Improvement in d-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering

Abstract

As the effects of climate change become apparent, metabolic engineers and synthetic biologists are exploring sustainable sources for transportation fuels. The design and engineering of microorganisms to produce gasoline, diesel, and jet fuel compounds from renewable feedstocks can significantly reduce our dependence on fossil fuels as well as lower the emissions of greenhouse gases. Over the past 2 decades, a considerable amount of work has led to the development of microbial strains for the production of advanced fuel compounds from both C5 and C6 sugars. In this work, we combined two strategies—adaptive laboratory evolution and rational metabolic engineering—to improve the yeast Saccharomyces cerevisiae’s ability to utilize d-xylose, a major C5 sugar in biomass, and produce the advanced biofuel isobutanol. Whole genome resequencing of several evolved strains followed by reverse engineering identified two single nucleotide mutations, one in CCR4 and another in TIF1, that improved the yeast’s specific growth rate by 23% and 14%, respectively. Neither one of these genes has previously been implicated to play a role in utilization of d-xylose. Fine-tuning the expression levels of the bottleneck enzymes in the isobutanol pathway further improved the evolved strain’s isobutanol titer to 92.9 ± 4.4 mg/L (specific isobutanol production of 50.2 ± 2.6 mg/g DCW), a 90% improvement in titer and a 110% improvement in specific production over the non-evolved strain. We hope that our work will set the stage for an economic route to the advanced biofuel isobutanol and enable efficient utilization of xylose-containing biomass.

Co-Production of Isobutanol and Ethanol from Prairie Grain Starch Using Engineered Saccharomyces cerevisiae

“…Greater yields may also be possible when the yeast is engineered to effectively assimilate pentose sugars, which make up 1-2% of the sugars in the present in the mash. Some progress has been made on improving the conversion of xylose to isobutanol in S. cerevisiae but to date yields remain low and there has been no effective demonstration of the conversion of pentose sugars from a mixed carbon feedstock [9,42,43]. This may also open the possibility of using cellulosic and hemicellulosic materials as feedstock.…”

Recent advances in the microbial production of C4 alcohols by metabolically engineered microorganisms

“…[111] Comprehensive research on isobutanol production using S. cerevisiae as a host strain has also been reported, but isobutanol production remains low (see Generoso et al, 2015 for a detailed review). [112][113][114][115][116][117] A relatively high titer of isobutanol (1.62 g L -1 ) was achieved by introducing the aforementioned mutated NADH-dependent pathway to S.…”

Biorefinery: The Production of Isobutanol from Biomass Feedstocks

“…E2 in S. cerevisiae. Overexpressing RKI1, RPE1, TKL1, TAL1 and XYL3 in this background increased xylose assimilation and facilitated adaptive laboratory evolution [52]. Boles' group [53] further optimized the use of xylose in this strain by knocking out gene PHO13.…”