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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING BIOCHEMICAL PROCESSES FOR THE PRODUCTION OF SUSTAINABLE FUELS AND CHEMICALS

Location: Renewable Product Technology Research Unit

Title: Production of a yeast artificial chromosome for stable expression of a synthetic xylose isomerase-xylulokinase polyprotein in a fuel ethanol yeast strain

Authors
item Hughes, Stephen
item Cox, Elby
item Bang, Sookie -
item Pinkelman, Rebecca -
item Qureshi, Nasib
item Johnson, Tylor -
item Bischoff, Kenneth
item Liu, Siqing
item Rich, Joseph
item Cotta, Michael

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 18, 2013
Publication Date: N/A

Technical Abstract: Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. A yeast artificial chromosome (YAC) was engineered to contain a polyprotein gene construct expressing xylose isomerase (XI) and xylulokinase (XKS) simultaneously for production of a stable ethanologenic yeast strain with enhanced xylose-utilizing capability. The Pml1 to Xba1 fragment of the TRP1 gene of pYAC4 was removed and replaced with the gene construct expressing XI –XKS polyprotein. A SUMO tag preceding the gene constructs ensures rapid high-level expression of properly folded XI and XKS proteins. Protein expression utilizes the TRP1 promoter and the trp1 terminator of pYAC4. When the INVSc1 yeast strain was transformed with this artificial chromosome, it grew on all cellulosic sugars including glucose, galactose, xylose, arabinose, and mannose. Western blot analysis demonstrated expression of the polyprotein. The full processing of the polyprotein into XI and XKS was also documented. In glucose medium, the doubling time of the recombinant yeast strain (2.8 hours) was comparable to that of wild-type S. cerevisiae (4.12 hours). In xylose medium, the wild-type strain did not grow; the doubling time of the recombinant strain was 22.6 hours. In whole corn acid hydrolysate, the doubling time of the recombinant yeast strain was 5.05 hours; that of the wild-type strain was 7.07 hours. On equivalent amounts of whole corn hydrolysate, ethanol production after 96 hours for the recombinant strain was 6.2 g/L compared to 4.1 g/L for the wild-type strain. The recombinant strain metabolizes but does not ferment xylose allowing the remaining sugars to be used for fermentation rather than cell growth. Metabolism of xylose by the recombinant strain causes a significant and unexpected increase in the pH of the medium from 4.18 to 6.65, while that of the wild-type strain remains essentially unchanged at 4.28.

Last Modified: 7/28/2014
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