Overcoming the thermodynamic equilibrium of an isomerization reaction through oxidoreductive reactions for biotransformation

Abstract

Isomerases perform biotransformations without cofactors but often cause an undesirable mixture of substrate and product due to unfavorable thermodynamic equilibria. We demonstrate the feasibility of using an engineered yeast strain harboring oxidoreductase reactions to overcome the thermodynamic limit of an isomerization reaction. Specifically, a yeast strain capable of consuming lactose intracellularly is engineered to produce tagatose from lactose through three layers of manipulations. First, GAL1 coding for galactose kinase is deleted to eliminate galactose utilization. Second, heterologous xylose reductase (XR) and galactitol dehydrogenase (GDH) are introduced into the ∆gal1 strain. Third, the expression levels of XR and GDH are adjusted to maximize tagatose production. The resulting engineered yeast produces 37.69 g/L of tagatose from lactose with a tagatose and galactose ratio of 9:1 in the reaction broth. These results suggest that in vivo oxidoreaductase reactions can be employed to replace isomerases in vitro for biotransformation.

Fig. 1

Galactose accumulation in lactose-consuming strain EJ2 with GAL1 deletion (EJ2g). a Growth profile as shown by dry cell weight (DCW), b lactose consumption, c ethanol production, and d galactose production by the EJ2g strain (filled circles) as compared with the EJ2 control stain (open circles) under aerobic conditions. Data are presented as mean value and standard deviations of three independent biological replicates. Source data are provided as a Source Data file

Fig. 2

Galactitol production after xylose reductase (XR) introduction into EJ2g. a Growth profile as shown by dry cell weight (DCW), b lactose consumption, c galactose production, and d galactitol produciton by the EJ2g strain with XR overexpression in the pRS42K plasmid (EJ2g_pX, filled circles), and EJ2g with empty plasmid pRS42K as control (open circles) on YP medium with 40 g/L lactose under aerobic conditions. Data presented as mean values and standard deviations of three independent biological replicates. Source data are provided as a Source Data file

Fig. 3

Tagatose production after the introduction of galactitol-2-dehydrogenase (GDH). a Growth curve as shown by DCW, b lactose consumption, c galactose production, and d galactitol production, and e tagatose production by the EJ2g_pXpG strain (filled circles) and the EJ2g_pX strain with p42H empty plasmid as control (open circles) under aerobic condition. Data are presented as mean value and standard deviations of three independent biological replicates. Source data are provided as a Source Data file

Fig. 4

Improved production of tagatose by increasing the XR and GDH gene copy numbers. a Galactose yield, b galactitol yield, and c tagatose yield. Data are presented as mean value and standard deviations of three independent biological replicates. Source data are provided as a Source Data file

Fig. 5

Tagatose production by the EJ2g_iXiG_pXpG strain in a 2 L bioreactor. a Profiles of lactose, galactose, galactitol, and tagatose concentrations, and b the growth of the EJ2g_iXiG_pXpG strain and the volume changes of bioreactor during the fed-batch fermentation. Source data are provided as a Source Data file

Fig. 6

Schematic diagram of the production of tagatose from lactose in engineered S. cerevisiae. Heterologous expression of cdt-1 (cellobiose transporter), gh1-1 (β-glucosidase), XR (xylose reductase), GDH (galactitol-2-dehydrogeanse), and deletion of Gal1 (galactose kinase) were combined in S. cerevisiae to produce tagatose directly from lactose