Engineering Saccharomyces cerevisiae and controlling conditions for 2,3-butanediol production from glycerol

Abstract

2,3-Butanediol (2,3-BDO) has gained much attention due to its bulk chemical uses in numerous applications such as the production of pharmaceuticals, cosmetics, synthetic rubber, inks, resins, perfumes, foodstuffs, fuel additives, and aviation fuel. Additionally, 2,3-BDO can be used as an antifreeze, enhancer of plant drought resistance, plant growth stimulator, and plant pest-virus control agent. Engineered Saccharomyces cerevisiae has exhibited superb potential for converting glucose to 2,3-BDO, but it lacks the native capability to do so from glycerol. Glycerol, an affordable by-product of biodiesel, is a green solvent with high permittivity and low inhibitory effects, making it a cost-effective option for lignocellulose biorefinery. Therefore, we aimed to further engineer and expand substrate utilization to glycerol or co-utilization of glycerol with glucose for 2,3-BDO production using the engineered S. cerevisiae SK-FGG4 strain, which was previously engineered to convert glycerol to ethanol. The 2,3-BDO biosynthesis pathway, consisting of codon-optimized Bacillus subtilis acetolactate synthase (BsAlsS), acetolactate decarboxylase (BsAlsD), and S. cerevisiae butanediol dehydrogenase (ScBDH), was assembled into one module and integrated into the genomic DNA of SK-FGG4, generating the SK-MA1 strain. Interestingly, SK-MA1 completely shifted glycerol conversion under microaerobic conditions from ethanol to a mixture of acetoin and 2,3-BDO at 0.43 g_{acetoin+2,3-BDO} g_glycerol⁻¹, with 2,3-BDO accounting for 0.28 g_2,3-BDO g_glycerol⁻¹. The SK-MA2, SK-MA3, and SK-MA4 strains, which were generated using CRISPR by replacing alcohol dehydrogenase ScADH1, ScADH2, and ScADH5 enzymes individually with the 2,3-BDO biosynthesis enzymes, exhibited enhanced conversion parameters on a complex medium under aerobic conditions compared to the SK-MA1 strain. Double replacement of ScADH1 and ScADH5 with 2,3-BDO biosynthesis enzymes in the SK-MA7 strain promoted higher accumulation of 2,3-BDO over other strains in aerobic conditions, with total conversion reaching 0.41 g_{acetoin+2,3-BDO} g_{glucose+glycerol}⁻¹, which is over 3.2-fold higher than the reference SK-MA6 strain, a native strain (D452-2) with ScADH5 replaced with 2,3-BDO biosynthesis enzymes. Under these aerobic conditions, acetoin was over 2-fold of the 2,3-BDO. Lowering the aeration to microaerobic levels decreased acetoin accumulation and increased the production of 2,3-BDO. Strain SK-MA3 produced 0.34 g_2,3-BDO g_{glucose+glycerol}⁻¹ with a total conversion of 0.455 g_{acetoin+2,3-BDO} g_{glucose+glycerol}⁻¹, representing 92% of the theoretical yield at 96 hours. This study is the first to explore the conversion of glycerol to 2,3-BDO using engineered S. cerevisiae and supports broadening glycerol-based biorefinery approaches.