Project description:BackgroundWith the development of metabolic engineering and synthetic biology, the biosynthesis of aromatic compounds has attracted much attention. Cinnamylamine is an aromatic compound derived from L-phenylalanine, which is used in the synthesis of biologically active molecules, including drugs, and energetic materials. Cinnamylamine has been mainly synthesized by chemical methods to date, and few reports have focused on the biosynthesis of cinnamylamine. Therefore, it is desirable to establish an efficient biosynthesis method for cinnamylamine.ResultsThe ω-aminotransferase Cv-ωTA from Chromobacterium violaceum has been demonstrated to have high enzyme activity in the conversion of cinnamaldehyde to cinnamylamine. To prevent the preferable conversion of cinnamaldehyde to cinnamyl alcohol in wild-type Escherichia coli, the E. coli MG1655 strain with reduced aromatic aldehyde reduction (RARE) in which six aldehyde ketone reductase and alcohol dehydrogenase genes have been knocked out was employed. Then, the carboxylic acid reductase from Neurospora crassa (NcCAR) and phosphopantetheinyl transferase (PPTase) from E. coli were screened for a high conversion rate of cinnamic acid to cinnamaldehyde. To shift the equilibrium of the reaction toward cinnamylamine, saturation mutagenesis of Cv-ωTA at key amino acid residues was performed, and Cv-ωTA Y168G had the highest conversion rate with 88.56 mg/L cinnamylamine obtained after 4 h of fermentation. Finally, by optimizing the substrates and the supply of the cofactors, PLP and NADPH, in the fermentation, the yield of cinnamylamine in engineered E. coli reached 523.15 mg/L.ConclusionWe achieved the first biosynthesis of cinnamylamine using cinnamic acid as the precursor in E. coli using a combinatorial metabolic engineering strategy. This study provides a reference for the biosynthesis of other amine compounds and lays a foundation for the de novo synthesis of cinnamylamine.
Project description:Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment-resistant depression and major depressive disorder. The psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot-scale protocol for recombinant PsiK that yielded 150 mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost.
Project description:A novel biocatalytic system to access a wide variety of β-hydroxydioxinones from β-ketodioxinones employing commercial engineered ketoreductases has been developed. This practical system provides a remarkably straightforward solution to limitations in accessing certain chemical scaffolds common in β-hydroxydioxinones that are of great interest due to their diversification capabilities. A few highlights of this system are that it is high yielding, highly enantioselective, and chromatography-free. We have demonstrated both a wide substrate scope and a high degree of scalability.
Project description:An enzyme-catalyzed synthesis of rhododendrol, an intermediate in the production of raspberry ketone, was investigated. The approach involves the enzymatic hydrolysis of rhododendrol glycosides into rhododendrol and a glycosidic residue. Rhododendrol glycosides, which are naturally derived from the inner bark of birch trees-a renewable resource-vary considerably in composition depending on the origin of the plants. In this study, mixtures of betuloside and apiosylrhododendrin from natural resources were used in different proportions. An in-depth study was conducted to assess the feasibility of the process. A mathematical model was developed based on studies of the kinetics and operational stability of the enzyme. The model for betuloside hydrolysis catalyzed by β-glucosidase was validated in batch, repetitive batch, and ultrafiltration membrane reactors. The highest productivity, ranging from 83.9 to 94.5 g L-1 day-1, was achieved in the latter. After screening nearly 50 enzymes, RAPIDASE emerged as a solution for the hydrolysis of apiosylrhododendrin, and the model was validated in a batch reactor. Model-based optimization enabled the prediction of input parameters for different compositions of biogenic rhododendrol glycosides to obtain consistent process output metrics.
Project description:Mechanism-based kinetic models are rigorous tools to analyze enzymatic reactions, but their extension to actual conditions of the biocatalytic synthesis can be difficult. Here, we demonstrate (mechanistic-empirical) hybrid modeling for systematic optimization of the sucrose phosphorylase-catalyzed glycosylation of glycerol from sucrose, to synthesize the cosmetic ingredient α-glucosyl glycerol (GG). The empirical model part was developed to capture nonspecific effects of high sucrose concentrations (up to 1.5 M) on microscopic steps of the enzymatic trans-glycosylation mechanism. Based on verified predictions of the enzyme performance under initial rate conditions (Level 1), the hybrid model was expanded by microscopic terms of the reverse reaction to account for the full-time course of GG synthesis (Level 2). Lastly (Level 3), the application of the hybrid model for comprehensive window-of-operation analysis and constrained optimization of the GG production (~250 g/L) was demonstrated. Using two candidate sucrose phosphorylases (from Leuconostoc mesenteroides and Bifidobacterium adolescentis), we reveal the hybrid model as a powerful tool of "process decision making" to guide rational selection of the best-suited enzyme catalyst. Our study exemplifies a closing of the gap between enzyme kinetic models considered for mechanistic research and applicable in technologically relevant reaction conditions; and it highlights the important benefit thus realizable for biocatalytic process development.
Project description:Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.
Project description:Aliphatic α,ω-dicarboxylic acids (DCAs) are a class of useful chemicals that are currently produced by energy-intensive, multistage chemical oxidations that are hazardous to the environment. Therefore, the development of environmentally friendly, safe, neutral routes to DCAs is important. We report an in vivo artificially designed biocatalytic cascade process for biotransformation of cycloalkanes to DCAs. To reduce protein expression burden and redox constraints caused by multi-enzyme expression in a single microbe, the biocatalytic pathway is divided into three basic Escherichia coli cell modules. The modules possess either redox-neutral or redox-regeneration systems and are combined to form E. coli consortia for use in biotransformations. The designed consortia of E. coli containing the modules efficiently convert cycloalkanes or cycloalkanols to DCAs without addition of exogenous coenzymes. Thus, this developed biocatalytic process provides a promising alternative to the current industrial process for manufacturing DCAs.
Project description:Ribonucleosides are essential building blocks used extensively in antiviral and oligonucleotide therapeutics. A major challenge in the further development of nucleoside analogues for therapeutic applications is access to scalable and environmentally sustainable synthetic strategies. This study uses the type II nucleoside 2'-deoxyribosyltransferase from Lactobacillus leichmannii (LlNDT-2) to prepare a suite of ribonucleoside analogues using naturally-occurring uridine and cytidine sugar donors. Crystal structure and mutational analyses are used to define the substrate tolerance of the nucleobase exchange and the 2'-substituent of the nucleoside sugar donor. Nucleobase profiling identified acceptance of both purine and pyrimidine nucleobases. Finally, the scalability of the approach is showcased, enabling the preparation of ribonucleosides on millimolar scales. This biocatalytic strategy opens up opportunities to establish chemoenzymatic routes to prepare nucleoside analogues incorporating 2' modifications that are of therapeutic importance.
Project description:Peptidic natural products (PNPs) represent a rich source of lead compounds for the discovery and development of therapeutic agents for the treatment of a variety of diseases. However, the chemical synthesis of PNPs with diverse modifications for drug research is often faced with significant challenges, including the unavailability of constituent nonproteinogenic amino acids, inefficient cyclization protocols, and poor compatibility with other functional groups. Advances in the understanding of PNP biosynthesis and biocatalysis provide a promising, sustainable alternative for the synthesis of these compounds and their analogues. Here we discuss current progress in using native and engineered biosynthetic enzymes for the production of both ribosomally and nonribosomally synthesized peptides. In addition, we highlight new in vitro and in vivo approaches for the generation and screening of PNP libraries.