Production of (2R, 3R)-2,3-butanediol using engineered Pichia pastoris: strain construction, characterization and fermentation

Zhiliang Yang, Zisheng Zhang

2,3-butanediol (2,3-BD) is a bulk platform chemical with various potential applications such as aviation fuel. 2,3-BD has three optical isomers: (2R, 3R)-, (2S, 3S)- and meso-2,3-BD. Optically pure 2,3-BD is a crucial precursor for the chiral synthesis and it can also be used as anti-freeze agent due to its low freezing point. 2,3-BD has been produced in both native and non-native hosts. Several pathogenic bacteria were reported to produce 2,3-BD in mixture of its optical isomers includingKlebsiella pneumoniaeandKlebsiella oxytoca. Engineered hosts based on episomal plasmid expression such asEscherichia coli,Saccharomyces cerevisiaeandBacillus subtilisare not ideal for industrial fermentation due to plasmid instability. Pichia pastoris is generally regarded as safe and a well-established host for high-level heterologous protein production. To produce pure (2R, 3R)-2,3-BD enantiomer, we developed aP. pastorisstrain by introducing a synthetic pathway. ThealsS andalsD genes fromB. subtiliswere codon-optimized and synthesized. The BDH1 gene fromS. cerevisiaewas cloned. These three pathway genes were integrated into the genome ofP. pastorisand expressed under the control of GAP promoter. Production of (2R, 3R)-2,3-BD was achieved using glucose as feedstock. The optical purity of (2R, 3R)-2,3-BD was more than 99%. The titer of (2R, 3R)-2,3-BD reached 12 g/L with 40 g/L glucose as carbon source in shake flask fermentation. The fermentation conditions including pH, agitation speeds and aeration rates were optimized in batch cultivations. The highest titer of (2R, 3R)-2,3-BD achieved in fed-batch fermentation using YPD media was 45 g/L. The titer of 2,3-BD was enhanced to 74.5 g/L through statistical medium optimization. The potential of engineeringP. pastorisinto a microbial cell factory for biofuel production was evaluated in this work using (2R, 3R)-2,3-BD as an example. EngineeredP. pastoriscould be a promising workhorse for the production of optically pure (2R, 3R)-2,3-BD.