight chain ketose, which increases the separation cost; the adopted The price of the substrate dihydroxyacetone is relatively high, which increases the production cost; so it is very necessary to develop a branched-chain ketose synthesis method that uses cheap substrates as raw materials, is environmentally friendly, and has a single product

Method used

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  • Biological synthesis method of branched ketose
  • Biological synthesis method of branched ketose
  • Biological synthesis method of branched ketose

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, L-rhamnosan-1-phosphate aldolase characterization

[0040] 1. Construction of L-rhamnosan-1-phosphate aldolase recombinant strain

[0041] Primers were designed according to the L-rhamnosan-1-phosphate aldolase gene (SEQ ID NO: 5) derived from Escherichia coli MG1655 in Genbank to amplify the L-rhamnosan-1-phosphate derived from Escherichia coli 1-phosphate aldolase gene (rhaD) fragment, rhaD and pET21a were simultaneously digested with restriction enzymes NdeI and HindIII, and then ligated with T4 DNA ligase to obtain L-rhamnosugar-1-phosphate aldolase The carrier plasmid of the enzyme gene rhaD is named pET21-RhaD.

[0042] 2. The above-mentioned recombinant plasmid pET21-RhaD was transformed into Escherichia coli BL21(DE3) by chemical transformation, and two recombinant strains of Escherichia coli 21RhaD were obtained.

[0043] 3. Preparation of L-rhamnosan-1-phosphate aldolase

[0044] First, culture and induce Escherichia coli recombinant strain 21...

Embodiment 2

[0050] Example 2, multi-enzyme in vitro cascade catalyzes the synthesis of branched-chain ketose from dihydroxyacetone

[0051] Construct a multi-enzyme system composed of dihydroxyacetone kinase, L-rhamnose-1-phosphate aldolase, fructose 1-phosphatase and polyphosphate kinase to catalyze the synthesis of branched chain ketose from dihydroxyacetone, including The following steps:

[0052] 1. Construction of recombinant dihydroxyacetone kinase strain

[0053] Primers were designed according to the dihydroxyacetone kinase gene (SEQ ID NO: 10) derived from Citrobacter freundii in Genbank to amplify the dihydroxyacetone kinase gene (dhaK) fragment, and dhaK was simultaneously treated with restriction endonucleases NdeI and HindIII Digested with pET21a, and then ligated with T4 DNA ligase to obtain a vector plasmid containing dihydroxyacetone kinase gene dhaK, which was named pET21-DhaK.

[0054] 2. Construction of fructose 1-phosphatase recombinant strain

[0055] Primers were ...

Embodiment 3

[0064] Embodiment 3, construction Corynebacterium glutamicum recombinant strain SY30

[0065] Construction of Recombinant Strain SY30 of Corynebacterium glutamicum

[0066] Corynebacterium glutamicum recombinant strain SY6 with triose phosphate isomerase gene knockout, its construction method is described in patent 201410055300.X, L-rhamnogum-1-phosphate aldolase gene was introduced into recombinant strain SY6, Phosphorylase gene and dihydroxyacetone phosphate phosphatase to obtain the recombinant strain of Corynebacterium glutamicum, named as bacterial strain S30, the construction scheme is as follows image 3 shown.

[0067] The specific construction process is as follows:

[0068] 1.1. According to the L-rhamnosan-1-phosphate aldolase gene (SEQ ID NO:5) and dephosphorylase (YqaB) gene (SEQ ID NO:6) derived from Escherichia coli MG1655 in Genbank ), derived from the sequence of dihydroxyacetone phosphate phosphatase (SEQ ID NO: 7) of Corynebacterium glutamicum, primers we...

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Abstract

The invention discloses a branched ketose method. According to the invention, the new function of known aldolase is provided, wherein the aldolase can catalyze an aldol condensation reaction between dihydroxyacetone phosphate and dihydroxyacetone to synthesize branched ketose, so that the invention discloses a method for synthesizing branched ketose by constructing an in-vitro multi-enzyme cascadereaction system to catalyze dihydroxyacetone on the basis, and the conversion rate is 90%; the invention also discloses a construction method of a corynebacterium glutamicum recombinant strain for producing branched ketose, wherein the branched ketose is synthesized by fermenting a cheap substrate glucose or glycerol with the obtained recombinant strain, and the yield reaches 36.3 g / L; and compared with the existing chemical method for synthesizing branched ketose, the branched ketose synthesis method provided by the invention has the advantages of cheap substrate, environmental friendliness,high product synthesis efficiency, single product and convenience in separation, has industrial application potential, and provides a basis for synthesis of 4-hydroxymethylfurfural.

Description

technical field [0001] The invention belongs to the field of biomanufacturing, and in particular relates to a biosynthesis method of branched chain ketose. Background technique [0002] Rare sugar is a type of monosaccharide and its derivatives that exist in nature but are very low in content (defined by the International Rare Sugar Society ISRS in 2002). Branched-chain ketose is one of the rare sugars that rarely exist in nature. 4-Hydroxymethylfurfural is a very important pharmaceutical intermediate. This compound can be used to prepare cantharidin. Furfural can also be used to prepare anti-schizophrenia drugs. In addition, this compound can also be used to prepare 2,4-dimethylfuran and C9-C15 branched alkanes for liquid fuels. The traditional method of synthesizing 4-hydroxymethylfurfural is mainly through chemical modification of the furan ring, but this method requires very harsh reaction conditions, and the solution used is also very expensive; studies have reported ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N1/21C12P19/02C12R1/15
CPCC12N9/16C12N9/88C12N9/90C12P19/02C12Y301/03011C12Y401/02019C12Y503/01001
Inventor 杨建刚曾艳朱玥明孙媛霞
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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