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Microorganisms Having Enhanced Resistance To Acetate And Related Compositions And Methods of Use

a technology of acetate and microorganisms, which is applied in the field of microorganisms and genetic modification thereof, can solve the problems of increasing the cost of ethanol, reducing the ethanol production rate and the total ethanol yield, and recalcitrance of biomass breakdown

Inactive Publication Date: 2010-11-18
UT BATTELLE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016]In a specific embodiment, the microbial strain is a Z. mobilis strain, wherein the 5′ upstream region of the nhaA gene is modified such that a nucleotide sequence of 1000 bp or less containing SEQ ID NO: 4 is deleted, while the nucleotide sequence of SEQ ID NO: 4 immediately 5′ to the nhaA coding sequence remains intact. Such Z. mobilis strain displays constitutive and elevated expression of nhaA, and enhanced resistance to sodium acetate.

Problems solved by technology

A core challenge in coverting cellulosic material to biofuels such as ethanol and butanol is the recalcitrance of biomass to breakdown.
In addition, the metabolic byproducts such as ethanol, lactate, and acetate also impact the fermentation by slowing and potentially stopping the fermentation prematurely.
The increased lag phase and slower growth increases the ethanol cost due to both ethanol production rate and total ethanol yield decreases (Takahashi et al.
To overcome the issue of inhibition caused by pretreatment processes, there are two approaches, one is to remove the inhibitor after pretreatment from the biomass physically or chemically, which requires extra equipment and time leading to increased costs.
One drawback to using wild-type Z. mobilis is its narrow substrate utilization range.
Acetate removal processes have been described but they are energy or chemical-intensive and their impact on processing costs have yet to be determined (McMillan 1994).

Method used

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  • Microorganisms Having Enhanced Resistance To Acetate And Related Compositions And Methods of Use
  • Microorganisms Having Enhanced Resistance To Acetate And Related Compositions And Methods of Use
  • Microorganisms Having Enhanced Resistance To Acetate And Related Compositions And Methods of Use

Examples

Experimental program
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example 1

[0064]This example describes the materials and methods used in the experiments described in the subsequent examples.

Strains and Culture Conditions

[0065]Bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains were cultured on Luria-Bertani (LB) broth or agar plates for cloning and strain maintenance. Z. mobilis ZM4 was obtained from the American Type Culture Collection (ATCC 31821). AcR is the Z. mobilis ZM4 acetate tolerant strain as described previously (Joachimstahl et al. 1998). ZM4 and AcR were cultured in RM medium at 30° C.

[0066]S. cerevisiae wild-type, deletion mutant and GST-fusion ORF over-expression strains were obtained through Open Biosystems (Huntsville, Ala.). S. cerevisiae strains were cultured in rich media (YPD media) and minimum complete medium (CM). CM media with 2% glucose was used for S. cerevisiae wild-type and S. cerevisiae deletion mutants. CM media with 2% glucose minus uracil was used for S. cerevisiae GST-over expressing st...

example 2

[0082]This example describes the results from the experiments conducted to determine the genetic basis for the acetate tolerance observed with the mutant Z. mobilis strain, AcR.

[0083]Using microarray comparative genome sequencing, next generation 454-pyroresequencing, and Sanger sequencing approaches, it was identified and confirmed that the genomic differences between the wild-type Z. mobilis strain, ZM4, and the acetate mutant AcR strain. The genetic changes in the mutant included a 1.5-kb deletion (FIG. 1) and single nucleotide polymorphisms (SNPs). Expression of the nhaA sodium proton anti-porter gene in AcR was found to be constitutive and significantly higher than in the wild-type strain under all the conditions tested (FIG. 2). Whole genome expression profiles were analyzed for mutant and wild-type exponential and stationary phase cells under sodium acetate and sodium chloride control conditions. A summary of these data is presented in FIG. 2 and Table 2.

TABLE 2Expression dat...

example 3

[0084]This example describes experiments conducted to test the hypothesis that the 1.5-kb deletion in the AcR genome resulted in increased nhaA expression that conferred increased acetate tolerance in the mutant.

[0085]A new Gateway® cloning compatible vector pBBR3-DEST42 was constructed. This vector contained the tetracycline resistance gene (FIG. 3) for candidate gene over-expression in ZM4 due to intrinsic, broad Z. mobilis antibiotic resistance. The anti-porter nhaA gene (FIG. 4) was cloned into the vector resulting in a nhaA over-expression vector p42-0119, which was then transformed into the wild-type ZM4 strain through conjugation to generate a strain over-expressing nhaA, which was named as “ZM4(p42-0119)”. In addition, a deletion mutant was constructed to mimic the 1.5-kb deletion region (FIG. 1) of the AcR acetate tolerant strain using the pJK100 system (Denef et al. 2006). Since the deletion covers most of the hypothetical protein ZMO0117 and the promoter region of ZMO0119...

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Abstract

The present invention provides isolated or genetically modified strains of microorganisms that display enhanced resistance to acetate as a result of increased expression of a sodium proton antiporter. The present invention also provides methods for producing such microbial strains, as well as related promoter sequences and expression vectors. Further, the present invention provides methods of producing alcohol from biomass materials by using microorganisms with enhanced resistance to acetate.

Description

CROSS REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit of priority from U.S. Provisional Application No. 61 / 173,649, filed on Apr. 29, 2009.[0002]This invention was made with government support under Contract Number DE-AC05-00OR22725 between the United States Department of Energy and UT-Battelle, LLC. The U.S. Government has certain rights in this invention.FIELD OF THE INVENTION[0003]This invention generally relates to the field of microorganism and genetic modification thereof. In particular, the invention relates to microorganisms that display enhanced resistance to acetate as a result of increased expression of an antiporter gene, and are therefore advantageous for use in fermentation of biomass materials to produce biofuels such as ethanol. Related compositions, including promoter sequences, expression vectors, genetically engineered microbial strains, as well as methods of making and using the strains, are also provided by the invention.BACKGROUND OF T...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P7/16C12N1/00C12N1/20C12N1/14C12N1/21C12N1/15C12N1/19C12N15/63C07H21/04C12P7/10C12P7/04
CPCC07K14/39C07K14/395C12P7/04C12P7/065Y02E50/10C12P7/16Y02E50/16Y02E50/17C12P7/02C12P7/10
Inventor BROWN, STEVEN D.YANG, SHIHUI
Owner UT BATTELLE LLC
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