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.

Industrially Relevant Enzyme Cascades for Drug Synthesis and Their Ecological Assessment.

Environmentally friendly and sustainable processes for the production of active pharmaceutical ingredients (APIs) gain increasing attention. Biocatalytic synthesis routes with enzyme cascades support many stated green production principles, for example, the reduced need for solvents or the biodegradability of enzymes. Multi-enzyme reactions have even more advantages such as the shift of the equilibrium towards the product side, no intermediate isolation, and the synthesis of complex molecules in one reaction pot. Despite the intriguing benefits, only a few enzyme cascades have been applied in the pharmaceutical industry so far. However, several new enzyme cascades are currently being developed in research that could be of great importance to the pharmaceutical industry. Here, we present multi-enzymatic reactions for API synthesis that are close to an industrial application. Their performances are comparable or exceed their chemical counterparts. A few enzyme cascades that are still in development are also introduced in this review. Economic and ecological considerations are made for some example cascades to assess their environmental friendliness and applicability.

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.

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.

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.

Estimating environmental impacts of early-stage bioprocesses.

The use of bioprocesses in industrial production promises resource- and energy-efficient processes starting from renewable, nonfossil feedstocks. Thus, the environmental benefits must be demonstrated, ideally in the early development phase with standardized methods such as life cycle assessment (LCA). Herein we discuss selected LCA studies of early-stage bioprocesses, highlighting their potential and contribution to estimating environmental impacts and decision support in bioprocess development. However, LCAs are rarely performed among bioprocess engineers due to challenges such as data availability and process uncertainties. To address this issue, recommendations are provided for conducting LCAs of early-stage bioprocesses. Opportunities are identified to facilitate future applicability, for example, by establishing dedicated bioprocess databases that could enable the use of LCAs as standard tools for bioprocess engineers.

Bivariate One Strain Many Compounds Designs Expand the Secondary Metabolite Production Space in Corallococcus coralloides.

The scarcely investigated myxobacterium Corallococcus coralloides holds a large genome containing many uncharacterized biosynthetic gene clusters (BGCs) that potentially encode the synthesis of entirely new natural products. Despite its promising genomic potential, suitable cultivation conditions have not yet been found to activate the synthesis of new secondary metabolites (SMs). Finding the right cultivation conditions to activate BGCs in the genome remains a major bottleneck, and its full biosynthetic potential has so far not been determined. We therefore applied a bivariate "one strain many compounds" (OSMAC) approach, using a combination of two elicitor changes at once, for the activation of BGCs and concomitant SM production by C. coralloides. The screening was carried out in Duetz-System 24-well plates, applying univariate and bivariate OSMAC conditions. We combined biotic additives and organic solvents with a complex growth medium for univariate conditions and with minimal medium for bivariate conditions. The success in the activation of BGCs was evaluated by determining the number of new mass features detected in the respective extracts. We found synergistic effects in the bivariate OSMAC designs, evidenced by the detection of completely new mass features in the bivariate OSMAC experiments, which were not detected in the univariate OSMAC designs with only one elicitor. Overall, the bivariate OSMAC screening led to 55 new mass features, which were not detected in the univariate OSMAC design. Molecular networks revealed that these new mass features embody potential novel natural compounds and chemical derivatives like the N-acyl fatty amine N-pentyloctadecanamide and possibly sulfur-containing natural products. Hence, the presence of multiple elicitors in the bivariate OSMAC designs successfully activated the biosynthetic potential in C. coralloides. We propose bivariate OSMAC designs with a complex combination of elicitors as a straightforward strategy to robustly expand the SM space of microorganisms with large genomes.

Long-term follow-up after rituximab for steroid-dependent idiopathic nephrotic syndrome.

BACKGROUND: In patients with refractory steroid-sensitive nephrotic syndrome (SSNS), treatment with rituximab has shown encouraging results; however, long-term follow-up data are not available. METHODS: We performed a retrospective analysis of 37 patients (25 boys) with steroid-dependent nephrotic syndrome who were treated with rituximab (375 mg/m(2) given weekly for one to four courses). Long-term follow-up data (>2 years, median 36, range 24-92.8 months) are available for 29 patients (12 boys). RESULTS: Twenty-six of 37 (70.3%) patients remained in remission after 12 months. Relapses occurred in 24 (64.8%) patients after a median of 9.6 (range 5.2-64.1) months. Time to first relapse was significantly shorter in patients receiving one or two compared to three or four initial infusions. In the 29 patients with long-term follow-up for >2 years, 12 (41%) patients remained in remission after the initial rituximab course for >24 months, 7 (24.1%) patients without further maintenance immunosuppression. Nineteen children received two to four repeated courses of rituximab increasing the total number of patients with long-term remission to 20 (69%), remission including 14 (48%) patients off immunosuppression. The proportion of patients with long-term remission was not related to the number of initial rituximab applications. No serious side effects were noted. CONCLUSION: Rituximab is an effective treatment option in the short- and long-term control of treatment refractory SSNS. Further controlled studies are needed to address optimal patient selection, dose and safety of rituximab infusions.

Recent Advances in Biocatalysis for Drug Synthesis.

Biocatalysis is constantly providing novel options for the synthesis of active pharmaceutical ingredients (APIs). In addition to drug development and manufacturing, biocatalysis also plays a role in drug discovery and can support many active ingredient syntheses at an early stage to build up entire scaffolds in a targeted and preparative manner. Recent progress in recruiting new enzymes by genome mining and screening or adapting their substrate, as well as product scope, by protein engineering has made biocatalysts a competitive tool applied in academic and industrial spheres. This is especially true for the advances in the field of nonribosomal peptide synthesis and enzyme cascades that are expanding the capabilities for the discovery and synthesis of new bioactive compounds via biotransformation. Here we highlight some of the most recent developments to add to the portfolio of biocatalysis with special relevance for the synthesis and late-stage functionalization of APIs, in order to bypass pure chemical processes.

Identification and Expression of New Unspecific Peroxygenases - Recent Advances, Challenges and Opportunities.

In 2004, the fungal heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs) was first described in the basidiomycete Agrocybe aegerita. As UPOs naturally catalyze a broad range of oxidative transformations by using hydrogen peroxide as electron acceptor and thus possess a great application potential, they have been extensively studied in recent years. However, despite their versatility to catalyze challenging selective oxyfunctionalizations, the availability of UPOs for potential biotechnological applications is restricted. Particularly limiting are the identification of novel natural biocatalysts, their production, and the description of their properties. It is hence of great interest to further characterize the enzyme subfamily as well as to identify promising new candidates. Therefore, this review provides an overview of the state of the art in identification, expression, and screening approaches of fungal UPOs, challenges associated with current protein production and screening strategies, as well as potential solutions and opportunities.

Triaging of Culture Conditions for Enhanced Secondary Metabolite Diversity from Different Bacteria.

Over the past decade, the one strain many compounds (OSMAC) approach has been established for the activation of biosynthetic gene clusters (BGCs), which mainly encode the enzymes of secondary metabolite (SM) biosynthesis pathways. These BGCs were successfully activated by altering various culture conditions, such as aeration rate, temperature, and nutrient composition. Here, we determined the biosynthetic potential of 43 bacteria using the genome mining tool antiSMASH. Based on the number of BGCs, biological safety, availability of deposited cultures, and literature coverage, we selected five promising candidates: Bacillus amyloliquefaciens DSM7, Corallococcus coralloides DSM2259, Pyxidicoccus fallax HKI727, Rhodococcus jostii DSM44719, and Streptomyces griseochromogenes DSM40499. The bacteria were cultivated under a broad range of OSMAC conditions (nutrient-rich media, minimal media, nutrient-limited media, addition of organic solvents, addition of biotic additives, and type of culture vessel) to fully assess the biosynthetic potential. In particular, we investigated so far scarcely applied OSMAC conditions to enhance the diversity of SMs. We detected the four predicted compounds bacillibactin, desferrioxamine B, myxochelin A, and surfactin. In total, 590 novel mass features were detected in a broad range of investigated OSMAC conditions, which outnumber the predicted gene clusters for all investigated bacteria by far. Interestingly, we detected mass features of the bioactive compounds cyclo-(Tyr-Pro) and nocardamin in extracts of DSM7 and DSM2259. Both compounds were so far not reported for these strains, indicating that our broad OSMAC screening approach was successful. Remarkably, the infrequently applied OSMAC conditions in defined medium with and without nutrient limitation were demonstrated to be very effective for BGC activation and for SM discovery.