As a microbial host for cellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway. However, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (pho13) has been reported to be a crucial genetic perturbation for improving xylose fermentation. A confirmed mechanism of the pho13-positive effect on xylose fermentation is that the deletion of PHO13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the present study, we reported that a pho13-positive effect was not observed from a couple of engineered strains, among the many others we have examined. To extend our knowledge of pho13-mediated metabolic regulation, we performed genome sequencing of pho13-negative strains. We identified a loss-of-function mutation in GCR2 responsible for the pho13-negative phenotype. Gcr2 is a transcriptional activator of the lower glycolytic pathway. Thus, the deletion of GCR2 (gcr2) led to deactivation of lower glycolysis as confirmed by RNA-seq. Also, gcr2 resulted in the up-regulation of PPP genes, which explains the improved xylose fermentation of gcr2 mutants. As pho13 and gcr2 cause similar transcriptional changes with PPP genes, there was no synergistic effect between pho13 and gcr2 for improving xylose fermentation. The present study identified GCR2 as a new knockout target to improve xylose fermentation and cellulosic biofuel production.
β-carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce β-carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a β-carotene biosynthetic pathway containing crtYB, crtI and crtE from Xanthophyllomyces dendrorhous were introduced into a xylose-fermenting S. cerevisiae. The resulting strain produced β-carotene from xylose at a titer three-fold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate (MVA) pathway and β-carotene production in yeast when glucose is used, did not further improve the production of β-carotene from xylose. Through fermentation profiling, metabolites analysis and transcriptional studies, we found the advantages of using xylose as a carbon source instead of glucose for β-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl-CoA pool, and up-regulated expression levels of rate-limiting genes in the β-carotene producing pathway, including ACS1 and HMG1. As a result, 772.81 mg/L of β-carotene was obtained in a fed-batch bioreactor culture with xylose feeding. Considering the inevitable production of xylose at large scales when cellulosic biomass-based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.
Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns on limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources towards engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Although many challenges remain, these research investments have facilitated efficient and rapid fermentation of xylose, simultaneous co-consumption of xylose with carbon sources in lignocellulosic hydrolysates, and enhanced production of a wide range of valuable chemicals from xylose. In particular, understanding of xylose-induced metabolic rewiring in engineered yeast has stimulated the use of xylose as a preferred carbon source for the production of various non-ethanol bioproducts. Here, we summarize recent advances in metabolic engineering in yeast to address bottlenecks of xylose assimilation, and to enable simultaneous co-utilization of xylose and other cellulosic carbon sources. We also highlight distinct characteristics of xylose metabolism which can be harnessed for the production of advanced biofuels and chemicals.