Systems biology of bacterial methylotrophy for biotechnological products from methanol

Microorganisms are widely used in industrial biotechnology for sustainable production of medicines, biomaterials, food, and health products. The raw materials used are dominantly sugars and molasses derived from plants and thus requiring use of cultivable land in competition with food production. Methanol can be produced from CO2 and natural gas, and it is regarded an alternative and potentially attractive raw material for industrial biotechnology. The methylotrophs are specialized bacteria that can utilize methanol as raw material, and these bacteria thus have potential in industrial biotechnology to make useful products from methanol. MetAPP has used a systems biology driven research approach on two different methylotrophic bacteria; Methylobacterium extorquens and Bacillus methanolicus, to develop genetically modified bacteria for sustainable conversion of methanol into useful chemicals. The focus for SINTEF has been research on the bacterium B. methanolicus, including establishment of up-scaled and industry-relevant cultivations in bioreactors, as well as design and construction of genetically modified mutants that overproduce high amounts of two industrially important compounds; cadaverine for uses in bioplastics production and GABA, which has several applications including in bioplastics and medicine. Totally, 10 previously not studied wild type isolates of B. methanolicus has been genome sequenced and the collected information has provided new genetic insight of methylotrophy, highly useful for better design of genetically modified mutants. The MetAPP research has also made a major contribution to extending the genetic toolbox, including new plasmids and adjustable promoter systems, and will thus have a high impact for further biotechnological applications of this organism in industrial biotechnology. Efforts were invested to establish CRISPR/Cas technology for B. methanolicus enabling targeted gene editing on the entire chromosome. This was not successful within the project period; however, useful experience, tools and methodologies were developed and brought further in the new ERA-project that MetAPP partners have established in the project period. When this eventually works, it will further widen the potential for bringing methylotrophic bacteria into important workhorses in industrial biotechnology. Summarized, MetAPP has made a major contribution to systems biology knowledge and methodologies, as well as development of methylotrophic mutant strains that overproduce two industrially important chemicals, cadaverine and GABA, from methanol.

A systems-level understanding of microorganisms is a prerequisite for their rational engineering and efficient use as microbial cell factories in biotechnology. There is a high societal need for a sustainable production of key chemistry, food and health care compounds. Methanol is abundant and regarded as alternative highly attractive non-feed raw material in microbial fermentation. Methylotrophy, the ability of microorganisms to use methanol as their sole source of carbon and energy for growth, bears the potential to build value from methanol through production of special, fine, bulk, and fuel chemicals. The US makes strong efforts towards utilizing shale gas: here we provide and European alternative. Nature evolved different solutions to harness methanol for the purpose of energy generation and biomass formation. These are reflected best by the two facultative methylotrophic model bacteria Methylobacterium extorquens and Bacillus methanolicus. M. extorquens is a mesophilic Gram-negative that shows flexible carbon source utilization of several one-carbon compounds and possess the serine cycle and ethylmalonyl-CoA pathway for carbon assimilation. It is intensely pigmented due to production of carotenoids. B. methanolicus is a thermophilic Gram-positive which possesses the ribulose monophosphate cycle as key pathway and is the first example of plasmid-dependent methylotrophy. B. methanolicus is a natural overproducer of amino acids, a trait that will be further exploited in this project. Our vision is the first application of systems biology to bacterial methylotrophy in order to gain systems-level understanding of evolutionary alternatives of a key metabolic trait. The project MetApp encompasses genome-scale modelling, quantitative multi-Omics and high-throughput genetic analysis, tests of orthogonality, data management, and model refinement and abstraction to deduce and experimentally evaluate strategies for methanol-based production of sought-after chemicals.