Engineering a Cytidine Aminotransferase for Biocatalytic Production of the Antiviral Molnupiravir

The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of a cytidine aminotransferase for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >100,000 turnovers in less than 30 minutes, operates at 180 g/L substrate loading and benefits from in situ crystallization of the N-hydroxy-cytidine product (>90% yield), which can be converted to Molnupiravir by a selective 5’-acylation using Novozym® 435.

Identification of copper-containing oxidoreductases in the secretomes of three Colletotrichum species with a focus on copper radical oxidases for the biocatalytic production of fatty aldehydes

Copper Radical Alcohol Oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time-course analyses of the secretomes of three Colletotrichum species ( C. graminicola , C. tabacum and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29-52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11-AA13, AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavors and fragrances ingredients. CRO-AlcOx have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial application to alleviate various costs pertaining to biocatalysts production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative-stressful conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, LPMOs) usually assigned to “plant-cell wall degradation”, despite the absence of any plant-biomass mimicking compound, and only little amount of CRO-AlcOx were secreted, pointing out at recombinant expression as the most promising path for their biocatalytic application.

The Biocatalytic Production of 3-Hydroxypropionaldehyde and Evaluation of Its Stability

3-Hydroxypropionaldehyde (3-HPA, reuterin) is a broad-spectrum natural antimicrobial agent used in the food industry and other fields. The low yield from the industrial production of 3-HPA using Lactobacillus reuteri and the spontaneous conversion of 3-HPA to acrolein have limited its more widespread use. We isolated L. reuteri BR201 as a biocatalyst for 3-HPA production and confirmed the effect of each factor in the two-step procedure for 3-HPA bioconversion. After initial cultivation for 8 h (late exponential phase), this isolate produced 378 mM of 3-HPA in 1 h at a concentration of OD600 nm 100, 30 °C, and an initial glycerol concentration of 500 mM. This is the highest reported biocatalytic yield of 3-HPA from a glycerol aqueous solution without additives. We confirmed that 4 mM of 3-HPA had antimicrobial activity against five pathogens. The degradation of 3-HPA to acrolein was greater at high temperatures, and there was little degradation when 3-HPA was maintained at 4 °C for 4 weeks. Our results may be useful for future applications of 3-HPA.

Biocatalytic Conversion of Short-Chain Fatty Acids to Corresponding Alcohols in Escherichia coli

Advanced biofuels possess superior characteristics to serve for gasoline substitutes. In this study, a whole cell biocatalysis system was employed for production of short-chain alcohols from corresponding fatty acids. To do so, Escherichia coli strain was equipped with a biocatalytic pathway consisting of endogenous atoDA and Clostridium acetobutylicum adhE2. The strain was further reprogrammed to improve its biocatalytic activity by direction the glycolytic flux to acetyl-CoA and recycling acetate. The production of 1-propanol and n-pentanol were exemplified with the engineered strain. By substrate (glucose and propionate) feeding, the strain enabled production of 5.4 g/L 1-propanol with productivity reaching 0.15 g/L/h. In addition, the strain with a heavy inoculum was implemented for the n-pentanol production from n-pentanoic acid. The production titer and productivity finally attained 4.3 g/L and 0.86 g/L/h, respectively. Overall, the result indicates that this developed system is useful and effective for biocatalytic production of short-chain alcohols.

Prospects of Using Biocatalysis for the Synthesis and Modification of Polymers

Trends in the dynamically developing application of biocatalysis for the synthesis and modification of polymers over the past 5 years are considered, with an emphasis on the production of biodegradable, biocompatible and functional polymeric materials oriented to medical applications. The possibilities of using enzymes not only as catalysts for polymerization but also for the preparation of monomers for polymerization or oligomers for block copolymerization are considered. Special attention is paid to the prospects and existing limitations of biocatalytic production of new synthetic biopolymers based on natural compounds and monomers from biomass, which can lead to a huge variety of functional biomaterials. The existing experience and perspectives for the integration of bio- and chemocatalysis in this area are discussed.