Aldolase mutant and its application in production of 1, 3-propylene glycol

A mutant, phosphate aldolase technology for applications, microbial-based methods, enzymes, etc., capable of solving problems such as restricted 3-HPA

Active Publication Date: 2021-06-08
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this pathway is limited by the efficiency of decarboxylation of HOBA to 3-HPA (Wang et al., 2019)

Method used

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  • Aldolase mutant and its application in production of 1, 3-propylene glycol
  • Aldolase mutant and its application in production of 1, 3-propylene glycol
  • Aldolase mutant and its application in production of 1, 3-propylene glycol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Screening efficient DERA for novel formaldehyde and acetaldehyde to PDO production pathway

[0055]Different sources of DERA were constructed on the expression vector pRSFduet-1-DhaT with the gene encoding DhaT, respectively: deoxyribose-5-phosphate aldolase pRSFduet-DERA derived from Escherichia coli ECO -DhaT; deoxyribose-5-phosphate aldolase pRSFduet-DERA derived from Thermotoga maritima TMA -DhaT(DERA TMA The amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.3); deoxyribose-5-phosphate aldolase pRSFduet-DERA derived from Geobacillus thermodenitrificans GTH -DhaT; Deoxyribose-5-phosphate aldolase pRSFduet-DERA derived from Pyrobaculum aerophilum PAE -DhaT; deoxyribose-5-phosphate aldolase pRSFduet-DERA derived from Staphylococcus epidermidis SEP -DhaT. The above expression vectors were respectively transformed into the E. coli expression host E.coli BL21(DE3) (Control did not express any DERA, only expressed...

Embodiment 2

[0057] Example 2: For DERA TMA Enzyme activity assay results for semi-rational design mutations

[0058] From Example 1, we can screen the deoxyribose-5-phosphate aldolase derived from Thermotoga maritima with the best performance in this biotransformation system. Then, we carried out a semi-rational mutation design on the enzyme, and carried out enzyme activity determination on the obtained mutants, the results are as follows: image 3 , its S233D mutant obtained a catalytic activity 51% higher than that of the wild type, and its specific activity was 1.89±0.04U / mg. The amino acid sequence of the S233D mutant is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID Shown in NO.4.

Embodiment 3

[0059] Example 3: Validation of novel formaldehyde and ethanol production pathways to PDO

[0060] The HpADH1 gene (shown in SEQ ID NO.6) derived from Hansenula polymorpha and the deoxyribose-5-phosphate aldolase mutant DERA derived from Thermotoga maritima TMA The S233D gene (shown in SEQ ID NO.4) and the DhaT gene (shown in SEQ ID NO.5) derived from Klebsiella pneumoniae were constructed on the expression vector pRSFduet-1. The above expression vectors were respectively transformed into the E. coli expression host E.coli BL21(DE3), single clones were picked and cultured in LB medium at 37°C for 8h, and then transferred to 400mL containing 50μg / mL kanamycin LB medium to OD 600 After reaching 0.6-0.8, add 0.2mM IPTG to induce gene expression, continue culturing at 30°C for 10 hours, measure the absorbance value of the bacteria at 600nm, collect a certain amount of bacteria and centrifuge to remove the supernatant, then buffer with a certain amount of 50mM phosphate Resuspend...

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Abstract

The invention relates to the technical field of synthetic biology and microbial fermentation, in particular to modification of deoxyribose-5-phosphate aldolase through semi-rational design and overexpression of four enzymes in recombinant escherichia coli, namely methanol dehydrogenase, ethanol dehydrogenase, deoxyribose-5-phosphate aldolase, 1, 3-propylene glycol oxidoreductase or 1, 3-propylene glycol oxidoreductase isozyme, to construct unnatural 1, 3-propylene glycol from methanol, a formaldehyde-carbon compound, ethanol and acetaldehyde. The activity of the DERATMAS233D mutant screened by the invention is improved by 51% compared with that of a wild type enzyme; When the conversion from formaldehyde and ethanol to PDO by using escherichia coli, the yield of PDO is increased by 21.8%; when the formaldehyde and the acetaldehyde are converted into the PDO, the yield of the PDO is increased by 9.2%, the synthesis route of the PDO is greatly shortened, and the yield of the PDO is increased.

Description

Technical field: [0001] The present invention relates to the field of synthetic biology and microbial fermentation technology, specifically, the present invention relates to semi-rational design to the transformation of enzyme and overexpress four kinds of enzymes in recombinant escherichia coli: methanol dehydrogenase, alcohol dehydrogenase, deoxyribose -5-Phosphate aldolase, 1,3-propanediol oxidoreductase or 1,3-propanediol oxidoreductase isoenzyme, construct non-natural conversion of methanol, formaldehyde one carbon compound and ethanol, acetaldehyde into 1,3 - Method for the production route of propylene glycol. Background technique: [0002] Formaldehyde can be derived from methanol (Methanol), formic acid (Formate) and methane (Methane), and is an important metabolic intermediate of one-carbon compounds, which can be used for the growth of microorganisms and the synthesis of chemicals (Bennett et al., 2018; Hwang et al., 2018; Pieja et al., 2017; Zhang et al., 2018)....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/53C12N15/70C12N15/81C12N1/19C12N1/21C12P7/18C12R1/19C12R1/865
CPCC12N9/88C12N9/0006C12N15/70C12N15/81C12P7/18C12Y401/02004C12Y101/01001C12Y101/01244C12Y101/01202
Inventor 曾安平孟豪任杰
Owner BEIJING UNIV OF CHEM TECH
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