Multi-level engineering to overcome limitations of whole cell bioreductions

Enantiomeric purity is a key determinant of efficacy and safety of modern drugs. The pharmaceutical industry therefore strives for chiral catalysts in process development. A prominent route to introduce chirality is the asymmetric reduction of oxo-containing molecules into chiral alcohols. Exquisite stereoselectivities of reductases increasingly outperform counterpart chemo-catalytic reductions of prochiral ketones, making biocatalysis the method of choice. In general, the biocatalyst employed for ketone reduction can be a whole cell system or a purified protein preparation. The proposed project is a follow up of the Hertha Firnberg-project Harnessing aldo- keto reductases for biocatalysis`. We focused on the synthesis of industrially relevant, chiral alcohols by whole cell reduction of the corresponding ketones. The high toxicity of organic substrates, especially those that displayed high hydrophobicity, limited productivity and product yield due to fast catalyst deactivation. Biocatalyst stability is a major concern in virtually all bioprocesses and strategies to address the problem of poor catalyst stability would therefore be important from basic and applied points of view. A detailed study of the impact of hydrophobic substrates on each process level to formulate criteria for an integrated process solution is still missing. This project proposes to address the problem of poor catalyst stability by enhancing performance at the enzyme, cell and process level. Aldo-keto reductase 2B5, xylose reductase from Candida tenuis (CtXR), turned out as broadly specific and highly stereoselective catalyst for the NADH-dependent reduction of a variety of ketones. Loop engineering was used to expand the substrate scope of the enzyme to bulky-bulky ketones. The designed enzymes showed >100-fold increased turnover numbers for the reduction of aromatic ?-keto esters as compared to the wild-type. Insights into the mechanism of rate acceleration in CtXR variants will be of general relevance considering the use of aldo-keto reductases for the production of chiral alcohols. Besides easy catalyst preparation and improved reductase stability, whole cell reductions offer the advantage that the supply of NADH or NADPH is accomplished by the metabolism of a co-substrate. We plan to compare engineered and natural whole cell catalysts based on CtXR in the reductions of aromatic ketones with respect to stereoselectivities, initial rates and stabilities. The dependence of catalyst stability on hydrophobic substrate and product concentrations will be determined and guide process options such as substrate feeding and in situ product removal. Developed processes will be evaluated considering process metrics such as productivity and product concentration. Multi-variance and complexity of problems in whole cell reductions require a window of operation analysis to visualise constraints from the process, biological boundaries and its correlations.

Numbers one, two, three and nine out of the 10 top-selling drugs in history are small, single-enantiomer molecules and, as reckoned by analysts, enantiopure pharmaceuticals will still play a leading role on blockbuster drug lists by 2020 (data from 2013, 2014). A single-enantiomer drug is one of two molecules that are mirror images of each other and are non-superimposable. The concept is explicable by left and right hands that are eventually the same except for their opposite orientation. Owing to the enantiopurity of most biological molecules (proteins, sugars, etc.) individual enantiomers can have markedly different effects: One may have the desired pharmacologic activity while the other may be less active, not active or may even be toxic. Enantiomers that approach near absolute enantiopurity are best obtained from enzyme catalyzed reactions. Time pressure in pharmaceutical industry prevents, however, in-depth investigation of biocatalytic reactions and optimization is oftentimes based on trial-and-error principle. The project made the thorough investigation of a biocatalytic reaction from enzyme to reaction scale-up possible. The bioreduction of o-chloroacetophenone became one of the best described biocatalytic reactions that are found in literature. Complex dependencies were by and by resolved during the project. Design problems were assigned to the enzyme, catalyst, reaction or process level and solutions were published in peer-reviewed articles. We provided, for the first time, an algorithm that helps to identify the most straight-forward strategy for the production of a specific chiral alcohol by a bioreduction. Diagnostic parameters for the characterization and bottleneck identification of bioreductions were introduced. The breaking down of a complex reaction into basic problems that can be assigned to the enzyme, catalyst, reaction or process stage is a universal approach and generally applicable to biocatalytic reactions.