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 normalgCDW−1, product yield on substrate (Yp/s) of 0.52 mol mol−1 and product yield on catalyst (Yp/x) of 2.23g normalgCDW−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 strain tolerated high concentrations of substrate and product to achieve high STY of the chiral sugar l‐erythrulose. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:99–106, 2018

Whole cell biosynthetic activity of Komagataella phaffii (Pichia pastoris) GS115 strains engineered with transgenes encoding Chromobacterium violaceum ω‐transaminase alone or combined with native transketolase

“…Previously, Wei et al used pAO815 as the backbone for construction of the plasmid, pAOX0150‐TK, which encodes a gene for expression of a native K. phaffii transketolase (Genbank: CAY67980.1) under control of the methanol‐inducible AOXI promoter. For DNA assembly purposes, we used standard molecular biology techniques to perform Dpn I‐mediated, site directed mutagenesis of pAOX0150‐TK to remove a Bgl II site from the transketolase ORF while preserving amino acid sequence encoding.…”

“…Transketolases are enzymes capable of catalyzing the transfer of a two‐carbon group from a ketose sugar to an aldose sugar forming an asymmetric carbon–carbon bond. They have been used for whole cell biocatalysis in Escherichia coli , and Komagataella phaffii cells, for asymmetrization of hydroxypyruvate and glycolaldehyde to generate the ketoalcohol, L‐erythrulose. Transketolases require two cofactors; thiamine pyrophosphate (TPP) and a divalent cation such as magnesium (II).…”

“…K. phaffii can use methanol as its only carbon and energy source, making its cultivation economically favorable. We previously engineered a recombinant K. phaffii strain for overexpression of a native transketolase and characterized its whole‐cell biocatalysis performance for the production of the chiral sugar L‐erythrulose from the prochiral compounds hydroxypyruvate (HPA) and glycolaldehyde (GA).…”

“…Yeasts such as K. phaffii are in theory a viable alternative, with no pathogens equivalent to bacteriophage and a broader range of pH tolerance, from pH 3 to pH 7.11. To validate K. phaffii as an alternative biocatalytic host cell, we further modify the transketolase K. phaffii strain of Wei et al by inserting transgenes encoding the native transketolase and the C. violaceum transaminase CV2025, shown by others to be the best‐performing transaminase for the production of ABT within the equivalent de novo pathway in E. coli whole cell biocatalysis (Figure ). We also engineer a further K. phaffii strain to harbor a transgene encoding the C. violaceum transaminase CV2025 only.…”

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

“…Recently, asymmetric carbon–carbon bond formation reaction was reported using P. pastoris as a host for transketolase whole‐cell biocatalysis . A transketolase‐overexpressing strain was constructed to catalyze the formation of l ‐erythrulose from β‐hydroxypyruvic acid and glycolaldehyde.…”