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AN INNOVATIVE MICROBIAL CELL FACTORY FOR NATURAL VANILLIN

Periodic Reporting for period 1 - BELLOSSOM (AN INNOVATIVE MICROBIAL CELL FACTORY FOR NATURAL VANILLIN)

Reporting period: 2020-10-01 to 2022-01-31

EV Biotech develops sustainable microbial production methods for fine chemicals. The company has created the first computational pipeline that merges biology with computational modelling and artificial intelligence (AI) to create one-of-a-kind microbial production strains. Before starting any lab experiments, the Dry Lab team models and predicts which type of microorganism and genetic modifications will give the highest yield and quality. The Dry Lab, a virtual laboratory, saves time, resources and energy and can generate a limitless repertoire of microbial production strains for different compounds.

EV Biotech is developing different proof-of-concept microbial production strains, one of which is for vanillin. Vanilla is one of the most expensive spices worldwide and is a popular ingredient in fragrances and the food industry. Due to vanilla’s high price, chemically synthesised vanillin (the main compound in vanilla) is used as an alternative, but the market demand for chemically synthesised vanillin is declining due to excessive use of fossil resources and solvents that severely impact the environment. However, natural vanilla could never meet the demand due to the limited production capacity, natural disasters, and high costs. There is thus a need for a cheap and scalable production method for vanillin of biological origin, which can be realised by microbial production.

In microbial production strain design, it is important to design a strain that produces a significant amount of the desired product. To get from a starting block like glucose to the end product - in this case vanillin - many proteins are involved with a specific function. Sometimes such a function needs to be improved, removed or changed. The search space (number of possible mutations) depends on the size of the protein. Bigger proteins increase the search space, making this task impossible to perform in the lab for more than one protein. In order to design specific proteins that improve the strain, molecular modelling techniques were developed. Many of these tools are based on molecular dynamics (MD), which is a computer simulation method for analysing the physical movements of atoms and molecules. This technique allows us to analyse each possible mutation in a relatively small time compared with the lab procedure. Thus, we can design a protein in less than a week instead of years of work in the lab.

The main objective of this project was to design a molecular dynamics modelling infrastructure (platform) for proteins integrated into the current microbial strain development workflow. The platform was then used for optimisation of a vanillin-producing microbial strain that is efficient, cheap, environmentally friendly, and thus competitive with the chemical synthesis of vanillin from fossil resources.
The result of this project is a platform with several computational tools which can be applied in any protein design project, not just microbial vanillin production. These tools include modules that generate the 3D structure of proteins from incomplete experimental data, aid in improving protein solubility, measure thermostability, analyse MD trajectories, and several user interfaces that ensure that the platform can be used by non-experts. Each module can connect to the others to generate a pipeline and evaluate thousands of potential mutants. The platform can be applied to design any protein depending on the function that is required.
The project has not only resulted in an MD platform that can process many structures for protein design, but also in other tools like a thermostability measure that can evaluate thousand of mutations in just days. These tools allow design of proteins that can improve the microbial production strains used to create green fine chemicals. We expect that the platform can be further improved by applying machine learning methods, in order to create novel proteins and thus improve the strains even more. New production methods created with this technique can be designed in months rather than in years, thereby increasing the number of green fine chemicals in the market and thus contributing to a more sustainable chemical industry and society at large.