Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production

Scientific Achievement

The following two novel heterologous genes were integrated into the genome of the chassis cell BSIF: the mutant MGT05196^N360F, which encodes a xylose-specific, glucose-insensitive transporter and is derived from the Meyerozyma guilliermondii transporter gene MGT05196, and Ru-xylA (where Ru represents the rumen), which encodes a xylose isomerase (XI) with higher activity in S. cerevisiae. Additionally, endogenous modifications were also performed, including the overproduction of the xylulokinase Xks1p and the non-oxidative PPP (pentose phosphate pathway), and the inactivation of the aldose reductase Gre3p and the alkaline phosphatase Pho13p. These rationally designed genetic modifications, combined with alternating adaptive evolutions in xylose and SECS liquor (the leach liquor of steam-exploding corn stover), resulted in a final strain, LF1, with excellent xylose fermentation and enhanced inhibitor resistance. The specific xylose consumption rate of LF1 reached as high as 1.089 g g-1 h-1 with xylose as the sole carbon source. Moreover, its highly synchronized utilization of xylose and glucose was particularly significant; 77.6% of xylose was consumed along with glucose within 12 h, and the ethanol yield was 0.475 g g−1, which is more than 92% of the theoretical yield. Additionally, LF1 performed well in fermentations with two different lignocellulosic hydrolysates.

Significant and Impact

An efficient glucose-xylose co-fermentation diploid S. cerevisiae strain was constructed through integration of two novel proprietary heterologous genes (MGT05196^N360F and Ru-xylA) combined with other multiple genetic modifications and three rounds of adaptive evolution. LF1 exhibited highly synchronous utilization of glucose and xylose, also performed well in lignocellulosic hydrolysates, with ethanol yields of over 80.0% of the theoretical yields. These results highlight the great potential for the practical use of LF1 in the production of second-generation bioethanol.

Research Details

•A XR–XDH expressing S. cerevisiae was modified by a combination of diverse strategies to enhance xylose fermentation，and the feasibility of the strategies above was verified.

•A new xylose isomerase was screened from the contents of the bovine rumen metagenomics library and was functionally expressed in S. cerevisiae.

•A new transporter gene was screened from Meyerozyma guilliermondii, and its mutant MGT05196^N360F, which could specifically transported D-xylose without any glucose-inhibition, was obtained.

•A suitable strain BSIF, isolated from tropical fruit in Thailand, was selected, and was verified to be an excellent chassic strain for lignocellulosic ethanol production. Based on BSIF, an excellent glcose-xylose co-fermentation strain was constructed through multiple genetic modifications and evolution engineering.