Abstract
As the importance of reducing carbon emissions as a means to limit the serious effects of global climate change becomes apparent, synthetic biologists and metabolic engineers are looking to develop renewable sources for transportation fuels and petroleum-derived chemicals. In recent years, microbial production of high-energy fuels has emerged as an attractive alternative to the traditional production of transportation fuels. In particular, the Baker’s yeast Saccharomyces cerevisiae, a highly versatile microbial chassis, has been engineered to produce a wide array of biofuels. Nevertheless, a key limitation of S. cerevisiae is its inability to utilize xylose, the second most abundant sugar in lignocellulosic biomass, for both growth and chemical production. Therefore, the development of a robust S. cerevisiae strain that is able to use xylose is of great importance. Here, we engineered S. cerevisiae to efficiently utilize xylose as a carbon source and produce the advanced biofuel isobutanol. Specifically, we screened xylose reductase (XR) and xylose dehydrogenase (XDH) variants from different xylose-metabolizing yeast strains to identify the XR–XDH combination with the highest activity. Overexpression of the selected XR–XDH variants, a xylose-specific sugar transporter, xylulokinase, and isobutanol pathway enzymes in conjunction with the deletions of PHO13 and GRE3 resulted in an engineered strain that is capable of producing isobutanol at a titer of 48.4 ± 2.0 mg/L (yield of 7.0 mg/g d-xylose). This is a 36-fold increase from the previous report by Brat and Boles and, to our knowledge, is the highest isobutanol yield from d-xylose in a microbial system. We hope that our work will set the stage for an economic route for the production of advanced biofuel isobutanol and enable efficient utilization of lignocellulosic biomass.
Highlights
Renewable fuels have received significant interest in recent years due to environmental concerns and unsustainable energy demands (Peralta-Yahya et al 2012; Liao et al 2016)
Screening of optimal xylose reductase–xylose dehydrogenase pathway genes To engineer S. cerevisiae with the ability to grow on xylose, we first integrated the xylose-specific variant of the sugar transporter HXT7 (HXT7F79S) and a copy of the S. stipitis xylulokinase gene (XYL3) into BY4742, a laboratory strain of S. cerevisiae
While a gene encoding xylulokinase is present in S. cerevisiae (i.e. XKS1), its expression level is insufficient for efficient conversion of xylulose to xylulose-5-phosphate (Johansson et al 2001)
Summary
Renewable fuels have received significant interest in recent years due to environmental concerns and unsustainable energy demands (Peralta-Yahya et al 2012; Liao et al 2016). The use of starch or the sucrose fractions of agricultural crops as the carbon substrates for biomass conversion has become less attractive due to environmental, economical and ethical considerations (Mosier et al 2005). Instead, lignocellulosic biomass, such as agricultural residues and energy crops, offers a cheap and renewable alternative carbon source for the microbial production of biofuels. Since xylose is the second most abundant component of lignocellulosic biomass, enabling the yeast S. cerevisiae to efficiently utilize xylose and convert it into biofuels is of great importance and has been the focus of intense and highly competitive research (Li et al 2019)
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