Recombinant zymomonas mobilis capable of producing ethanol by using xylose and fermentation method thereof

A technology of Zymomonas and movement, applied in the field of bioengineering, can solve problems such as increasing the expression of exogenous genes, and achieve the effect of solving the energy crisis

Inactive Publication Date: 2010-07-21
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Escherichia coli rrnB operator T1T2 is a powerful transcription terminator, which can effectively prevent the read-through phenomenon in transcription and ensure the stability of the expression plasmid, thereby increasing the expression level of foreign genes in prokaryotic cells. rrnB of E. coli can increase The expression levels of exogenous genes in Escherichia coli and Bacillus subtilis (Rong 2005; Wang 1992); however, there is no report on the use of rrnB and xylose metabolism-related genes as operons in Zymomonas mobilis

Method used

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  • Recombinant zymomonas mobilis capable of producing ethanol by using xylose and fermentation method thereof
  • Recombinant zymomonas mobilis capable of producing ethanol by using xylose and fermentation method thereof
  • Recombinant zymomonas mobilis capable of producing ethanol by using xylose and fermentation method thereof

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Embodiment 1

[0044] Example 1 The construction of the shuttle expression vector pZ that can be replicated in Escherichia coli and Zymomonas mobilis: first extract the plasmid of Z.mobilis (ATCC10988) with SDS alkaline lysis method (Sam Brook, 2002), and use Ava I enzyme After cutting the plasmid, electrophoresis was performed to recover a 2.7kb plasmid linear fragment, and after ligation, the pZM2 plasmid of Zymomonas mobilis was obtained. Escherichia coli plasmid pACYC was extracted by SDS alkaline lysis method. Based on Escherichia coli plasmid pACYC and Zymomonas mobilis plasmid pZM2, it was digested with AvaI and ligated into plasmid pZ. pZ contains the origin of replication for Escherichia coli and Zymomonas mobilis ( figure 1 ), which can replicate in the above two host bacteria.

Embodiment 2

[0045] Example 2 Construction of the shuttle vector pXylose expressing xylose metabolism genes

[0046] Genomic DNA of Escherichia coli K12 and Zymomonas mobilis (ATCC NO.31821) were extracted with the bacterial genome extraction kit of Tiangen Company;

[0047] Using the genome of Zymomonas mobilis (ATCC NO.31821) as a template, using the sequences described in SEQ ID NO.1 and SEQ ID NO.2 in the sequence table as PCR primers, amplifying the promoter Pgap of Zymomonas mobilis;

[0048] Using Escherichia coli K12 genomic DNA as a template, using the sequences described in SEQ ID NO.3 and SEQ ID NO.4 in the sequence table as PCR primers, amplifying the genes encoding xylose isomerase XylA and xylokinase XylB and the untranslated regions , the amplified product was digested with Kpn I and Xba I and ligated into the pUC19 plasmid, then digested with Nsi I and Sma I, blunt the sticky ends and ligated to obtain the vector pXX;

[0049] Using pXX as a template, using the sequences d...

Embodiment 3

[0067] The acquisition of embodiment 3 recombinant strains

[0068] Plasmid pXylose was extracted with the large extraction plasmid kit of Promega Company. Inoculate the culture of Zymomonas mobilis CP4 (CICC10232) into 50ml RM liquid medium at 0.5% or 1.5% inoculum size, and culture statically at 30°C until OD 600 =0.5; centrifuge at 6000rpm for 5min, collect the thallus, wash 2 or 4 times with 10% aqueous glycerol solution in ice bath, suspend and mix evenly with 2ml 10% glycerin aqueous solution by volume, to obtain Competent cells: Take 40μl competent cells, add 1μg plasmid pXylose to mix, transfer to a sterile pre-cooled 1mm electric shock cup, 1800V electric shock, quickly add 450ul RM body culture medium, mix well and transfer to a 1.5ml sterile centrifuge tube In the medium, let stand at 30°C for 14 hours (12 hours or 17 hours can also be selected), spread the culture on the RM solid medium screening plate containing 20 μg / ml tetracycline, and place it upside down in ...

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Abstract

The invention discloses recombinant zymomonas mobilis capable of producing ethanol by using xylose and a fermentation method thereof. The recombinant zymomonas mobilis GX1 capable of producing the ethanol by using the xylose is preserved in China General Microbiological Culture Collection Center, and has a preservation number of CGMCC 3438. The controlling elements of Escherichia coli rrnB and related genes of xylose metabolism are fused to build plasmids with artificially fused operons for the first time; and recombinant zymomonas mobilis strains capable of producing the ethanol by using the xylose are obtained through a method of electrotransformation and acclimatization. The recombinant zymomonas mobilis can efficiently express exogenous genes, can efficiently produce the ethanol by using glucose and the xylose to ferment together at the temperature of between 30 and 34 DEG C, and can produce the ethanol by using corncob hydrolysate containing the xylose and the glucose. The strains have important significance for producing clean energy by effectively using waste lignocellulose and solving the current energy crisis.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a recombinant Zymomonas mobilis capable of producing ethanol from xylose and a fermentation method. Background technique [0002] With the depletion of fossil energy and the gradual emergence of the greenhouse effect, clean alternative energy has become a global research hotspot, and the research and development of fuel ethanol produced by fermentation of lignocellulose as raw material has attracted more attention. Renewable lignocellulose resources are extremely abundant in nature, and one of its main components is hemicellulose. 85%-90% of hemicellulose is xylose (Lachke 2002), so the use of xylose to ferment ethanol is of great significance to the effective utilization of lignocellulose and to solve the current energy crisis. [0003] However, high-efficiency ethanol-producing bacteria in nature can only use six-carbon sugars such as glucose to produce ethanol, and cannot use xylo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/74C12P7/06C12R1/01
CPCY02E50/17Y02E50/10
Inventor 马媛媛邹少兰张鲲洪解放刘成张敏华
Owner TIANJIN UNIV
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