Unlock instant, AI-driven research and patent intelligence for your innovation.

Construction body for biosynthesis of ethanol, strain and method for producing ethanol

A technology for biosynthesis and construct, which is applied in the biological field to achieve the effects of increasing pyruvic acid content, efficient synthesis and good feasibility

Pending Publication Date: 2021-09-17
JIANGSU UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, from the perspective of biosynthetic quantity, the actual yield obtained by using Synechocystis to synthesize biologically active substances is far lower than the theoretical target output value.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction body for biosynthesis of ethanol, strain and method for producing ethanol
  • Construction body for biosynthesis of ethanol, strain and method for producing ethanol
  • Construction body for biosynthesis of ethanol, strain and method for producing ethanol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Construction of copper ion elicitor PpetE plasmid PET01

[0042] Based on the pMD18-T vector purchased by Sangon Biotechnology Company (Shanghai, China), a 600bp homology arm of slr0168 upstream and downstream was inserted into pMD18-T to construct the vector PMD0168. The basic structure of the vector PMD0168 is as follows figure 2 shown. Using the extracted genomic DNA of Synechocystis PCC6803 as a template, primers slr0168Up-F (5'-GGCATGCCGAGCGGCACCACGGGGCACCACCGC-3') and slr0168Up-R (5'-GACGCGTCGGCGCACAGCAGCGTGCGACGTGTG-3'), slr0168Dw-F (5'-CTCTAGAGTGCCACTACCTGGCGTGCCGCTACC) were used as templates, respectively. -3') and slr0168Dw-R (5'-GGGGITACCCCGCATGACCAGCTGCCGCCCCAGC-3') amplified the 600bp homology arms upstream and downstream of slr0168 and cloned into the SphI / mLuI and XbaI / KpnI sites of pMD18-T. The spectinomycin gene was cloned into plasmid pMD0168 to construct plasmid PET406. The basic structure of plasmid PET406 is as follows image 3 shown. ...

Embodiment 2

[0043] Example 2: Construction of light-intensity super-promoter Pcpc560 plasmid PET02

[0044] Different from plasmid PET01, plasmid PET02 uses the genomic DNA of Synechocystis PCC6803 as a template, and primers Pcpc-F (5'-CGTCTAGAGGATCCCCTGTAGAGAAGAGTCCCTG-3') and Pcpc-R (5'-TTTCTCCTCTTTTGAATTAATCTCCTACTTGACTTTATGAG-3'), TrbcL-F (5'-CGCGTCGACCGGTGTTTGGATTGTCGGAGT-3') and TrbcL-R (5'-CCGACGCGTAAGCTTCCGGTAATTGGTAAATTGCTGTC-3') amplified the promoter and terminator genes and cloned them into the BamHI / SalI and SalI / HindIII sites of PET406 to obtain PET408. According to the codon preference of Synechocystis PCC6803, using online software (http: / / www.jcat.de / ), the pyruvate carboxylase gene (pdc) of Zymomonas mobilis and the NADPH-dependent gene of Escherichia coli The nucleotide sequence of the aldehyde reductase gene (yqhD) was optimized. The optimized sequences were synthesized by Sangon Biotechnology (Shanghai, China). The optimized synthetic strain was used as the template...

Embodiment 3

[0045] Example 3: Construction of plasmid PET03 knocking out phosphoenolpyruvate synthesis pathway

[0046] Using the extracted genomic DNA of Synechocystis PCC6803 as a template, primers slr0301Up-F (5'-ACATGCATGCATTAACTCCCGCAGAAAGGGA-3') and slr0301Up-R (5'-CGACGCGTCGACATCATGGGTGCCCACCTCTTCA-3'), slr0301Dw-F (5'-CTAGTCTAGACGGCTTCTCCATTGGCTCCAAT) were used respectively. -3') and slr0301Dw-R (5'-CGAGCTCGGTACCGCCCTCAACCTCTCCATTTCC-3') amplified the 600bp upstream and downstream homology arms of slr0301 and cloned them into the SphI / MluI and XbaI / KpnI sites of pMD18-T. The spectinomycin gene was cloned into plasmid pMD0301 to construct plasmid pMD-slr0301-Ω. In the same way, PET03 is constructed, and the basic structure of plasmid PET03 is as follows Image 6 As shown, its nucleotide sequence is shown in SEQ ID NO.8. The specific construction plasmids and primers are shown in Table 1.

[0047] Table 1. Plasmids and primers

[0048]

[0049]

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the technical field of biology, in particular to a construction body for biosynthesis of ethanol, a strain and a method for producing ethanol. A light intensity promoter Pcpc560 is utilized to drive pyruvate decarboxylase and NADPH dependent aldehyde reductase yqhd genes to express in one or more gene loci in cyanobacteria, and a molecular technology is utilized to knock out a cyanobacteria endogenous pathway reverse to pyruvic acid metabolism for the first time so as to construct and synthesize genetic engineering cyanobacteria with high ethanol yield. According to the method, a metabolic pathway for efficiently synthesizing the bioethanol is successfully constructed in the synechocystis PCC6803 for the first time, the content of pyruvic acid serving as an ethanol synthesis precursor can be increased, efficient synthesis of the ethanol in the synechocystis PCC6803 is realized, and the method has good feasibility.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a construct for biosynthesizing ethanol, a strain and a method for producing ethanol. Background technique [0002] With the depletion of fossil fuels, the development of renewable energy has received great attention. Cyanobacteria is a large single-celled prokaryotic organism capable of anaerobic photosynthesis, which uses water as an electron acceptor to release oxygen. It makes the whole earth's atmosphere develop from anaerobic state to aerobic state, thus giving birth to the evolution and development of all aerobic organisms. Cyanobacteria act as carbon fixation sites that can fix atmospheric CO 2 . At present, the use of engineered cyanobacteria to produce renewable biofuel ethanol has received more and more attention on the issue of renewable energy. Synechocystis sp. PCC6803 (Synechocystis sp) is a single-celled spherical cyanobacteria that can use light energy for autotro...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/74C12N15/65C12P7/06C12N1/21C12R1/01
CPCC12N15/74C12N15/65C12P7/065C12N15/52C12N9/88C12N9/0006C12Y401/01001C12Y101/01002C12Y401/01032Y02E50/10
Inventor 高恶斌朱杨杰叶鹏林
Owner JIANGSU UNIV