In order to achieve rapid xylose utilization in the presence of acetate, improved yeast strains were engineered for higher bioethanol production. First, a six-gene cluster, including XYL1/XYL2/XKS1/TAL1/PYK1/MGT05196, was generated by using an in-depth two-stage (glucose and xylose) transcription reprogramming strategy in an evolutionary adapted strain of CE7, resulting in two improved engineered strains WXY46 and WXY53. Through a combined screening of xylose and glucose stage-specific promoters between tricarboxylic acid (TCA)/HSP and constitutive types, respectively, WXY46 with the constitutive promoters showed a much higher ethanol yield than that of WXY53 with the TCA/HSP promoters. Second, an optimized strain WXY74 was obtained by using more copies of a six-gene cluster, which resulted in a higher ethanol yield of 0.500 g/g total sugars with acetate conditions. At last, simultaneous saccharification and co-fermentation were performed by using the evolved WXY74 strain, which produced 58.4 g/L of ethanol from wheat straw waste and outperformed previous haploid XR-XDH strains.

Despite a growing preference for second-generation (2G) ethanol in industries, its application is severely restricted owing to a major obstacle of developing a suitable yeast strain for fermentation using feedstock biomasses. In this study, a yeast strain, Saccharomyces cerevisiae A31Z, for 2G bioethanol production was developed from an industrial strain, Angel, using metabolic engineering by the incorporation of gene clusters involved in the xylose metabolism combined with adaptive evolution for evolving its anti-inhibitory properties. This strain outcompeted its ancestors in xylose utilization and subsequent ethanol production and manifested higher tolerance against common inhibitors from lignocellulosic hydrolysates, and also it lowered the production of glycerol by-product. Furthermore, A31Z outperformed in ethanol production using industrial hydrolysate from dried distillers grains with solubles and whole corn. Overall, this study provided a promising path for improving 2G bioethanol production in industries using S. cerevisiae.

Background: Saccharomyces cerevisiae has been widely used in the fermentation of plant-derived sugars to produce ethanol, called first-generation (1G) bioethanol, but made an impact on global food markets. Significant efforts have been therefore to employ non-food lignocellulosic feedstocks for bioethanol production, known as second-generation (2G) bioethanol. However, S. cerevisiae cannot naturally utilize xylose, a major component in lignocellulosic hydrolysates, and it has low tolerance to common carboxylic acid inhibitors present in lignocellulosic hydrolyzates. Metabolic engineering and evolutionary engineering have shown great power in strain improvement, which were also adopted here to solve these limiting factors in developing 2G bioethanol.Results: An efficient expression of a six-gene cluster, including XYL1/XYL2/XKS1/TAL1/PYK1/MGT05196, was achieved in the evolved S. cerevisiae diploid strain A21Z, showing the ability to use mixed glucose and xylose. The engineered strain A21Z expressing the six-gene cluster displayed a high xylose consumption after 96 h, reaching 90.7% of the theoretical yield in ethanol production. To investigate its industrial characteristics, A31Z was obtained by direct evolution of A21Z under the treatment of industrial hydrolysate from wheat straw. Under different fermentation conditions with 1G and 2G feedstock candidates, A31Z showed a markedly improved xylose fermentation performance. A31Z could produce more ethanol and less glycerol compared to the control Angel from corn starch during 120 h, with a final ethanol production at 122.32 g/L. The ability to produce higher ethanol production was also found under the fermentation using carbon source from hydrolysis of Dried Distillers Grains with Solubles (DDGS) or whole corn.Conclusions: Here, we report an effective strategy to improve xylose fermentation with an evolutionary engineering in the industrial S. cerevisiae diploid strain A31Z. This study demonstrated that a constructed A31Z has the higher xylose consumption and efficient ethanol production in mixed glucose and xylose with acetate. A31Z also gave a good ethanol production in 1G and 2G industrial feedstocks, indicating its significant contribution in the transition stage from the 1st generation to the 2nd generation bioethanol.