Design of novel biosynthetic pathways for the production of industrially valuable chemicals

Abstract

Metabolic engineering has gained much recognition as an alternative to conventional petrochemical processes by producing industrially valuable chemicals through genetically engineered microorganisms. The grand objective of metabolic engineering is to reduce the reliance of modern societies on the petroleum and its refinery processes that have caused devastating environmental effects. Although state of the art gene manipulation techniques keep emerging, enabling better control of the microbial phenotypes, it is still considerably difficult to determine which biosynthetic pathways to use for the overproduction of a target chemical. This problem becomes more complex when it comes down to the production of chemicals not producible in the widely used microbial hosts (e.g., Escherichia coli and Saccharomyces cerevisiae). To handle this problem, several computational systems that design novel biological pathways have been introduced Biochemical Network Integrated Computational Explorer (BNICE), DESHARKY and RetroPath. In the case of the BNICE, it was implemented to identify a wide range of possible reactions, including not only previously known reactions, but also novel ones for the biosynthesis of 3-hydroxypropanoate (3-HP), a high-value chemical in industry. However, many computational platform systems, including the BNICE, generate a tremendous number of candidate biosynthetic pathways based on the nature of their reaction rules, without systematic prioritization of the predicted pathways for their feasibility in a real cell. A large number of the generated candidate biosynthetic pathways are extremely difficult to compare with one another, and prioritize. In order to resolve this issue, we developed a pathway prediction systems called EnzMatcher, which not only generates novel biosynthetic pathways to produce non-natural chemicals based on the chemical reaction mechanism, but also examines their feasibility and efficiency by considering two prioritization factors, namely structural similarities of the involved chemicals and thermodynamic favorability. We demonstrate prediction capacity of the platform by generating biosynthetic pathways for the two industrially important chemicals, 6-aminocaproic and acrylic acids. Also, we show that this pathway designing system can suggest biosynthetic pathways of several target chemicals, which are known to be impossible to produce in biological system, such as primary amines, 2-butanone, three branched-carboxylic acids

isobutyrate, isovalerate and 2-methylbutyrate. In this work, we present the successful case study of design, appropriate selection and experimental validation process of novel biosynthetic pathways by in vivo production of non-natural chemicals including primary amines, 2-butanone, isobutyrate, isovalerate and 2-methylbutyrate, all industrially important chemicals and some of which is not reported to be produced in microorganisms. This pathway generation system should be a useful tool in systems metabolic engineering