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.

Stabilization and scale‐up of photosynthesis‐driven ω‐hydroxylation of nonanoic acid methyl ester by two‐liquid phase whole‐cell biocatalysis

“…Hydrophobic compounds with log P (octanol/water) values between 2 and 4 typically impair cell viability via intercalation into microbial membranes (Laane, Boeren, Vos, & Veeger, ; Sikkema, de Bont, & Poolman, ). Using the empirical equation for predicting the concentration leading to membrane dissociation as reported by Sikkema et al () and Kratzer et al () for heterotrophic microorganisms, NAME (log P = 3.9) and NA (log P = 3.4) are expected to become toxic at aqueous phase concentrations of 216 and 617 µM, respectively. Our results show an impaired growth at concentrations of 1 mM NAME (solubility in water is 133 µM) and 100 µM NA (Figure S7), indicating a higher sensitivity of Synechocystis sp.…”

Advanced Rhodococcus Biocatalysts for Environmental Biotechnologies

“…Both Rhodococcus whole cells and purified enzymes are currently used as biocatalysts. Enzymatic catalysts guarantee high reaction selectivity and the absence of side reactions, while whole cells perform multi-step bioconversions and allow easy cofactor regeneration [66]. In any case, the most important parameter is the economic viability of a biocatalytic system, which depends greatly on the system's stability.…”

“…Minimal inhibitory concentrations of heavy metals (Cd, Cs, Cr, Cu, Mo, Ni, Pb, Zn, and V) for Rhodococcus reached up to 250 mM; moreover, rhodococci were able to detoxify heavy metals and metalloids (As), changing their oxidation level [64,65].Both Rhodococcus whole cells and purified enzymes are currently used as biocatalysts. Enzymatic catalysts guarantee high reaction selectivity and the absence of side reactions, while whole cells perform multi-step bioconversions and allow easy cofactor regeneration [66]. In any case, the most important parameter is the economic viability of a biocatalytic system, which depends greatly on the system's stability.…”

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

“…Factors influencing as well as strategies tackling respective limitations have been investigated in great detail . Metabolic engineering‐derived whole‐cell biocatalysts, which can be a beneficial catalyst format, when reactions require energy, cofactor, or enzyme regeneration or constitute multistep pathways (iii‐v), have been described in depth with respect to activity restrictions and strategies to cope with those , but less extensively regarding catalyst stability. This review aims at a comprehensive discussion of different factors affecting whole‐cell biocatalyst stability and of knowledge‐based strategies to overcome respective limitations.…”

“…Factors influencing as well as strategies tackling respective limitations have been investigated in great detail [16][17][18][19]. Metabolic engineering-derived whole-cell biocatalysts, which can be a beneficial catalyst format, when reactions require energy, cofactor, or enzyme regeneration or constitute multistep pathways (iii-v), have been described in depth with respect to activity restrictions and strategies to cope with those [7,[20][21][22] , but less extensively regarding catalyst stability. This review aims at a comprehensive discussion of different factors affecting whole-cell biocata- Operational window, where the bioprocess performance is defined by four boundaries determined by downstream processing (DSP) demanding a minimal product titer, a maximally achievable reaction rate, toxicity/inhibition (of/by reactants) determining the maximally achievable amount of product per volume, and inherent biocatalyst stability determining a maximal process duration.…”