Unraveling the genetic basis of fast l‐arabinose consumption on top of recombinant xylose‐fermenting Saccharomyces cerevisiae

Abstract

One major challenge in the bioconversion of lignocelluloses into ethanol is to develop Saccharomyces cerevisiae strains that can utilize all available sugars in biomass hydrolysates, especially the d-xylose and l-arabinose that cannot be fermented by the S. cerevisiae strain naturally. Here, we integrated an l-arabinose utilization cassette (AUC) into the genome of an efficient d-xylose fermenting industrial diploid S. cerevisiae strain CIBTS0735 to make strain CIBTS1972. After evolving on arabinose, CIBTS1974 with excellent fermentation capacity was obtained. A comparison between genome sequences of strains CIBTS1974 and CIBTS1972 revealed that the copy number of the AUC had increased from 1 to 12. We then constructed the AUC null-mutant CIBTS1975 and gradually rescued the l-arabinose utilization defect by integrating AUC iteratively. On the other hand, the parental strain CIBTS0735 was able to acquire the same performance as CIBTS1974 by the direct introduction of 12 copies of the AUC; the performance was further improved by adding two more copies. Besides, we found that not the two transporters present in the AUC were both needed during l-arabinose utilization, GAL2 was necessary and STP2 was not essential. We have described for the first time that a high copy number of AUC is sufficient for the strain to metabolize l-arabinose efficiently independent of evolution.

High Gravity Fermentation of Sugarcane Bagasse Hydrolysate by Saccharomyces pastorianus to Produce Economically Distillable Ethanol Concentrations: Necessity of Medium Components Examined

“…Further, several recombinant yeast studies that have reported using high density (20 OD) inoculum [25,26] did not include in the ethanol yields or productivities the material and time required to generate the inoculum. In many other ethanol-from-lignocellulosic studies, it is common to use high inoculum, supplementing the medium with yeast extract and peptone [29,32,51,52], corn steep liquor [35], or other nutrients [54]. It is not always clear if the inocula or these added carbon/nutrient sources are included in the total carbon balances when yields are reported.…”

“…First, the yeast biomass production was accounted for in the conversion of glucose to ethanol. Conversely, most reported ethanol fermentations use yeast inoculums on the order of 10 to 50 g dcw/L (or 1.75 to 3.5 × 10 7 cell/mL) [18,30,32,[34][35][36]58,59]. In these high inoculum cases, it is not apparent that the yield from sugar to ethanol accounts for the sugar used to generate the biomass, which for the 10 g/L case would be at least 20 g/L of glucose.…”

“…Complementary approaches have coupled cellobiose utilization with xylose utilization in recombinant S. cerevisiae with improved xylose to ethanol conversion rates [30]. Recombinant approaches have not yet yielded a successful approach to xylose utilization that overall surpasses the ethanol productivities of native species on glucose; however, work is getting closer [8,14,[31][32][33][34][35][36].…”

Heterologous secretory expression of β-glucosidase from Thermoascus aurantiacus in industrial Saccharomyces cerevisiae strains

“…Conversion of L-arabinose into D-xylulose-5-phosphate is based on heterologous expression of bacterial genes encoding L-arabinose isomerase (AraA), L-ribulokinase (AraB), and L-ribulose-5-phosphate-4epimerase (AraD). Further improvements on alcoholic fermentation of these most abundant pentoses have been obtained through directed-evolution strategies aimed to accumulate spontaneous beneficial mutation [5][6][7]. A redox engineering study has revealed that deletion of S. cerevisiae genes encoding glycerol-3-phosphate dehydrogenase and expression of an acetylating acetaldehyde dehydrogenase from Escherichia coli (A-ALD) allow researchers to achieve conversion of inhibitory acetic acid to ethanol and to eliminate glycerol formation in anaerobic cultures of yeast [3].…”

Minimize the Xylitol Production in Saccharomyces cerevisiae by Balancing the Xylose Redox Metabolic Pathway

“…Although S. cerevisiae cannot utilize xylose naturally (Ma and Liu, 2010;Wang et al, 2019), it can use xylulose, an isomer of xylose, to produce ethanol, making it attractive to introduce xylose metabolism pathways derived from other microorganisms to S. cerevisiae (Zhang et al, 2012).…”