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Method for producing 1,3-propanediol by using methanol/formaldehyde and glucose as common substrates

A technology of propylene glycol and methanol dehydrogenase, applied in the field of microbial fermentation, can solve the problems of long synthetic pathway, difficult regulation and enhancement, and low yield of 1,3-PDO

Active Publication Date: 2019-04-12
HUAANTANG BIOTECH GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are problems such as long synthetic pathways, difficult regulation and enhancement, and low yield of 1,3-PDO.

Method used

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  • Method for producing 1,3-propanediol by using methanol/formaldehyde and glucose as common substrates
  • Method for producing 1,3-propanediol by using methanol/formaldehyde and glucose as common substrates
  • Method for producing 1,3-propanediol by using methanol/formaldehyde and glucose as common substrates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Screening and characterization of suitable aldolases for novel 1,3-PDO production pathways

[0075] The aldolase genes from different sources are: 2-oxo-4-hydroxybutyrate aldolase KHB derived from Escherichia coli; 2-keto-3 derived from Escherichia coli - Deoxy-L-rhamnolate aldolase YfaU; 2-oxo-4-hydroxyglutarate aldolase RnKHG from rat (Rattus norvegicus); 2-oxo from human (Homo sapiens) -4-hydroxyglutarate aldolase HsKHG; 2-oxo-4-hydroxyglutarate aldolase BtKHG derived from bovine (Bos taurus), constructed on the expression vector pET22b, respectively: pET22b-KHB; pET22b-YfaU; pET22b-RnKHG; pET22b-HsKHG; pET22b-BtKHG.

[0076] The above expression vectors were respectively transformed into E. coli expression host E.coli BL21(DE3), cultivated at 37°C until the OD reached 0.5-0.6, then added 0.2mM IPTG to induce gene expression, continued to cultivate at 30°C for 10h, and then collected the cells. After sonication, the supernatant was taken for use.

[0077...

Embodiment 2

[0078] Example 2: In vitro verification of formaldehyde to 1,3-PDO new biosynthetic pathway

[0079] The KHB coding gene (SEQ ID NO.2) derived from Escherichia coli (Escherichia coli) was constructed on the expression vector pET22b, and the KDC coding gene (SEQ ID NO.3) derived from Lactococcus lactis (Lactococcus lactis) was constructed to express On the vector pET22b, the DhaT gene (SEQ ID NO. 4) derived from Klebsiella pneumoniae was constructed on the expression vector pET22b. The above expression vectors were respectively transformed into E. coli expression host E.coli BL21(DE3), cultivated at 37°C until the OD reached 0.5-0.6, then added 0.2mM IPTG to induce gene expression, continued to cultivate at 30°C for 10h, and then collected the cells. After sonication, the supernatant was taken for use.

[0080] Add formaldehyde at a final concentration of 10 mM, 10 mM sodium pyruvate, 20 mM NADH, and 1 mM thiamine pyrophosphate (TPP) to a total volume of 1 mL of PBS buffer wit...

Embodiment 3

[0081] Example 3: Construction of formaldehyde to 1,3-PDO new biosynthetic pathway recombinant strain

[0082] Utilize kit In-fusion HD Cloning Kit (Clontech Laboratories, Inc, US) to construct plasmid pRSFduet-1-KHB-KDC-DhaT: first, plasmid vector pRSFduet-1 and KDC encoding gene (SEQ ID NO.3) were passed through NdeI, NcoI Restriction endonucleases were used for double digestion, and enzyme ligation was performed to obtain pRSFduet-1-KDC. Afterwards, the KHB coding gene (SEQ ID NO.2) was amplified by PCR using primers KHB-F and KHB-R, and the DhaT gene (SEQ ID NO.4) was amplified by PCR using primers DhaT-F and DhaT-R. The pRSFduet-1-KDC plasmid was amplified by PCR, and the primers were pRSFduet-1-KDC-F and pRSFduet-1-KDC-R, respectively. The fragments were connected with the In-fusion kit, and the newly synthesized plasmid was named pRSFduet-1- KHB-KDC-DhaT. (See Table 3 for primers)

[0083] Plasmid pRSFduet with 2-oxo-4-hydroxybutyrate aldolase (KHB), branched-chain-2...

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Abstract

The present invention belongs to the technical field of microbial fermentation and particularly relates to a method for producing 1,3-propanediol by using methanol / formaldehyde and glucose as common substrates. Four enzymes are over-expressed in recombinant Escherichia coli: methanol dehydrogenase (MDH), aldolase, 2-oxo decarboxylase and 1,3-propanediol dehydrogenase. A novel methanol and formaldehyde C1 compound converted 1,3-PDO synthetic pathway is established. The method is used to solve problems that utilization of methanol, formaldehyde and other C1 compounds is too dependent on ribulosemonophosphate pathway (RuMP) and regeneration of Ru5P receptors is inefficient, avoids an addition of a cofactor vitamin B12 or S-adenosylmethionine (SAM), also shortens the 1,3-PDO synthetic pathwayand increases yield of 1,3-PDO.

Description

Technical field: [0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for producing 1,3-propanediol by using methanol / formaldehyde and glucose as co-substrates. Background technique: [0002] Formaldehyde is an important metabolic intermediate of one-carbon compounds. Methanol, formic acid, and methane can all be converted to formaldehyde, which then enters central metabolic pathways to synthesize biomass for microbial growth and synthesis of chemicals (Bennett et al.2018; Zhang et al.2018; Hwang et al.2018; Pieja et al. 2017). The current study found that there are mainly three different natural metabolic pathways for formaldehyde utilization, including: (1) ribulose monophosphate pathway (RuMP, ribulose monophosphate pathway), (2) serine pathway (serine pathway) and (3) Calvin Cycle (CBB, Calvin-Benson-Bassham pathway) (Zhang et al. 2017; Ludmila 2011; Vorholt 2002). The most studied and effective formaldeh...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/53C12P7/18C12R1/19
CPCC12N9/0006C12N9/88C12P7/18C12Y101/01202C12Y101/01244C12Y401/01001
Inventor 曾安平任杰王闯
Owner HUAANTANG BIOTECH GRP CO LTD
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