Document Detail


Single amino acid substitutions in HXT2.4 from Scheffersomyces stipitis lead to improved cellobiose fermentation by engineered Saccharomyces cerevisiae.
MedLine Citation:
PMID:  23263959     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes from Neurospora crassa. Here, we report that an engineered S. cerevisiae strain expressing the putative hexose transporter gene HXT2.4 from Scheffersomyces stipitis and gh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when HXT2.4 is overexpressed in S. cerevisiae. However, cellobiose fermentation by the engineered strain expressing HXT2.4 and gh1-1 was much slower and less efficient than that by an engineered strain that initially expressed cdt-1 and gh1-1. The rate of cellobiose fermentation by the HXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved S. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher K(m) and 4-fold higher V(max) values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in S. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered S. cerevisiae strains.
Authors:
Suk-Jin Ha; Heejin Kim; Yuping Lin; Myoung-Uoon Jang; Jonathan M Galazka; Tae-Jip Kim; Jamie H D Cate; Yong-Su Jin
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-12-21
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  79     ISSN:  1098-5336     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-02-14     Completed Date:  2013-07-26     Revised Date:  2013-09-03    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1500-7     Citation Subset:  IM    
Affiliation:
Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Substitution*
Cellobiose / metabolism*
DNA Mutational Analysis
Fermentation
Metabolic Engineering*
Monosaccharide Transport Proteins / genetics*,  metabolism*
Mutant Proteins / genetics,  metabolism
Recombinant Proteins / genetics,  metabolism
Saccharomycetales / genetics*,  metabolism*
Sequence Analysis, DNA
Serial Passage
Chemical
Reg. No./Substance:
0/Monosaccharide Transport Proteins; 0/Mutant Proteins; 0/Recombinant Proteins; 16462-44-5/Cellobiose
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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