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Improved xylose utilization in recombinant zymomonas having increased ribose-5-phosphate activity

A technology for recombining bacteria and recombining host cells, used in the fields of microbiology and genetic engineering

Inactive Publication Date: 2013-03-20
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, xylose utilization is still not comparable to glucose utilization

Method used

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  • Improved xylose utilization in recombinant zymomonas having increased ribose-5-phosphate activity
  • Improved xylose utilization in recombinant zymomonas having increased ribose-5-phosphate activity
  • Improved xylose utilization in recombinant zymomonas having increased ribose-5-phosphate activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Ribulose-accumulating Zymomonas mobilis ZW801-4 strain

[0155] The recombinant Zymomonas mobilis ZW801-4 strain is described in US Patent 7,741,119, which is incorporated herein by reference. The ZW801-4 strain is derived from the ZW800 strain, which is derived from the ZW658 strain, both of which are described in US 7,741,119. Integration into the ZW1 (renaming of strain ZM4; ATCC #31821) genome by successive transposition events into the genome contains genes encoding xylose isomerase (xylA), xylulokinase (xylB), transaldolase (tal) and transaldolase (tal) Two operands (P gap xylAB and P gap taltkt), resulting in the X13L3 strain, which was renamed ZW641 and subsequently acclimatized on xylose-containing selection medium. ZW658 is deposited under the Budapest Treaty as ATCC #PTA-7858. In ZW658, the gene encoding glucose-fructose oxidoreductase was insertionally inactivated using host-mediated double-crossover homologous recombination with spectinomycin resistan...

Embodiment 2

[0158] Zymomonas mobilis with chimeric ribulose-5-phosphate isomerase gene Engineering modifications carried out

[0159] Expression of an extrachromosomal copy of a chimeric gene comprising the native Zymomonas mobilis ribulose-5-phosphate isomerase (RPI-A) coding region was performed by cloning the coding region into a shuttle vector adjacent to The glucose isomerase (GI) promoter of Actinomycetes mobilis is realized.

[0160] The Zymomonas mobilis RPI-A coding region (SEQ ID NO: 1870) was isolated from ZW1 strain (renamed of ZM4 strain; ATCC #31821 ) genomic DNA using PCR. Genomic DNA was prepared from ZW1 using the Puregene Genomic DNA Purification Kit according to the manufacturer's instructions (Gentra Systems, Minneapolis, Minnesota). PCR primers were designed to amplify the RPI-A coding region and incorporate appropriate restriction enzyme sites for cloning purposes. PCR primers were also designed to maintain the natural distance between the Shine-Delgarno sequen...

Embodiment 3

[0167] Expression of Zymomonas mobilis ribulose-5-phosphate isomerase in recombinant Zymomonas mobilis strains Improve the impact of expression

[0168] To determine whether recombinant Zymomonas mobilis strains carrying an extrachromosomal copy of the RPI-A gene have altered ribulose levels and improved xylose fermentation, individual transformations containing pZB-GI-RPI were screened from MRM3G5-Spec250 plates strain. Transformed strains (RPI-1 and RPI-2) were transferred into MRM3G5-Spec250 liquid medium at an initial OD600 of 0.1 and incubated at 30°C. After 14-16 hours of incubation, the strains were transferred to MRM3X10 medium (1% yeast extract, 15mM KH 2 PO 4 , 4mM MgSO 4 and 100g / L xylose) and incubated for 24 hours. Testing the effect of increased RPI-A gene copy number on fermentation performance began after a second transfer of the culture to fresh MRM3X10 medium as a measure to prevent glucose carryover in the original culture. Transformed strains carry...

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Abstract

Xylose-utilizing Zymomonas strains studied were found to accumulate ribulose when grown in xylose-containing media. Engineering these strains to increase ribose-5-phosphate isomerase activity led to reduced ribulose accumulation, improved growth, improved xylose utilization, and increased ethanol production.

Description

[0001] Statement of Government Rights [0002] This application claims the benefit of US Provisional Application 61 / 359445, filed June 29, 2010. [0003] This invention was made with United States Government support through Contract DE-FC36-07GO17056 awarded by the Department of Energy. The United States Government has necessary rights in this invention. field of invention [0004] The invention relates to the fields of microbiology and genetic engineering. More specifically, increasing ribose-5-phosphate isomerase activity in the xylose-utilizing Zymomonas mobilis (Z. mobilis) improved xylose utilization by the microorganism. Background of the invention [0005] The production of ethanol by microorganisms offers an alternative energy source to fossil fuels and is therefore an important area of ​​current research. Ideally, microorganisms that produce ethanol as well as other useful products would be able to utilize xylose as a carbon source since xylose is the major pent...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N1/21C12P7/06C12N1/22
CPCC12N1/20C12N9/90Y02E50/17C12P7/065C12N1/22Y02E50/16C12N9/92C12P7/06C12P7/10Y02E50/10
Inventor P.G.凯米L.麦科尔L.陶J-F.汤布P.V.维塔宁
Owner EI DU PONT DE NEMOURS & CO
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