Systems biology and pathway engineering enable Saccharomyces cerevisiae to utilize C-5 and C-6 sugars simultaneously for cellulosic ethanol production

摘要

Saccharomyces cerevisiae is a traditional industrial workhorse for ethanol production. However, conventional ethanologenic yeast is superior in fermentation of hexose sugars (C-6) such as glucose but unable to utilize pentose sugars (C-5) such as xylose richly embedded in lignocellulosic biomass. In order to efficiently utilizing biomass sugars for lower cost cellulosic ethanol production, a significant effort has been taken worldwide for decades to improve xylose utilization capability of S. cerevisiae by genetic engineering. Yet challenges remain strong since the efficiency of the improved C-5 utilization is low and insufficient for low-cost cellulosic ethanol production. Scientists at ARS using tolerant yeast strain NRRL Y-50049 as a mother host created six (6) new genotypes of S. cerevisiae NRRL Y-50049-YXI-XUT4, -XUT5, -XUT6, -XUT7, -RGT2, and -SUT4 applying systems biology and pathway engineering approaches. Instead of a commonly used traditional fungal xylose utilization pathway, we introduced a bacterial xylose isomerase pathway into the yeast. We first synthesized a novel sequence of xylose isomerase (YXI ; GenBank Accession No. JF261697) containing optimized transcription codons for our yeast expression, and then integrated it into a specific chromosomal locus of the yeast to obtain a high level of constitutive expression, resulting in a daughter host strain NRRL Y-50049-YXI. We cloned and characterized six xylose transporter genes from Scheffersomyces stipitis, a natural xylose utilization yeast, to aid xylose transport and uptake. These heterologous xylose transporter genes were genetically engineered into the daughter host resulting in a set of new genotypes of S. cerevisiae. These newly developed industrial yeast strains are able to grow on xylose as sole carbon source and produce ethanol. When mixed sugars of glucose and xylose were added in the medium, all these new strains displayed a simulteneous utilization of C-5 and C-6 sugars and significantly improved xylose uptake and utilization for ethanol conversion. Among which, genotypes S. cerevisiae NRRL Y-50049-YXI-XUT7, -RGT2, and -SUT4 demonstrated superior fermentation capability in utilizing both sugars. In contrast with poor results observed from lab model strains, our research established the first example of using industrial yeast as a host for the next-generation biocatalyst development for advanced biofuels production. All these US patented strains are available for interested parties in collaborative efforts.