Biotransformation of β-hydroxypyruvate and glycolaldehyde to l-erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase
Abstract
Transketolase is a proven biocatalytic tool for asymmetric carbon-carbon bond formation, both as a purified enzyme and within bacterial whole-cell biocatalysts. The performance of Pichia pastoris as a host for transketolase whole-cell biocatalysis was investigated using a transketolase-overexpressing strain to catalyze formation of l-erythrulose from β-hydroxypyruvic acid and glycolaldehyde substrates. Pichia pastoris transketolase coding sequence from the locus PAS_chr1-4_0150 was subcloned downstream of the methanol-inducible AOX1 promoter in a plasmid for transformation of strain GS115, generating strain TK150. Whole and disrupted TK150 cells from shake flasks achieved 62% and 65% conversion, respectively, under optimal pH and methanol induction conditions. In a 300 μL reaction, TK150 samples from a 1L fed-batch fermentation achieved a maximum l-erythrulose space time yield (STY) of 46.58 g L-1 h-1 , specific activity of 155 U gCDW-1, product yield on substrate (Yp/s ) of 0.52 mol mol-1 and product yield on catalyst (Yp/x ) of 2.23g gCDW-1. We have successfully exploited the rapid growth and high biomass characteristics of Pichia pastoris in whole cell biocatalysis. At high cell density, the engineered TK150 Pichia pastoris ...Continue Reading
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