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Microorganisms Having Enhanced Tolerance To Inhibitors and Stress

Inactive Publication Date: 2010-12-09
UT BATTELLE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]It has been identified in accordance with the present invention that increased expression of a protein of the Sm-like superfamily in a microorganism confers enhanced tolerance to stress and inhibitors such as sodium acetate, ammonium acetate, potassium acetate, vanillin, furfural, hydroxymethylfurfural (HMF) and H2O2. In accordance with the present invention, microorganisms can be genetically modified to increase the expression of a protein of the Sm-like superfamily to achieve enhanced tolerance to stress and inhibitors. Such genetically modified microorganisms are particularly useful for production of biofuels based on fermentation of biomass materials.

Problems solved by technology

A core challenge in converting 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 Tolerance To Inhibitors and Stress
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  • Microorganisms Having Enhanced Tolerance To Inhibitors and Stress

Examples

Experimental program
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example 1

[0068]This example describes the materials and methods used in the experiments described in the subsequent examples.

Strains and Culture Conditions

[0069]Bacterial strains and plasmids used in this study are listed in Table 2. E. coli strains were cultured using Luria-Bertani (LB) broth or plates. E. coli WM3064 was supplemented with 100 μg / mL diaminopimelic acid (DAP). Z. mobilis ZM4 was obtained from the

[0070]American Type Culture Collection (ATCC31821) and the Z. mobilis acetate tolerant strain AcR has been described previously (Joachimsthal et al. 2000). ZM4 and AcR were cultured in RM medium at 30° C. 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 YPD media. 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 strai...

example 2

[0077]This example describes the results of the experiments showing that overexpression of the Zymomonas mobilis global regulator gene hfq confers enhanced tolerance to sodium acetate.

[0078]The Z. mobilis hfq gene (SEQ ID NO: 1) was cloned into the vector pBBR3-DEST42 (FIG. 2A) and the resulting plasmid construct p42-0347 was transformed into the wild-type strain ZM4 and acetate mutant AcR through conjugation. In addition, an insertional mutant of Z. mobilis strain AcR hfq gene (ZMO0347) was created using the pKNOCK system (Brown 2006; Alexeyev 1999) and complemented with plasmid p42-0347. The hfq gene and the primer positions used for mutant construction and hfq gene over-expression are shown in FIG. 2B. An insertional mutant of hfq (ZMO0347) was generated in the AcR background and designated as strain “AcRIM0347”. The hfq gene was over-expressed via plasmid p42-0347 in both wild-type ZM4 and the acetate mutant AcR backgrounds, their susceptibilities to sodium acetate and other str...

example 3

[0080]This example describes the experiments performed to compare the negative effects of pretreatment inhibitors on Z. mobilis growth, and to demonstrate that Hfq overexpression confers tolerance to pretreatment inhibitors.

[0081]Pretreatment Inhibitors had Negative Effects on Z. mobilis Growth

[0082]The growth of Z. mobilis strains was reduced in the presence of acetate, vanillin, furfural, or HMF with increased lag phases and / or slower growth rates and / or final bacterial cell densities depending on the respective condition and strain (Tables 3-4; FIGS. 4A-4E and 5A-5E). Among the different forms of acetate counter-ions tested, sodium acetate had the most significant inhibitory effect on wild-type Z. mobilis growth. This was followed by potassium acetate and ammonium acetate, and sodium chloride had the least negative influence on wild-type Z. mobilis growth (Table 3; FIGS. 4A-4E). Wild-type ZM4 growth was completely inhibited when RM medium was supplemented with 195 mM sodium aceta...

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Abstract

The present invention provides genetically modified strains of microorganisms that display enhanced tolerance to stress and / or inhibitors such as sodium acetate and vanillin. The enhanced tolerance can be achieved by increasing the expression of a protein of the Sm-like superfamily such as a bacterial Hfq protein and a fungal Sm or Lsm protein. Further, the present invention provides methods of producing alcohol from biomass materials by using the genetically modified microorganisms of the present invention.

Description

CROSS REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit of priority from U.S. Provisional Application No. 61 / 184,961, filed on Jun. 8, 2009, the content of which in its entirety is incorporated herein by reference.[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 tolerance to stress and inhibitors as a result of increased expression of a protein of the Sm-like superfamily such as bacterial Hfq and yeast Sm or Lsm proteins. Such microorganisms are advantageous for use in fermentation of biomass materials to produce biofuels such as ethanol.BACKGROUND OF THE INVENTION[0004]Biomass-based bioenergy...

Claims

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Application Information

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IPC IPC(8): C12P7/04C12N1/21C12N1/19
CPCC07K14/47C12N1/22Y02E50/17Y02E50/16C12P7/10Y02E50/10
Inventor BROWN, STEVEN D.YANG, SHIHUI
Owner UT BATTELLE LLC
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