Rules for biocatalyst and reaction engineering to implement effective, NAD(P)H-dependent, whole cell bioreductions

Abstract

Access to chiral alcohols of high optical purity is today frequently provided by the enzymatic reduction of precursor ketones. However, bioreductions are complicated by the need for reducing equivalents in the form of NAD(P)H. The high price and molecular weight of NAD(P)H necessitate in situ recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme recycling mode and reaction conditions represent design options that strongly affect bioreduction efficiency. In this paper, each option was critically scrutinized and decision rules formulated based on well-described literature examples. The development chain was visualized as a decision-tree that can be used to identify the most promising route towards the production of a specific chiral alcohol. General methods, applications and bottlenecks in the set-up are presented and key experiments required to "test" for decision-making attributes are defined. The reduction of o-chloroacetophenone to (S)-1-(2-chlorophenyl)ethanol was used as one example to demonstrate all the development steps. Detailed analysis of reported large scale bioreductions identified product isolation as a major bottleneck in process design.

Keywords: Chiral alcohol; Cost analysis; Decision tree for bioreduction development; Design of Escherichia coli whole cell catalysts; Limitations of whole cell reductions; Scale-up.

Fig. 1

General scheme of bioreductions catalyzed by free enzymes or whole cells (gray oval indicates the cell envelope) (A). Whole cell reduction of o-chloroacetophenone catalyzed by recombinant E. coli based on CtXR and CbFDH (the dashed oval line depicts cell permeabilization, the blue hexagons illustrate isss and ispr by a water immiscible co-solvent). (B). Scheme of the multiphasic o-chloroacetophenone bioreduction at 0.5-L scale. The reaction was performed in a stirred tank reactor with pH and temperature control (gray points depict the biomass, blue drops show the hexane phase extracting o-chloroacetophenone and (S)-1-(2-chlorophenyl)ethanol). The three tubes show the extracted (S)-1-(2-chlorophenyl)ethanol that was obtained per batch (20 g) and that was further analyzed by chiral HPLC (C).

Fig. 2

Decision tree for the set-up of bioreductions. Rounded rectangles represent decision rules, blue squares show process solutions, inefficient branches are terminated by blue circles. (Process options for cell-free bioreduction systems are not further developed.)