ABSTRACT
BACKGROUND:
Sugarcane hemicellulosic material is a compelling source of usually neglected
xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as
xylitol. In this context,
Saccharomyces cerevisiae strains typically used in the Brazilian
bioethanol industry are a robust chassis for
genetic engineering, given their robustness towards harsh operational conditions and outstanding
fermentation performance. Nevertheless, there are no
reports on the use of these
strains for
xylitol production using
sugarcane hydrolysate.
RESULTS:
Potential single-guided
RNA off-targets were analyzed in two preeminent industrial
strains (PE-2 and SA-1), providing a database of 5'-NGG 20
nucleotide sequences and guidelines for the fast and
cost-effective
CRISPR editing of such
strains. After genomic integration of a
NADPH-preferring
xylose reductase (XR), FMYX (SA-1 hoΔxyl1) and CENPKX (CEN.PK-122 hoΔxyl1) were tested in varying cultivation conditions for
xylitol productivity to infer influence of the
genetic background. Near-theoretical yields were achieved for all
strains; however, the industrial consistently outperformed the
laboratory strain. Batch
fermentation of raw
sugarcane straw hydrolysate with remaining solid particles represented a challenge for
xylose metabolization, and 3.65 ± 0.16 g/L
xylitol titer was achieved by FMYX. Finally, quantification of
NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX.
CONCLUSIONS:
Although widely used in several
S. cerevisiae strains, this is the first
report of
CRISPR-Cas9 editing major
yeast of the Brazilian
bioethanol industry. Fermentative assays of
xylose consumption revealed that
NADPH availability is closely related to mutant
strains' performance. We also pioneer the use of
sugarcane straw as a substrate for
xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation
industry, given its inhibitor tolerance and better
redox environment that may favor
production of reduced
sugars.