Immobilization of transaminases creates promising biocatalysts for production of chiral amines in batch or continuous-flow mode reactions. E. coli cells containing overexpressed transaminases of ...various selectivities and hollow silica microspheres as supporting agent were immobilized by an improved sol-gel process to produce immobilized transaminase biocatalysts with suitable stability and mechanical properties for continuous-flow applications. The immobilized cell-based transaminase biocatalyst proved to be durable and easy-to-use in kinetic resolution of four racemic amines 1a–d. The batch and continuous-flow mode kinetic resolutions with transaminase biocatalyst of opposite stereopreference provided access to both enantiomers of the corresponding amines. By using the most suitable immobilized transaminase biocatalysts, this study describes the first transaminase-based approach for the production of both pure enantiomers of 1-(3,4-dimethoxyphenyl)ethan-1-amine 1d.
The nicotinamide adenine dinucleotide regeneration system present in Escherichia coli cells was exploited for the oxidation and deracemisation of secondary alcohols with the overexpressed alcohol ...dehydrogenase from Rhodococcus ruber DSM 44541 (E. coli/ADH‐A). Thus, various racemic alcohols were selectively oxidised with lyophilised or resting E. coli/ADH‐A cells without need for an external cofactor or co‐substrate. The addition of these substrates to the E. coli/ADH‐A cells in buffer afforded the corresponding ketones and the remaining enantioenriched (R)‐alcohols. This methodology was used for the desymmetrisation of a meso‐diol and for the synthesis of the highly valuable raspberry ketone. Moreover, a biocatalytic concurrent process was developed with the resting cells of E. coli/ADH‐A, ADH from Lactobacillus brevis, and glucose dehydrogenase for the deracemisation of various secondary alcohols, which afforded the desired enantiopure alcohols in more than 99 % ee starting from the racemic mixture. The reaction time of deracemisation of 1‐phenylethanol was estimated to be less than 30 min. The stereoinversion of (S)‐1‐phenylethanol to its pure (R)‐enantiomer was also achieved, which provided a biocatalytic alternative to the chemical Mitsunobu inversion reaction.
An integrated recycling system: A nicotinamide adenine dinucleotide regeneration system present in Escherichia coli cells is exploited for the oxidation of secondary alcohols with E. coli/alcohol dehydrogenase (ADH‐A). Racemic alcohols are oxidized selectively without need for an external cofactor or cosubstrate, which affords the remaining enantioenriched (R)‐alcohols. Deracemization, desymmetrization, and stereoinversion processes are developed, which leads to optically pure high value‐added compounds.
The antiglaucoma agent travoprost, which is an analogue of the prostaglandin PGF2α, was synthesized by means of a three‐component coupling utilizing chemoenzymatically generated building blocks in ...high enantiopurity.
Efficient together! A chemoenzymatic synthesis of the active agent travoprost by using alcohol dehydrogenases is described. These biocatalysts operate in a highly enantioselective fashion providing enantiopure building blocks, which were used for the synthesis of travoprost by means of a three‐component coupling.
A chemoenzymatic approach for the asymmetric total synthesis of the title compounds is described that employs an enantioselective oxidative C–C bond formation catalyzed by berberine bridge enzyme ...(BBE) in the asymmetric key step. This unique reaction yielded enantiomerically pure (R)-benzylisoquinoline derivatives and (S)-berbines such as the natural product (S)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates rac-1 required for the biotransformation were prepared in 4–8 linear steps using either a Bischler–Napieralski cyclization or a C1–Cα alkylation approach. The chemoenzymatic synthesis was applied to the preparation of fourteen enantiomerically pure alkaloids, including the natural products (S)-scoulerine and (R)-reticuline, and gave overall yields of up to 20% over 5–9 linear steps.
Prochiral bicyclic diketones were transformed to a single diastereomer of 3‐substituted cyclohexylamine derivatives via three consecutive biocatalytic steps. The two chiral centres were set up by a ...CC hydrolase (6‐oxocamphor hydrolase) in the first step and by an ω‐transaminase in the last step. The esterification of the intermediate keto acid was catalysed by a lipase in the second step if possible. For two substrates the CC hydrolytic step as well as the esterification could be run simultaneously in a one‐pot cascade in an organic solvent. In one example, the reaction mixture of the first two steps could be directly subjected to bio‐amination in an organic solvent without the need to change the reaction medium. Depending on the choice of the ω‐transaminase employed and the substrate the cis‐ as well as the trans‐diastereomers could be obtained in optically pure forms.
Abstract
Hydrogen transfer biocatalysts to prepare optically pure alcohols are in need, especially when it comes to sterically demanding ketones, whereof the bioreduced products are either essential ...precursors of pharmaceutically relevant compounds or constitute APIs themselves. In this study, we report on the biocatalytic potential of an anti-Prelog (
R
)-specific
Lactobacillus kefir
ADH variant (Lk-ADH-E145F-F147L-Y190C, named Lk-ADH Prince) employed as
E. coli
/ADH whole-cell biocatalyst and its characterization for stereoselective reduction of prochiral carbonyl substrates. Key enzymatic reaction parameters, including the reaction medium, evaluation of cofactor-dependency, organic co-solvent tolerance, and substrate loading, were determined employing the drug pentoxifylline as a model prochiral ketone. Furthermore, to tap the substrate scope of Lk-ADH Prince in hydrogen transfer reactions, a broad range of 34 carbonylic derivatives was screened. Our data demonstrate that
E. coli/
Lk-ADH Prince exhibits activity toward a variety of structurally different ketones, furnishing optically active alcohol products at the high conversion of 65–99.9% and in moderate-to-high isolated yields (38–91%) with excellent anti-Prelog (
R
)-stereoselectivity (up to >99% ee) at substrate concentrations up to 100 mM.
A novel one-pot tandem biohydrogen transfer process to concurrently obtain two enantiopure sec-alcohols is presented; thus, using a suitable single enzyme and a catalytic amount of cofactor, several ...interesting building blocks could be easily achieved in an enantiocomplementary fashion, minimizing dramatically the quantity of reagents usually employed in the “coupled-substrate” approach.
Media containing yeast extracts and other complex raw materials are widely used for the cultivation of microorganisms. However, variations in the specific nutrient composition can occur, due to ...differences in the complex raw material ingredients and in the production of these components. These lot-to-lot variations can affect growth rate, product yield and product quality in laboratory investigations and biopharmaceutical production processes. In the FDA's Process Analytical Technology (PAT) initiative, the control and assessment of the quality of critical raw materials is one key aspect to maintain product quality and consistency. In this study, the Respiration Activity Monitoring System (RAMOS) was used to evaluate the impact of different yeast extracts and commercial complex auto-induction medium lots on metabolic activity and product yield of four recombinant Escherichia coli variants encoding different enzymes.
Under non-induced conditions, the oxygen transfer rate (OTR) of E. coli was not affected by a variation of the supplemented yeast extract lot. The comparison of E. coli cultivations under induced conditions exhibited tremendous differences in OTR profiles and volumetric activity for all investigated yeast extract lots of different suppliers as well as lots of the same supplier independent of the E. coli variant. Cultivation in the commercial auto-induction medium lots revealed the same reproducible variations. In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times. Only by online monitoring of the cultures, a distinct cultivation phase (e.g. glycerol depletion) could be detected and chosen for comparable and reproducible offline analysis of the yield of functional product.
This work proves that cultivations conducted in complex media may be prone to significant variation in final product quality and quantity if the quality of the raw material for medium preparation is not thoroughly checked. In this study, the RAMOS technique enabled a reliable and reproducible screening and phenotyping of complex raw material lots by online measurement of the respiration activity. Consequently, complex raw material lots can efficiently be assessed if the distinct effects on culture behavior and final product quality and quantity are visualized.
Transaminases have become a key tool in biocatalysis to introduce the amine functionality into a range of molecules like prochiral α-ketoacids and ketones. However, due to the necessity of shifting ...the equilibrium towards the product side (depending on the amine donor) an efficient amination system may require three enzymes. So far, this well-established transformation has mainly been performed in vitro by assembling all biocatalysts individually, which comes along with elaborate and costly preparation steps. We present the design and characterization of a flexible approach enabling a quick set-up of single-cell biocatalysts producing the desired enzymes. By choosing an appropriate co-expression strategy, a modular system was obtained, allowing for flexible plug-and-play combination of enzymes chosen from the toolbox of available transaminases and/or recycling enzymes tailored for the desired application.
By using a two-plasmid strategy for the recycling enzyme and the transaminase together with chromosomal integration of an amino acid dehydrogenase, two enzyme modules could individually be selected and combined with specifically tailored E. coli strains. Various plug-and-play combinations of the enzymes led to the construction of a series of single-cell catalysts suitable for the amination of various types of substrates. On the one hand the fermentative amination of α-ketoacids coupled both with metabolic and non-metabolic cofactor regeneration was studied, giving access to the corresponding α-amino acids in up to 96% conversion. On the other hand, biocatalysts were employed in a non-metabolic, "in vitro-type" asymmetric reductive amination of the prochiral ketone 4-phenyl-2-butanone, yielding the amine in good conversion (77%) and excellent stereoselectivity (ee = 98%).
The described modularized concept enables the construction of tailored single-cell catalysts which provide all required enzymes for asymmetric reductive amination in a flexible fashion, representing a more efficient approach for the production of chiral amines and amino acids.
In contrast to kinetic resolution, where only 50% of the racemic starting material can be converted into the desired product and where the remaining ‘wrong' enantiomer has to be considered as waste, ...so‐called deracemisation processes allow the production of a single stereoisomeric product from racemic starting material. In this context, the use of environmentally benign methods for biocatalytic racemisation holds great potential. The small and largely overlooked group of racemases (EC 5.1.X.X), which are increasingly being used for dynamic kinetic resolution or in auxiliary biocatalytic recycling processes, are reviewed with respect to their properties, their substrate tolerance and their biocatalytic potential.
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