Recombinant Saccharomyces Cerevisiae Construction through Rapid DNA Assembly and Evolutionary Engineering and High-Efficiency Lignocellulose Hydrolysates Fermentation

The bioethanol fermentation from lignocellulose hydrolysates requires efficient hexose and pentose co-utilizing and inhibitor-tolerant microbes. The objective of the present work is to develop a recombinant Saccharomyces cerevisiae strain which can make full use of mixed sugars in the presence of inhibitors in lignocellulose hydrolysates through metabolic engineering. In this study, a mixture of expression cassettes containing bacterial xylose and arabinose utilizing genes were assembled and integrated in the genome of a haploid S. cerevisiae strain Juk36a strain. The best functional strain 36aS1 was selected from consecutive adaptive evolution in synthetic medium containing of xylose and inhibitors. In the end, a high-efficiency hexose and pentose co-utilizing and inhibitor-tolerant strain 36aS1.10.4 was isolated from strain library of 36aS1.10 on lignocellulose hydrolysates plates. Strain 36aS1.10 exhibited a maximum specific growth rate of 0.176 1/h and 0.140 1/h in synthetic medium with 50 g/L D-xylose and 20 g/L L-arabinose as sole carbon source, respectively. Fermentation of industrial biomass hydrolysates obtained from two different companies using strain 36aS1.10.4 with a low yeast inoculum, maximum specific D-xylose consumption rates of 0.537 g/g DW/h and 0.541 g/g DW/h were obtained. The corresponding final ethanol titers were 54.11 g/L and 50.36 g/L with ethanol yields of 0.44 g/g and 0.41 g/g, respectively. After supplement of more sugars in one of the industrial hydrolysates, the final ethanol titer reached 78.37 g/L with about 94% total sugar conversion. The above results suggest that strain 36aS1.10.4 has great potential in industrial cellulosic ethanol production using lignocellulosic biomass sugars.