Benchmarking of Processes for the Biosynthesis of Natural Products

Natural products constitute an extensive family of organic molecules with more than 200,000 compounds discovered in several natural sources (plants and microbes). Most of these compounds have a very complex structure, multiple chiral centers and can have different biological activities. These biological activities can be of interest for use in different sectors of chemical industry, in particular pharmaceutical industry where several drugs are derived or inspired by natural products structure.

However, the large scale production of natural products is hindered by its relatively poor abundance in nature, which makes extraction from natural sources an economically unfeasible technology in most of the cases. Chemical synthesis is also very difficult given the structural complexity and chirality of the molecules.

Synthetic biology offers very promising tools for production of natural products. Genetic engineering allows expressing the genes responsible for the biosynthesis of these natural products and to insert them into fermentable organisms like yeast or bacteria. And protein engineering offers the possibility to engineer the enzymes that perform the natural products’ biosynthesis, which allows the production of these complex molecules in single or multi-steps biocatalytic reactions.

In this thesis, a systematic approach for route selection and screening of the different process options to manufacture a natural product is presented. This methodological approach includes a set of evaluation tools to assess processes both from an economic and environmental perspectives and it is demonstrated with two case-studies.

For each case-study different tools are used to evaluate the process. The first case-study consists of the bioconversion of (R)-limonene to (R)-perillic acid by Pseudomonas putida GS1. (R)-perillic acid is a monoterpenoic acid with antimicrobial properties. It has a strong inhibitory effect on bacteria and fungus, which makes it an attractive compound to be used as a preservative for instance in cosmetic industry, but on the other hand makes the biosynthesis a complicated process to develop.

An environmental assessment of the different synthetic routes for (R)-perillic acid production showed that biosynthesis represents the most promising option. This process was further explored through an economic assessment and process modelling including a sensitivity analysis on key process metrics, which allowed the identification of the main process bottlenecks.

Product inhibition and substrate loss were identified as some of the main process limitations and strategies for improving them were suggested. The second case-study describes the production of a recombinant protein, brazzein, by fermentation of the yeast Pichia pastoris. Brazzein is a natural sweetener found in the fruits of the African plant Pentadiplandra brazzena Baillon, which is sweeter than regular sucrose by several orders of magnitude.

Here different tools were applied to evaluate the environmental profile of the process in comparison with other sweeteners. The main benefit of this early-stage evaluation is putting the biosynthesis of natural products into context in relation to demands of an industrially feasible chemical process.

Moreover, it can give very meaningful insight into process development and provides a good overview of the whole reaction and process. The proposed in silico approach can guide research and development and ultimately contribute to the implementation of more bioprocesses for the production of natural products.