Engineering of a second-generation Saccharomyces cerevisiae strain for the production of 2,3-butanediol from biomass

2,3-butanediol(2,3-BDO) is an important platform chemical with multiple applications in the production of synthetic rubber, plastics, flavor enhancers and fuel additive. Nearly all current 2,3-BDO production is from petroleum, which is the result of an energy intensive cracking process. The high cost of bio-based 2,3-BDO caused by expensive feedstocks and media, high fermentation productivity only possible with pathogenic microorganisms and low recovery upon purification impose restrictions on its application. In this project, low cost production of 2,3-BDO from inexpensive lignocellulosic biomass will be explored with yeast as host organism, which is generally regarded as a safe organism(GRAS). First, we will develop a 2,3-BDO producing S. cerevisiae strain from highly robust and stress-tolerant cellulosic industrial yeast strain MDS130 by overexpressing Bacillus subtilis α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD), as well as endogenous S. cerevisiae 2,3-BDO dehydrogenase (Bdh1). Ethanol production will be eliminated by deleting the PDC genes, which will establish growth rate as a selection criterion for high 2,3-BDO production. Second, the engineered strain will be further improved by applying two non-targeted strategies. Mutagen treatment with EMS will be used to introduce random mutations and superior mutants will be selected and recombined through recursive genome shuffling to further increase performance. Third, at least two causative mutant genetic elements supporting high 2,3-BDO production will be identified by applying the powerful polygenic analysis technology of the host laboratory. A haploid strain showing the highest 2,3-BDO production will be selected and the underlying quantitative trait loci (QTLs) will be mapped using pooled-segregant whole-genome sequence analysis after crossing with a reference strain. The causative genetic elements will be identified by allele exchange between the parent strains and used to further improve the performance of the best strain. Finally, the fermentation process will be optimized changing parameters, like temperature, pH, feeding strategy and fermentation mode. A good balance of oxygen supply will be established to promote cell growth as well as 2,3-BDO production. Medium composition will be optimized to achieve prolonged and stable 2,3-BDO production. As the parent strain can tolerate high sugar concentrations, a batch mode will be selected to obtain high 2,3-BDO production in a bio-reactor. The final aim of this project is to develop a yeast strain producing at least 100 g/L 2,3-BDO with a yield of 0.45 g/g glucose and a productivity of 1.70g/L.h), which can meet the requirements for industrial production.