Generation of Aurachin Derivatives by Whole-Cell Biotransformation and Evaluation of Their Antiprotozoal Properties.

The natural product aurachin D is a farnesylated quinolone alkaloid, which is known to possess activity against the causative agent of malaria, Plasmodium spp. In this study, we show that aurachin D inhibits other parasitic protozoa as well. While aurachin D had only a modest effect on Trypanosoma brucei rhodesiense, two other trypanosomatids, T. cruzi and Leishmania donovani, were killed at low micromolar and nanomolar concentrations, respectively, in an in vitro assay. The determined IC50 values of aurachin D were even lower than those of the reference drugs benznidazole and miltefosine. Due to these promising results, we set out to explore the impact of structural modifications on the bioactivity of this natural product. In order to generate aurachin D derivatives with varying substituents at the C-2, C-6 and C-7 position of the quinolone ring system, we resorted to whole-cell biotransformation using a recombinant Escherichia coli strain capable of aurachin-type prenylations. Quinolone precursor molecules featuring methyl, methoxy and halogen groups were fed to this E. coli strain, which converted the substrates into the desired analogs. None of the generated derivatives exhibited improved antiprotozoal properties in comparison to aurachin D. Obviously, the naturally occurring aurachin D features already a privileged structure, especially for the inhibition of the causative agent of visceral leishmaniasis.

Cell-Free Protein Synthesis for the Screening of Novel Azoreductases and Their Preferred Electron Donor.

Azoreductases are potent biocatalysts for the cleavage of azo bonds. Various gene sequences coding for potential azoreductases are available in databases, but many of their gene products are still uncharacterized. To avoid the laborious heterologous expression in a host organism, we developed a screening approach involving cell-free protein synthesis (CFPS) combined with a colorimetric activity assay, which allows the parallel screening of putative azoreductases in a short time. First, we evaluated different CFPS systems and optimized the synthesis conditions of a model azoreductase. With the findings obtained, 10 azoreductases, half of them undescribed so far, were screened for their ability to degrade the azo dye methyl red. All novel enzymes catalyzed the degradation of methyl red and can therefore be referred to as azoreductases. In addition, all enzymes degraded the more complex and bulkier azo dye Brilliant Black and four of them also showed the ability to reduce p-benzoquinone. NADH was the preferred electron donor for the most enzymes, although the synthetic nicotinamide co-substrate analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) was also accepted by all active azoreductases. This screening approach allows accelerated identification of potential biocatalysts for various applications.

Biotechnological production of cyclic dinucleotides-Challenges and opportunities.

Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in prokaryotic and eukaryotic cells. As strong agonists of the stimulator of interferon genes, they are of great interest for pharmaceutical applications. In particular, cyclic-GMP-AMP and related synthetic CDNs are promising candidates in preclinical work and even some in clinical phase 1 and 2 studies. The comparison of chemical and biocatalytic synthesis routes elucidated that biological CDN synthesis offers some advantages, such as shorter synthesis time, avoiding complex protective group chemistry, and the access to a new spectrum of CDNs. However, the synthesis of CDNs in preparative quantities is still a challenge, since the chemical synthesis of CDNs suffers from low yields and complex synthetic routes and the enzymatically catalyzed synthesis is limited by low product titers and process stability. We aim to review the latest discoveries and recent trends in chemical and biocatalytic synthesis of CDNs with a focus on the synthesis of a huge variety of CDN derivatives. We furthermore consider the most promising biotechnological processes for CDN production by evaluating key figures of the currently known processes.

Cascades of Evolved Enzymes for the Synthesis of Complex Molecules.

Thanks to advances in enzyme discovery and protein engineering combined with the development of enzymatic multistep reaction cascades, new efficient routes for drug synthesis have been created that are superior to chemical syntheses. This supports the goal of the chemical and pharmaceutical industries to move to more sustainable and environmentally friendly processes. Recently described outstanding examples include the biocatalytic cascade syntheses of the cyclic dinucleotide MK-1454, molnupiravir, and islatravir, as well as the efficient fixation of CO2 to make starch using an artificial enzyme cascade.

Investigation of Vitamin D2 and Vitamin D3 Hydroxylation by Kutzneria albida.

The active vitamin D metabolites 25-OH-D and 1α,25-(OH)2 -D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D2 (VD2 ) and vitamin D3 (VD3 ) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25-OH-D2 and 25-OH-D3 was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100-fold higher product concentrations of 25-OH-D3 and tenfold higher concentrations of 25-OH-D2 after addition of 5 % (w/v) 2-hydroxypropyl ß-cyclodextrin (2-HPßCD) were reached. Besides the single-hydroxylation products, the human double-hydroxylation products 1,25-(OH)2 -D2 and 1,25-(OH)2 -D3 and various other potential single- and double-hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD2 and VD3 metabolites.

Recombinant expression and characterization of novel P450s from Actinosynnema mirum.

Cytochrome P450 monooxygenases (P450s) are the major contributor in the metabolism of xenobiotics, including therapeutic agents. Thus, P450s find broad application in the pharmaceutical industry to synthesize metabolites of new active pharmaceutical ingredients in order to evaluate toxicity and pharmacokinetics. As an alternative to human hepatic P450s, microbial P450s offer several advantages, such as an easier and more efficient heterologous expression as well as higher stability under process conditions. Recently, the wild-type strain Actinosynnema mirum has been reported to catalyze hydroxylation reactions with high activity on a broad range of substrates. In this study, one of these substrates, ritonavir, was used to analyze the transcriptional response of the wild-type strain. Analysis of the differential gene expression pattern allowed the assignment of genes potentially responsible for ritonavir conversion. Heterologous expression of these candidates and activity testing led to the identification of a novel P450 that efficiently converts ritonavir resembling the activity of the human CYP3A4.

Environmental Assessment of Enzyme Production and Purification.

The importance of bioprocesses has increased in recent decades, as they are considered to be more sustainable than chemical processes in many cases. E factors can be used to assess the sustainability of processes. However, it is noticeable that the contribution of enzyme synthesis and purification is mostly neglected. We, therefore, determined the E factors for the production and purification of 10 g enzymes. The calculated complete E factor including required waste and water is 37,835 gwaste·genzyme-1. This result demonstrates that the contribution of enzyme production and purification should not be neglected for sustainability assessment of bioprocesses.

Biosynthetic Plasticity Enables Production of Fluorinated Aurachins.

Enzyme promiscuity has important implications in the field of biocatalysis. In some cases, structural analogues of simple metabolic building blocks can be processed through entire pathways to give natural product derivatives that are not readily accessible by chemical means. In this study, we explored the plasticity of the aurachin biosynthesis pathway with regard to using fluoro- and chloroanthranilic acids, which are not abundant in the bacterial producers of these quinolone antibiotics. The incorporation rates of the tested precursor molecules disclosed a regiopreference for halogen substitution as well as steric limitations of enzymatic substrate tolerance. Three previously undescribed fluorinated aurachin derivatives were produced in preparative amounts by fermentation and structurally characterized. Furthermore, their antibacterial activities were evaluated in comparison to their natural congener aurachin D.

Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2'-3'-Linked Cyclic Dinucleotides.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that catalyzes the synthesis of the cyclic GMP-AMP dinucleotide 2'3'-cGAMP. 2'3'-cGAMP functions as inducer for the production of type I interferons. Derivatives of this important second messenger are highly valuable for pharmaceutical applications. However, the production of these analogues requires complex, multistep syntheses. Herein, human cGAS is shown to react with a series of unnatural nucleotides, thus leading to novel cyclic dinucleotides. Most substrate derivatives with modifications at the nucleobase, ribose, and the α-thio phosphate were accepted. These results demonstrate the catalytic promiscuity of human cGAS and its utility for the biocatalytic synthesis of cyclic dinucleotide derivatives.

A Gram-Scale Limonene Production Process with Engineered Escherichia coli.

Monoterpenes, such as the cyclic terpene limonene, are valuable and important natural products widely used in food, cosmetics, household chemicals, and pharmaceutical applications. The biotechnological production of limonene with microorganisms may complement traditional plant extraction methods. For this purpose, the bioprocess needs to be stable and ought to show high titers and space-time yields. In this study, a limonene production process was developed with metabolically engineered Escherichia coli at the bioreactor scale. Therefore, fed-batch fermentations in minimal medium and in the presence of a non-toxic organic phase were carried out with E. coli BL21 (DE3) pJBEI-6410 harboring the optimized genes for the mevalonate pathway and the limonene synthase from Mentha spicata on a single plasmid. The feasibility of glycerol as the sole carbon source for cell growth and limonene synthesis was examined, and it was applied in an optimized fermentation setup. Titers on a gram-scale of up to 7.3 g·Lorg-1 (corresponding to 3.6 g·L-1 in the aqueous production phase) were achieved with industrially viable space-time yields of 0.15 g·L-1·h-1. These are the highest monoterpene concentrations obtained with a microorganism to date, and these findings provide the basis for the development of an economic and industrially relevant bioprocess.

The Development of Biocatalysis as a Tool for Drug Discovery.

Enzymes are versatile biocatalysts capable of performing selective reactions. The advantages of enzymes in comparison to classical chemistry including chemical catalysts are the generally milder process conditions and avoidance of harmful reactants. Their high selectivity and specificity are especially beneficial for the enzymatic synthesis of new products with potential applications in drug research. Therefore, in the past decades, the utilization of isolated enzymes or whole-cell biocatalysts has spread through a growing number of biotechnological industries. The applications comprise the production of chiral building blocks for the pharmaceutical and fine chemical industry, the enzymatic synthesis of drug metabolites for testing of toxicity, function, biological activity, degradation and the production of biocatalytically modified natural products, which all play a role in drug discovery. Especially Oreste Ghisalba's contributions, which paved the way for the industrial use of enzymes, will be considered in this review.

Recent advances in heme biocatalysis engineering.

Heme enzymes have the potential to be widely used as biocatalysts due to their capability to perform a vast variety of oxidation reactions. In spite of their versatility, the application of heme enzymes was long time-limited for the industry due to their low activity and stability in large scale processes. The identification of novel natural biocatalysts and recent advances in protein engineering have led to new reactions with a high application potential. The latest creation of a serine-ligated mutant of BM3 showed an efficient transfer of reactive carbenes into C═C bonds of olefins reaching total turnover numbers of more than 60,000 and product titers of up to 27 g/L-1 . This prominent example shows that heme enzymes are becoming competitive to chemical syntheses while being already advantageous in terms of high yield, regioselectivity, stereoselectivity and environmentally friendly reaction conditions. Advances in reactor concepts and the influencing parameters on reaction performance are also under investigation resulting in improved productivities and increased stability of the heme biocatalytic systems. In this mini review, we briefly present the latest advancements in the field of heme enzymes towards increased reaction scope and applicability.

Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis.

The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski's horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.

A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

The First Biocatalytic Carbon-Silicon Bond Formation.

Linking two worlds: As a further expansion of the biocatalytic repertoire, carbene insertion into Si-H bonds catalyzed by the heme protein cytochrome c was recently reported. This new biocatalyst holds great promise because it enables the highly selective incorporation of silicon into molecules without prior protection of existing functional groups.

Human FMO2-based microbial whole-cell catalysts for drug metabolite synthesis.

BACKGROUND: Getting access to authentic human drug metabolites is an important issue during the drug discovery and development process. Employing recombinant microorganisms as whole-cell biocatalysts constitutes an elegant alternative to organic synthesis to produce these compounds. The present work aimed for the generation of an efficient whole-cell catalyst based on the flavin monooxygenase isoform 2 (FMO2), which is part of the human phase I metabolism. RESULTS: We show for the first time the functional expression of human FMO2 in E. coli. Truncations of the C-terminal membrane anchor region did not result in soluble FMO2 protein, but had a significant effect on levels of recombinant protein. The FMO2 biocatalysts were employed for substrate screening purposes, revealing trifluoperazine and propranolol as FMO2 substrates. Biomass cultivation on the 100 L scale afforded active catalyst for biotransformations on preparative scale. The whole-cell conversion of trifluoperazine resulted in perfectly selective oxidation to 48 mg (46% yield) of the corresponding N (1)-oxide with a purity >98%. CONCLUSIONS: The generated FMO2 whole-cell catalysts are not only useful as screening tool for human metabolites of drug molecules but more importantly also for their chemo- and regioselective preparation on the multi-milligram scale.

Complementation of biotransformations with chemical C-H oxidation: copper-catalyzed oxidation of tertiary amines in complex pharmaceuticals.

The isolation, quantitation, and characterization of drug metabolites in biological fluids remain challenging. Rapid access to oxidized drugs could facilitate metabolite identification and enable early pharmacology and toxicity studies. Herein, we compared biotransformations to classical and new chemical C-H oxidation methods using oxcarbazepine, naproxen, and an early compound hit (phthalazine 1). These studies illustrated the low preparative efficacy of biotransformations and the inability of chemical methods to oxidize complex pharmaceuticals. We also disclose an aerobic catalytic protocole (CuI/air) to oxidize tertiary amines and benzylic CH's in drugs. The reaction tolerates a broad range of functionalities and displays a high level of chemoselectivity, which is not generally explained by the strength of the C-H bonds but by the individual structural chemotype. This study represents a first step toward establishing a chemical toolkit (chemotransformations) that can selectively oxidize C-H bonds in complex pharmaceuticals and rapidly deliver drug metabolites.

Amino acid balancing for the prediction and evaluation of protein concentrations in cell-free protein synthesis systems.

Cell-free protein synthesis (CFPS) systems are an attractive method to complement the usual cell-based synthesis of proteins, especially for screening approaches. The literature describes a wide variety of CFPS systems, but their performance is difficult to compare since the reaction components are often used at different concentrations. Therefore, we have developed a calculation tool based on amino acid balancing to evaluate the performance of CFPS by determining the fractional yield as the ratio between theoretically achievable and experimentally achieved protein molar concentration. This tool was applied to a series of experiments from our lab and to various systems described in the literature to identify systems that synthesize proteins very efficiently and those that still have potential for higher yields. The well-established Escherichia coli system showed a high efficiency in the utilization of amino acids, but interestingly, less considered systems, such as those based on Vibrio natriegens or Leishmania tarentolae, also showed exceptional fractional yields of over 70% and 90%, respectively, implying very efficient conversions of amino acids. The methods and tools described here can quickly identify when a system has reached its maximum or has limitations. We believe that this approach will facilitate the evaluation and optimization of existing CFPS systems and provides the basis for the systematic development of new CFPS systems.

Comparative Life Cycle Assessment of Chemical and Biocatalytic 2'3'-Cyclic GMP-AMP Synthesis.

Life cycle assessments (LCAs) can provide insights into the environmental impact of production processes. In this study, a comparative LCA was performed for the synthesis of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) in an early development stage. The cyclic dinucleotide (CDN) is of interest for pharmaceutical applications such as cancer immunotherapy. CDNs can be synthesized either by enzymes or chemical catalysis. It is not known which of the routes is more sustainable as both routes have their advantages and disadvantages, such as a poor yield for the chemical synthesis and low titers for the biocatalytic synthesis. The synthesis routes were compared for the production of 200 g 2'3'-cGAMP based on laboratory data to assess the environmental impacts. The biocatalytic synthesis turned out to be superior to the chemical synthesis in all considered categories by at least one magnitude, for example, a global warming potential of 3055.6 kg CO2 equiv. for the enzymatic route and 56454.0 kg CO2 equiv. for the chemical synthesis, which is 18 times higher. This study demonstrates the value of assessment at an early development stage, when the choice between different routes is still possible.