Metabolic Engineering of Yeast for Enhanced Biofuel Production from Lignocellulosic Biomass

Abstract

Second-generation bioethanol depends on a fermenting organism that can do something Saccharomyces
cerevisiae does not do naturally: consume the pentose sugars, principally xylose, that make up a substantial
fraction of the sugar released from pretreated lignocellulosic biomass, while remaining tolerant of the inhibitory
byproducts that pretreatment inevitably generates. We engineered an industrial S. cerevisiae strain by integrating
a fungal xylose isomerase pathway together with overexpression of the non-oxidative pentose phosphate
pathway, followed by fifteen rounds of adaptive laboratory evolution under increasing xylose concentration and
sequential exposure to furfural and acetic acid, the two dominant inhibitors present in dilute-acid-pretreated wheat
straw hydrolysate. The evolved strain, designated Y-XI15, achieved a xylose consumption rate of 0.82 g/L/h in
defined medium, a nine-fold improvement over the unevolved parental construct, and fermented undetoxified
wheat straw hydrolysate containing 4.1 g/L furfural and 6.8 g/L acetic acid to 87% of theoretical ethanol yield
within 48 hours, compared with 34% for the parental strain under identical conditions. Whole-genome
resequencing of Y-XI15 identified a recurring set of mutations across independently evolved lineages, including a
gain-of-function allele in the transcription factor HAA1 associated with acetic acid tolerance and a loss-of-function
mutation in ISU1 correlated with improved xylose flux, both of which were individually reintroduced into the
unevolved background and shown to recapitulate a substantial fraction of the evolved phenotype. Fed-batch
fermentation at 2-litre scale using undetoxified hydrolysate produced 68 g/L ethanol at a volumetric productivity of
1.4 g/L/h, approaching the productivity benchmarks reported for optimised first-generation glucose fermentation.
These results indicate that combined rational pathway engineering and targeted adaptive evolution can close
much of the performance gap between engineered pentose-fermenting yeast and the glucose fermentation it was
originally selected for over millennia of domestication.