Synthetic biology approach to engineer and compartmentalise methanol utilisation pathway in E. coli. – ECOMUT

To carry out this project, we combined synthetic biology approach with a systems level understanding of methylotrophy. Specifically, we use model as the starting point to analyse metabolism and ‘omics’ technologies (of that system biology consists). we learn mostly from comparing predictions generated through modelling with experiments. The results might be congruent and models might confirm experiments and vice versa. However, we are also looking forward to discrepancies between predictions and data from which we will be able to gain new scientific insight into methylotrophy. Comparing synthetic methylotrophs with known natural methylotrophs as the benchmark will teach us how far advanced our biological knowledge in the field is. We are also exploring and testing “new” solutions for instance by creating modules composed of enzymes so far not known to act together in a pathway (of that synthetic biology consists) ; additionally, we apply directed evolution strategies. Such experiments will allow new insights into enzymatic steps and regulatory phenomena involved in methylotrophy.

Based on genomics and experimental knowledge from different natural methylotrophic organisms, in silico modeling was used to design ideal combinations of genes and minimal sets of modules allowing methylotrophy to occur in non methylotrophic host. From this analysis, we selected a synthetic pathway composed by two enzymes, i.e. a methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das), which ultimately produce dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P), metabolites which can enter the glycolysis. We then screened a combinatorial library of Mdh and Das orthologs (256 combinations in total) and applied high-throughput 13C labelling experiments to evaluate the functionality in vivo of these combinations and select the best one. Then, the carbon flux through the biosynthetic pathway was increased by knocking out and/or overexpressing key enzymes of the DHA metabolism. Finally the synthetic methylotrophic strain was subjected to long-term adaptive evolution in order to select variants able to grow solely on methanol as carbon and energy source.

In this project, we will not only generate new a fundamental knowledge of C1 metabolism but will also provide a biological platform capable of transforming methanol in any molecule of interest. This research will make a significant contribution towards unleashing the potential of methanol as a raw material in a vast range biotechnological applications in any industrialised location.

For the moment, this work lead to the publication of one article, one patent, 3 oral communications and one poster presentation.

With a worldwide production capacity of more than 100 million tons and a decreasing commodity price, methanol is regarded as a highly attractive alternative non-food raw material for biotechnology sector. The supply of methanol comes from both fossil and renewable resources, rendering it a highly flexible and sustainable raw material. C1 compounds are used by specialized groups of microorganisms i.e. the methylotrophs as their sole source of carbon and energy. While progress to use natural methylotrophs in biotechnology is on-going, ECOMUT propose to launch an alternative strategy by integrating methylotrophy into the established bacterial production host Escherichia coli. The synthetic biology approach we plan will be combined with a systems level understanding of methylotrophy. This will not only generate new a fundamental knowledge of C1 metabolism but will also provide a biological platform capable of transforming methanol in any molecule of interest. This research will make a significant contribution towards unleashing the potential of methanol as a raw material in a vast range biotechnological applications in any industrialised location.