Biotransformation of b-Hydroxypyruvate and Glycolaldehyde to L-Erythrulose by Pichia pastoris Strain GS115 Overexpressing Native Transketolase Yu-Chia Wei Dept. of Biochemical Engineering, University College London, Bernard Katz Building, London WC1E 6BT, U.K. Stephanie Braun-Galleani School of Medicine, Conway Institute, University College Dublin, Dublin 4, Ireland Maria Jos e Henr  ıquez Dept. of Biochemical Engineering, University College London, Bernard Katz Building, London WC1E 6BT, U.K. Sahan Bandara Dept. of Biochemical Engineering, University College London, Bernard Katz Building, London WC1E 6BT, U.K. Darren Nesbeth Dept. of Biochemical Engineering, University College London, Bernard Katz Building, London WC1E 6BT, U.K. DOI 10.1002/btpr.2577 Published online November 20, 2017 in Wiley Online Library (wileyonlinelibrary.com) 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 b- 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% conver- sion, respectively, under optimal pH and methanol induction conditions. In a 300 lL reac- tion, TK150 samples from a 1L fed-batch fermentation achieved a maximum L-erythrulose space time yield (STY) of 46.58 g L 21 h 21 , specific activity of 155 U g 21 CDW , product yield on substrate (Y p/s ) of 0.52 mol mol 21 and product yield on catalyst (Y p/x ) of 2.23g g 21 CDW . We have successfully exploited the rapid growth and high biomass characteristics of Pichia pas- toris 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 chi- ral sugar L-erythrulose. V C 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:99–106, 2018 Keywords: Pichia pastoris, transketolase, whole cell biocatalyst, L-erythrulose, product inhibition Introduction Small molecule pharmaceuticals, comprising one or more chiral centers, remain a major product type in modern medi- cine, comprising two of the top five selling drugs globally in 2016, and five of the top 15. 1 Optically pure synthesis of small molecule pharmaceuticals by conventional chemi- synthetic routes tends to require multiple steps, each of which is typically defined by low product yield. As such, complex small molecules with multiple chiral centers tend to have high production costs due to the need for multiple chemi-synthetic steps. This challenge has led to the development of alternate “toolbox” platforms 2,3 including biological routes to con- trolled, stepwise assembly of the chemical species required to synthesize a final, desired molecule. Such biological routes typically involve the application of enzymes in the place of chemical catalysis, for “biocatalysis” to produce the final compound, over one or more steps. One advantage of enzymes is their promiscuity with respect to the molecules they engage with as substrates, which can enable a given enzyme to be used for a broad range of chemistries. 4 Enzymes can also provide exquisite regio-, chemo-, and stereo-selectivity for the synthetic steps they catalyze. In comparison to chemi-catalytic conditions, which can require extremes of pH and temperature, biocatalysis is Correspondence concerning this article should be addressed to Darren Nesbeth at D.nesbeth@ucl.ac.uk. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. V C 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers 99