Affiliation:
1. Department of Biology, Middle Tennessee State University , Murfreesboro, Tennessee, USA
Abstract
ABSTRACT
Scheffersomyces stipitis
is a yeast that robustly ferments the 5-carbon sugar xylose, making the yeast a valuable candidate for lignocellulosic ethanol fermentation. However, the non-canonical codon usage of
S. stipitis
is an obstacle for implementing molecular tools that were developed for other yeast species, thereby limiting the molecular toolset available for
S. stipitis
. Here, we developed a series of molecular tools for
S. stipitis
including BLINCAR, a Bio-Luminescent Indicator that is Nullified by Cas9-Actuated Recombination, which can be used repeatedly to add different exogenous DNA payloads to the wild-type
S. stipitis
genome or used repeatedly to remove multiple native
S. stipitis
genes from the wild-type genome. Through the use of BLINCAR tools, one first produces antibiotic-resistant, bioluminescent colonies of
S. stipitis
whose bioluminescence highlights those clones that have been genetically modified; then second, once candidate clones have been confirmed, one uses a transient Cas9-producing plasmid to nullify the antibiotic resistance and bioluminescent markers from the prior introduction, thereby producing non-bioluminescent colonies that highlight those clones which have been re-sensitized to the antibiotic and are therefore susceptible to another round of BLINCAR implementation.
IMPORTANCE
Cellulose and hemicellulose that comprise a large portion of sawdust, leaves, and grass can be valuable sources of fermentable sugars for ethanol production. However, some of the sugars liberated from hemicellulose (like xylose) are not easily fermented using conventional glucose-fermenting yeast like
Saccharomyces cerevisiae
, so engineering robust xylose-fermenting yeast that is not inhibited by other components liberated from cellulose/hemicellulose will be important for maximizing yield and making lignocellulosic ethanol fermentation cost efficient. The yeast
Scheffersomyces stipitis
is one such yeast that can ferment xylose; however, it possesses several barriers to genetic manipulation. It is difficult to transform, has only a few antibiotic resistance markers, and uses an alternative genetic code from most other organisms. We developed a genetic toolset for
S. stipitis
that lowers these barriers and allows a user to deliver and/or delete multiple genetic elements to/from the wild-type genome, thereby expanding
S. stipitis’s
potential.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology