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Intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli

A technology of Escherichia coli and xylitol, applied in the field of microbial metabolic engineering, can solve problems such as low xylitol yield and inability to transport xylose into cells

Active Publication Date: 2021-08-24
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, relying on metabolic engineering technology, two important factors cannot be avoided when using Escherichia coli to produce xylitol in a mixed carbon source of glucose and xylose
First, the carbon catabolism repression effect (referred to as CCR effect) produced by the presence of glucose inhibits the expression of xylose transporter, resulting in the inability of xylose to be successfully transported into the cell; secondly, xylitol is also phosphorylated by xylulokinase to produce 5 - Xylitol phosphate, which produces feedback inhibition on the xylose transporter
This is also the reason for the low production of xylitol
[0004] Although there are currently existing methods to overcome the CCR effect and the inhibition of xylitol 5-phosphate in E. coli genetically engineered strains to realize the conversion of xylitol, but it is necessary to add enough glucose to E. coli to ensure that the cells are resistant to reducing power. continued demand for

Method used

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  • Intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli
  • Intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli
  • Intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Knockout in E. coli JM109(DE3) wxya and xylE, Completely remove the xylose transport ability in the strain and limit the metabolism of xylose by the strain. Knockout of Escherichia coli native xylose transporter by λred homologous recombination wxya and xyE , the purpose is to verify the role of SecY non-specific sugar transport channel in genetically engineered strains.

[0030] The specific knockout process includes the following steps:

[0031] Design PCR primers containing homology arm sequences respectively and use the pkD13 plasmid as a template for PCR amplification to obtain a KANA fragment containing homology arms and FRT recognition sites at both ends. Sugar-induced JM109 (DE3) competent cells were cultured at 37°C. Use the genome sequence to design PCR primers to verify that the KANA fragment (its nucleotide sequence is shown in SEQ ID NO.6) was successfully replaced wxya gene and xyE After transfecting the pCP20 plasmid, it is used to specifical...

Embodiment 2

[0034] Example 2 Construction Construction and expression of SecY non-specific sugar transport channel

[0035] Select JM109(DE3)ΔXylF-ΔXylE Escherichia coli as the chassis host, and co-express the double expression plasmid pETDuet-1 and pRSFDuet-1 through the recombinant vector secY(ΔP) , secE , secG and sCVE four genes.

[0036] the above secY(ΔP) The acquisition of genes includes the following methods: using overlapping PCR genetic engineering methods to construct Escherichia coli K family strains secY The mutated gene after the 60-74 amino acids in the gene are replaced by a flexible fragment including four amino acids of GlySerGlySer secY(ΔP) , secY(ΔP) The nucleotide sequence of the gene is shown in SEQ ID NO.1 . secY Gene NCBI Gene ID: 947799. The primers involved in overlapping PCR are as follows:

[0037]

[0038] the above sCVE The acquisition of genes includes the following methods: according to the preference of Escherichia coli encoded amino ac...

Embodiment 3

[0045] Embodiment 3 constructs and expresses the synthetic pathway of xylitol, overexpression wxya Gene

[0046] wxya gene from Candida boidinii , the gene is artificially synthesized after codon optimization, after optimization wxya The nucleotide sequence of the gene is shown in SEQ ID NO.5. Will wxya The gene was ligated into the recombinant vector prsfDuet- secG - sCVE The recombinant vector prsfDuet- secG - sCVE - wxya .

[0047] Transfect the recombinant vector pETDuet- secY (Δp)- secE and prsfDuet- secG - sCVE - cbxR. Successfully overexpressed in E. coli cells wxya After exogenous gene, obtain SecY engineered Escherichia coli, that is, JM109(DE3)ΔXylF-ΔXylE carrying pETDuet- secY (Δp)- secE and prsfDuet- secG - sCVE - wxya . In LB medium (5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl), it was initially verified whether xylitol could be synthesized. 50 mM xylose was added to the culture medium, and the culture inducer IPTG co...

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Abstract

The invention provides an intelligent regulation and control method for carbon metabolism flow of xylitol produced by escherichia coli, and aims to realize efficient synthesis of xylitol in a process of preparing xylitol by utilizing a cell factory. According to the invention, a strain of SecY engineered Escherichia coli is successfully constructed by introducing a SecY non-specific sugar transport channel and simultaneously overexpressing heterologous xylose reductase (CbXR). The strain can overcome the CCR effect through self-regulation and relieve the inhibition effect of the 5-phosphate xylitol on xylose transported by the strain. By taking the change of an external carbon source as a response signal, the CCR effect and the inhibition effect of the xylitol 5-phosphate are converted into two important regulating switches in a carbon metabolism network, and the metabolic capability of cells on glucose and xylose is regulated. The method can completely metabolize glucose and xylose in any proportion in a substrate, ensures the maximum conversion efficiency of energy to a target product, and better meets the requirements of green biological manufacturing.

Description

technical field [0001] The invention belongs to the field of microbial metabolism engineering, and in particular relates to an intelligent control method for carbon metabolism flow of xylitol produced by Escherichia coli. Background technique [0002] Biorefinery is beneficial to protect the environment and preserve non-renewable fossil fuels, and industrial biotechnology can convert carbon sources from agricultural waste into valuable chemicals, materials and biofuels for sustainable economic development. Xylitol is a five-carbon sugar alcohol that is as sweet as sucrose and occurs naturally in fruits, vegetables and mushrooms. Due to its low-calorie, tooth-protecting, anti-diabetic and anti-carcinogenic properties, xylitol is considered a valuable derivative in the food, pharmaceutical and chemical industries. The chemical production of xylitol is mainly artificially produced from xylan. On the contrary, the production of xylitol by microbial fermentation is more environ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/70C12P7/18C12R1/19
CPCC07K14/245C12N15/70C12P7/18
Inventor 范立海郭强郑辉东
Owner FUZHOU UNIV
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