Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae

Authors: QUANZHOU, FENG - Tsinghua University; Liu, Zonglin; WEBER, SCOTT - Former ARS Employee; LI, SHIZHONG - Tsinghua University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2018
Publication Date: 4/5/2018
Citation: Quanzhou, F., Liu, Z.L., Weber, S.A., Li, S. 2018. Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae. PLoS One. 13(4):e0195633. doi: 10.1371/journal.pone.0195633.
DOI: https://doi.org/10.1371/journal.pone.0195633

Interpretive Summary: The native Saccharomyces cerevisiae, also known as Brewer’s yeast, is superb in consumption of glucose but limited in uptake and utilization of five carbon sugars such as xylose. This drawback has been a major obstacle for efficient advanced biofuels production from lignocellulosic biomass. Laboratory model strains of S. cerevisiae are commonly used for genetic engineering to enable yeast utilizing xylose but little is known about the industrial yeast. ARS scientists enriched the genetic background of the industrial yeast with a yeast xylose isomerase gene integrated into its genome and expressed 4 other xylose-utilization facilitating genes. Using comparative gene expression analysis assays, this research found that the constitutive and unique heterologous gene expression initiated xylose reduction in the genetically engineered industrial yeast. Four sugar transforming genes were highly activated in the non-oxidative pentose phosphate pathway to enhance a serial of interactions for sugar transformation, driving the xylose metabolism into the glycolysis for ethanol conversion. An important biosynthesis gene family maintained a healthy life cycle and biosynthesis pathways for the yeast. Findings of this research provide the genetic basis underlying mechanisms of the xylose utilization by the genetically engineered industrial yeast. New knowledge obtained by this research aids future R&D efforts for the next-generation biocatalyst development for production of advanced biofuels from lignocellulosic materials.

Technical Abstract: Background: The limited xylose utilizing ability of native Saccharomyces cerevisiae has been a major obstacle for efficient cellulosic ethanol production from lignocellulosic materials. Haploid laboratory strains of S. cerevisiae are commonly used for genetic engineering to enable its xylose utilization but little is known about the industrial yeast. Genetically engineered strain NRRL Y-50463 with an industrial yeast background is able to grow on xylose and ferment ethanol on mixed sugars of glucose-xylose in the presence of fermentation inhibitors furfural and HMF. However, mechanisms of the improved xylose utilization for the industrial yeast are not fully understood. Results: Using pathway-based qRT-PCR array assay analysis, we identified three distinct signature expression patterns that enabled Y-50463 for the improved xylose utilization under aerobic and anaerobic conditions. A set of five heterologous genes, including a codon optimized YXI genetically integrated into chromosome XV and a plasmid-carried XUT4, XUT6, XYL2 and XKS1, enriched genetic background of Y-50463. The high level of constitutive expression of YXI alone with the four genes served a driving force for xylose reduction in Y-50463. Enhanced expression of 10 genes in the non-oxidative pentose phosphate pathway, especially for two transketolase genes TKL1 and TKL2 and two transaldolase genes TAL1 and NQM1, played critical roles for the accelerated xylose metabolism. These genes were actively involved in a serial of sugar transformation reactions through complex interactions to facilitate the efficient metabolism of xylose. It pushed the metabolic flow through the glycolysis for enhanced ethanol conversion. The entire PRS gene family consisting of PRS1, PRS2, PRS3, PRS4 and PRS5 showed unique elevated levels of expression in the presence of xylose under both aerobic and anaerobic conditions. Highly activated expression of the PRS family contributed to the sound life cycle and superpathway of biosynthesis of nucleotide and amino acids for Y-50463. Conclusions: The signature expression revealed by this investigation provides the genetic basis underlying mechanisms of the enabled xylose-utilization capability for the industrial yeast S. cerevisiae. New knowledge and the pathway insight obtained from this study using Y-50463 as an example aids future efforts of the next-generation biocatalyst development for efficient lignocellulose-to-advanced biofuels production.