Mutant strain for increasing content of ethanol produced by fermentation of synthesis gas and application of mutant strain

A mutant strain and ethanol production technology, which is applied in the biological field to achieve the effects of optimizing fermentation conditions, increasing proportion and increasing ethanol production

Active Publication Date: 2021-08-10
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows researchers to create new microorganisms with improved ability to produce certain compounds through chemical reactions without producing unwanted substances or side products during their process. By deleting genes involved in these processes, this method could improve efficiency and yield compared to traditional methods such as breeding techniques.

Problems solved by technology

This patented describes various ways to modify certain types of organisms called clostraids like those from crops used worldwide to make them better suited for use as biofuels due to their unique structure. However, current techniques have limitations including low yields and excessive costs associated with modifying these species themselves. Therefore, it would be necessary to develop improved processes for making higher levels of specific compounds while reducing waste generated through conventional means.

Method used

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  • Mutant strain for increasing content of ethanol produced by fermentation of synthesis gas and application of mutant strain
  • Mutant strain for increasing content of ethanol produced by fermentation of synthesis gas and application of mutant strain
  • Mutant strain for increasing content of ethanol produced by fermentation of synthesis gas and application of mutant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Construction of Clostridium yondhal DSM 13528 2,3-butanediol dehydrogenase deletion mutant 1. Construction of gene knockout plasmid

[0075]Use restriction endonucleases SalI and XhoI to double digest the plasmid pMTLcas-pta, recover the 9000bp long fragment, and obtain the linearized pMTLcas vector backbone;

[0076] Design a guide RNA targeting the 20nt target region of the CLJU_c23220 gene (2,3-butanediol dehydrogenase) in Clostridium yondhal DSM 13528. The gene sequence is shown in Gene ID: 45179669, and then use pMTLcas-pta as a template, sgRNA -F / sgRNA-R is a primer, and the sgRNA scaffold is obtained by polymerase chain reaction (PCR).

[0077] The polymerase chain reaction system was 50 μl (2×KAPA Mix, 25 μl; 1 μL each of sgRNA-F / sgRNA-R (10 μM), 1 μL (50 ng) of pMTLcas-pta carrier, added water to make up to 50 μl).

[0078] PCR amplification conditions, ①pre-denaturation at 95°C for 3 min, ②denaturation at 98°C for 20 sec, ③annealing at 60°C for 20 ...

Embodiment 2

[0104] Example 2: Construction of 2,3-butanediol dehydrogenase deletion mutant of Clostridium ethanologenum DSM 10061 1. Construction of gene knockout plasmid

[0105] Use restriction endonucleases SalI and XhoI to double digest the plasmid pMTLcas-pta to obtain the linearized pMTLcas vector fragment;

[0106] Using pMTLcas-pta as a template and sgRNA-F / sgRNA-R as primers, sgRNA was obtained by polymerase chain reaction (PCR), targeting the 20nt target region of the gene of Clostridium ethanogenum DSM 10061CAETHG_0385, the gene sequence is as Gene ID: 33107402 shown;

[0107] Genomic DNA of Clostridium ethanogenum DSM 10061 was used as a template, HA-upstream-F / HA-upstream-R and HA-downstream-F / HA-downstream-R were used as primers, respectively, and the open reader located at CAETHG_0385 was obtained by polymerase chain reaction Two homology arms (HA-upstream and HA-downstream) on both sides of the box;

[0108] By overlapping extension PCR, HA-upstream and HA-downstream wer...

Embodiment 3

[0116] Example 3: Construction of Clostridium yondhal DSM 13528 pyruvate decarboxylase gene instead of 2,3-butanediol dehydrogenase gene expression mutant

[0117] 1. Construction of gene replacement plasmid

[0118]Pyruvate decarboxylase converts pyruvate to acetaldehyde. Specifically, the pyruvate decarboxylase gene is from Zymomonas mobilis subsp. mobilis ATCC 29191.

[0119] In order to optimize the heterologous expression of the pyruvate decarboxylase gene (CP003704.1:1958606-1960312; Locus tag: ZZ6_1712) derived from Zymomonas mobilis, according to the preferred codons of the host Clostridium yondhal DSM 13528, Codon optimization was carried out on the pyruvate deacidase gene (codon optimization was entrusted to Huada Genomics Co., Ltd.), and the optimized gene sequence was synthesized, see SEQID NO.1 for details.

[0120] Use restriction endonucleases SalI and XhoI to double digest the plasmid pMTLcas-pta to obtain a linearized 9000bp pMTLcas vector backbone;

[0121...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a mutant strain for increasing the content of ethanol produced by fermentation of synthesis gas, construction and an application of the mutant strain and a method for increasing the content of ethanol produced by fermentation of synthesis gas of the mutant strain obtained by genetic engineering means. The mutant strain is mutation obtained by deletion or inactivation of 2, 3-butanediol dehydrogenase in clostridium aerovorans. According to the deletion mutant constructed by the invention, the yield of a byproduct 2, 3-butanediol and the proportion of the byproduct 2, 3-butanediol in a total fermentation organic solvent are remarkably reduced under the condition that the normal growth of a strain is hardly influenced. The clostridium yangdaei is taken as an example, the yield of the 2, 3-butanediol is reduced to 2.4 g/L from 16.6 g/L, and the generation of ethanol is basically not influenced. According to the method, the content of the byproduct 2, 3-butanediol in the total fermentation solvent is reduced in a gene deletion manner, and meanwhile, the proportion of ethanol in the total fermentation solvent is increased, so that the separation cost of the fermentation liquor is reduced, and the method has important significance on industrial implementation of fuel ethanol production through synthesis gas fermentation.

Description

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Claims

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Application Information

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Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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