Evolution of Biocatalysis at Novartis over the last 40 Years.

The fortieth anniversary of biocatalysis started at Ciba-Geigy and later at Novartis is a great time to pause and reflect on development of science and technology in this field. Enzyme-based synthesis became a highly valued enabling tool for pharmaceutical research and development over the last decades. In this perspective we aim to discuss how the scientific approaches and trends evolved over the time and present future challenges and opportunities.

Absorption, Distribution, Metabolism, and Excretion of Capmatinib (INC280) in Healthy Male Volunteers and In Vitro Aldehyde Oxidase Phenotyping of the Major Metabolite.

Capmatinib (INC280), a highly selective and potent inhibitor of the MET receptor tyrosine kinase, has demonstrated clinically meaningful efficacy and a manageable safety profile in patients with advanced non-small-cell lung cancer harboring MET exon 14-skipping mutations. We investigated the absorption, distribution, metabolism, and excretion of capmatinib in six healthy male volunteers after a single peroral dose of 600 mg 14C-labeled capmatinib. The mass balance, blood and plasma radioactivity, and plasma capmatinib concentrations were determined along with metabolite profiles in plasma, urine, and feces. The metabolite structures were elucidated using mass spectrometry and comparing with reference compounds. The parent compound accounted for most of the radioactivity in plasma (42.9% ± 2.9%). The extent of oral absorption was estimated to be 49.6%; the Cmax of capmatinib in plasma was reached at 2 hours (median time to reach Cmax). The apparent mean elimination half-life of capmatinib in plasma was 7.84 hours. Apparent distribution volume of capmatinib during the terminal phase was moderate-to-high (geometric mean 473 l). Metabolic reactions involved lactam formation, hydroxylation, N-dealkylation, formation of a carboxylic acid, hydrogenation, N-oxygenation, glucuronidation, and combinations thereof. M16, the most abundant metabolite in plasma, urine, and feces was formed by lactam formation. Absorbed capmatinib was eliminated mainly by metabolism and subsequent biliary/fecal and renal excretion. Excretion of radioactivity was complete after 7 days. CYP phenotyping demonstrated that CYP3A was the major cytochrome P450 enzyme subfamily involved in hepatic microsomal metabolism, and in vitro studies in hepatic cytosol indicated that M16 formation was mainly catalyzed by aldehyde oxidase. SIGNIFICANCE STATEMENT: The absorption, distribution, metabolism, and excretion of capmatinib revealed that capmatinib had substantial systemic availability after oral administration. It was also extensively metabolized and largely distributed to the peripheral tissue. Mean elimination half-life was 7.84 hours. The most abundant metabolite, M16, was formed by imidazo-triazinone formation catalyzed by cytosolic aldehyde oxidase. Correlation analysis, specific inhibition, and recombinant enzymes phenotyping demonstrated that CYP3A is the major enzyme subfamily involved in the hepatic microsomal metabolism of [14C]capmatinib.

Evolved Aliphatic Halogenases Enable Regiocomplementary C-H Functionalization of a Pharmaceutically Relevant Compound.

Non-heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline-derived fragment. Using structure-guided evolution, a halogenase variant with a more than 290-fold higher total turnover number and a 400-fold higher apparent kcat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non-heme iron halogenase beyond the native indole-alkaloid-type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late-stage halogenation.

Cladosporin Derivatives Obtained by Biotransformation Provide Guidance for the Focused Derivatization of this Antimalarial Lead Compound.

Cladosporin, a natural product known for decades, has recently been discovered to display potent and selective antiplasmodial activity by inhibition of lysyl-tRNA synthetase. It was subjected to a panel of oxidative biotransformations with one fungal and two actinomycetes strains, as well as a triple mutant bacterial CYP102A1, yielding eight, mostly hydroxylated, derivatives. These new compounds covered a wide chemical space and contained two pairs of epimers in the tetrahydropyran ring. Although less potent than the parent compound, all analogues showed activity in a cell-based synthetase assay, thus demonstrating uptake and on-target activity in living cells with varying degrees of selectivity for the enzyme lysyl-tRNA synthetase from Plasmodium falciparum and highlighting sites suitable for synthesis of future cladosporin analogues. Compounds with adjacent hydroxy functions showed different MS/MS fragmentation that can be explained in terms of an, in some cases, regioselective loss of water followed by a retro-Diels-Alder reaction.

Metabolism and Disposition of Siponimod, a Novel Selective S1P1/S1P5 Agonist, in Healthy Volunteers and In Vitro Identification of Human Cytochrome P450 Enzymes Involved in Its Oxidative Metabolism.

Siponimod, a next-generation selective sphingosine-1-phosphate receptor modulator, is currently being investigated for the treatment of secondary progressive multiple sclerosis. We investigated the absorption, distribution, metabolism, and excretion (ADME) of a single 10-mg oral dose of [14C]siponimod in four healthy men. Mass balance, blood and plasma radioactivity, and plasma siponimod concentrations were measured. Metabolite profiles were determined in plasma, urine, and feces. Metabolite structures were elucidated using mass spectrometry and comparison with reference compounds. Unchanged siponimod accounted for 57% of the total plasma radioactivity (area under the concentration-time curve), indicating substantial exposure to metabolites. Siponimod showed medium to slow absorption (median Tmax: 4 hours) and moderate distribution (Vz/F: 291 l). Siponimod was mainly cleared through biotransformation, predominantly by oxidative metabolism. The mean apparent elimination half-life of siponimod in plasma was 56.6 hours. Siponimod was excreted mostly in feces in the form of oxidative metabolites. The excretion of radioactivity was close to complete after 13 days. Based on the metabolite patterns, a phase II metabolite (M3) formed by glucuronidation of hydroxylated siponimod was the main circulating metabolite in plasma. However, in subsequent mouse ADME and clinical pharmacokinetic studies, a long-lived nonpolar metabolite (M17, cholesterol ester of siponimod) was identified as the most prominent systemic metabolite. We further conducted in vitro experiments to investigate the enzymes responsible for the oxidative metabolism of siponimod. The selective inhibitor and recombinant enzyme results identified cytochrome P450 2C9 (CYP2C9) as the predominant contributor to the human liver microsomal biotransformation of siponimod, with minor contributions from CYP3A4 and other cytochrome P450 enzymes.

Draft Genome Sequences of Three Actinobacteria Strains Presenting New Candidate Organisms with High Potentials for Specific P450 Cytochromes.

The three Actinobacteria strains Streptomyces platensis DSM 40041, Pseudonocardia autotrophica DSM 535, and Streptomyces fradiae DSM 40063 were described to selectively oxyfunctionalize several drugs. Here, we present their draft genomes to unravel their gene sets encoding promising cytochrome P450 monooxygenases associated with the generation of drug metabolites.

Definition of culture conditions for Arxula adeninivorans, a rational basis for studying heterologous gene expression in this dimorphic yeast.

The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l(-1) was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l(-1). Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h(-1) and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.

Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis.

Cytochrome P450 monooxygenases (CYPs) are important enzymes in the metabolism of xenobiotics. Therefore, several approaches to clone and overexpress the human isoforms have been made. In addition to microsomes or S9 preparations, these recombinant human isoforms have found diverse application in drug development. We discuss and give examples of the use of bacterial whole cell systems with rec. human CYPs for the preparative scale synthesis of drug metabolites.

Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation.

Industrial biocatalytic reduction processes require the efficient regeneration of reduced cofactors for the asymmetric reduction of prochiral compounds to chiral intermediates which are needed for the production of fine chemicals and drugs. Here, we present a new engineering strategy for improved NADH regeneration based on the Pichia pastoris methanol oxidation pathway. Studying the kinetic properties of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) and using the derived kinetic data for subsequent kinetic simulations of NADH formation rates led to the identification of FLD activity to constitute the main bottleneck for efficient NADH recycling via the methanol dissimilation pathway. The simulation results were confirmed constructing a recombinant P. pastoris strain overexpressing P. pastoris FLD and the highly active NADH-dependent butanediol dehydrogenase from S. cerevisiae. Employing the engineered strain, significantly improved butanediol production rates were achieved in whole-cell biotransformations.

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes.

For redox reactions catalyzed by microbial cells the analysis of involved cofactors is of special interest since the availability of cofactors such as NADH or NADPH is often limiting and crucial for the biotransformation efficiency. The measurement of these cofactors has usually been carried out using spectrophotometric cycling assays. Today LC-MS/MS methods have become a valuable tool for the identification and quantification of intracellular metabolites. This technology has been adapted to measure all four nicotinamide cofactors (NAD, NADP, NADH, and NADPH) during a whole cell biotransformation process catalyzed by recombinant Escherichia coli cells. The cells overexpressing an alcohol dehydrogenase from Lactobacillus brevis were used for the reduction of methyl acetoacetate (MAA) with substrate-coupled cofactor regeneration by oxidation of 2-propanol. To test the reliability of the measurement the data were evaluated using a process model. This model was derived using the measured concentrations of reactants and cofactors for initiation as well as the kinetic constants from in vitro measurements of the isolated enzyme. This model proves to be highly effective in the process development for a whole cell redox biotransformation in predicting both the right concentrations of cofactors and reactants in a batch and in a CSTR process as well as the right in vivo expression level of the enzyme. Moreover, a sensitivity analysis identifies the cofactor regeneration reaction as the limiting step in case for the reduction of MAA to the corresponding product (R)-methyl 3-hydroxybutyrate. Using the combination of in vitro enzyme kinetic measurements, measurements of cofactors and reactants and an adequate model initiated by intracellular concentrations of all involved reactants and cofactors the whole cell biotransformation process can be understood quantitatively.

Continuous asymmetric ketone reduction processes with recombinant Escherichia coli.

The reduction of methyl acetoacetate was carried out in continuously operated biotransformation processes catalyzed by recombinant Escherichia coli cells expressing an alcohol dehydrogenase from Lactobacillus brevis. Three different cell types were applied as biocatalysts in three different cofactor regeneration approaches. Both processes with enzyme-coupled cofactor regeneration catalyzed by formate dehydrogenase or glucose dehydrogenase are characterized by a rapid deactivation of the biocatalyst. By contrast the processes with substrate-coupled cofactor regeneration by alcohol dehydrogenase catalyzed oxidation of 2-propanol could be run over a period of 7 weeks with exceedingly high substrate and cosubstrate concentrations of up to 2.5 and 2.8 mol L(-1), respectively. Even under these extreme conditions, the applied biocatalyst showed a good stability with only marginal leakage of intracellular cofactors.

Metabolomics: current state and evolving methodologies and tools.

In recent years, metabolomics developed to an accepted and valuable tool in life sciences. Substantial improvements of analytical hardware allow metabolomics to run routinely now. Data are successfully used to investigate genotype-phenotype relations of strains and mutants. Metabolomics facilitates metabolic engineering to optimise mircoorganisms for white biotechnology and spreads to the investigation of biotransformations and cell culture. Metabolomics serves not only as a source of qualitative but also quantitative data of intra-cellular metabolites essential for the model-based description of the metabolic network operating under in vivo conditions. To collect reliable metabolome data sets, culture and sampling conditions, as well as the cells' metabolic state, are crucial. Hence, application of biochemical engineering principles and method standardisation efforts become important. Together with the other more established omics technologies, metabolomics will strengthen its claim to contribute to the detailed understanding of the in vivo function of gene products, biochemical and regulatory networks and, even more ambitious, the mathematical description and simulation of the whole cell in the systems biology approach. This knowledge will allow the construction of designer organisms for process application using biotransformation and fermentative approaches making effective use of single enzymes, whole microbial and even higher cells.

High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank bioreactors.

This study demonstrates the applicability of pressurized stirred tank bioreactors for oxygen transfer enhancement in aerobic cultivation processes. The specific power input and the reactor pressure was employed as process variable. As model organism Escherichia coli, Arxula adeninivorans, Saccharomyces cerevisiae and Corynebacterium glutamicum were cultivated to high cell densities. By applying specific power inputs of approx. 48kWm(-3) the oxygen transfer rate of a E. coli culture in the non-pressurized stirred tank bioreactor was lifted up to values of 0.51moll(-1)h(-1). When a reactor pressure up to 10bar was applied, the oxygen transfer rate of a pressurized stirred tank bioreactor was lifted up to values of 0.89moll(-1)h(-1). The non-pressurized stirred tank bioreactor was able to support non-oxygen limited growth of cell densities of more than 40gl(-1) cell dry weight (CDW) of E. coli, whereas the pressurized stirred tank bioreactor was able to support non-oxygen limited growth of cell densities up to 225gl(-1) CDW of A. adeninivorans, 89gl(-1) CDW of S. cerevisiae, 226gl(-1) CDW of C. glutamicum and 110gl(-1) CDW of E. coli. Compared to literature data, some of these cell densities are the highest values ever achieved in high cell density cultivation of microorganisms in stirred tank bioreactors. By comparing the specific power inputs as well as the k(L)a values of both systems, it is demonstrated that only the pressure is a scaleable tool for oxygen transfer enhancement in industrial stirred tank bioreactors. Furthermore, it was shown that increased carbon dioxide partial pressures did not remarkably inhibit the growth of the investigated model organisms.

Biocatalytic ketone reduction--a powerful tool for the production of chiral alcohols--part I: processes with isolated enzymes.

Enzymes are able to perform reactions under mild conditions, e.g., pH and temperature, with remarkable chemo-, regio-, and stereoselectivity. Because of this feature, the number of biocatalysts used in organic synthesis has rapidly increased during the last decades, especially for the production of chiral compounds. The present review highlights biotechnological processes for the production of chiral alcohols by reducing prochiral ketones. These reactions can be catalyzed by either isolated enzymes or whole cells that exhibit ketone-reducing activity. The use of isolated enzymes is often preferred because of a higher volumetric productivity and the absence of side reactions. Both types of catalysts have also deficiencies limiting their use in synthesis of chiral alcohols. Because reductase-catalyzed reactions are dependent on cofactors, one major task in process development is to provide an effective method for regeneration of the consumed cofactors. In this paper, strategies for cofactor regeneration in biocatalytic ketone reduction are reviewed. Furthermore, different processes carried out on laboratory and industrial scales using isolated enzymes are presented. Attention is turned to process parameters, e.g., conversion, yield, enantiomeric excess, and process strategies, e.g., the application of biphasic systems or methods of in situ (co)product recovery. The biocatalytic production of chiral alcohols utilizing whole cells is presented in part II of this review.

Biocatalytic ketone reduction--a powerful tool for the production of chiral alcohols-part II: whole-cell reductions.

Enzymes are able to perform reactions under mild conditions, e.g., pH and temperature, with remarkable chemo-, regio-, and stereoselectivity. Due to this feature the number of biocatalysts used in organic synthesis has rapidly increased during the last decades, especially for the production of chiral compounds. The present review highlights biotechnological processes for the production of chiral alcohols by reducing prochiral ketones with whole cells. Microbial transformations feature different characteristics in comparison to isolated enzymes. Enzymes that are used in whole-cell biotransformations are often more stable due to the presence of their natural environment inside the cell. Because reductase-catalyzed reactions are dependent on cofactors, one major task in process development is to provide an effective method for regeneration of the consumed cofactors. Many whole-cell biocatalysts offer their internal cofactor regeneration that can be used by adding cosubstrates, glucose or, in the case of cyanobacteria, simply light. In this paper, various processes carried out on laboratory and industrial scales are presented. Thereby, attention is turned to process parameters, e.g., conversion, yield, enantiomeric excess, and process strategies, e.g., the application of biphasic systems. The biocatalytic production of chiral alcohols utilizing isolated enzymes is presented in part I of this review.