Methylorubrum extorquens – a microbial cell factory for production of chemicals from methanol

Methanol represents an attractive, very flexible feedstock for industrial biotechnology. It is efficiently synthesized via syngas from cheap natural gas and other fossil resources, but can also be derived from renewables such as wood or biogas. As a biotech feedstock, methanol does not interfere with the use of arable land for food and nutrition, and being a liquid it is easier to supply and control in bioprocesses than gaseous substrates such as syngas, methane, CO2 or H2. The host organism of our methylotrophic cell factory is Methylorubrum extorquens which assimilates methanol via the serine cycle, whereas the ethylmalonyl-CoA pathway (EMCP) is necessary for glyoxylate regeneration during growth on methanol. Intermediates of the EMCP include acetoacetyl-CoA and dicarboxylic acid CoA esters usually not found in primary metabolism of conventional host strains and represent starting intermediates for the synthesis of e.g. dicarboxylic acids and terpenoids.

SynBioTech - Synergistic development of biotechnological and chemical processes for value creation based on decentralized one-carbon compounds

In the SynBioTech joint project as part of the BioBall innovation area, biogenic CO₂ is hydrogenated to methanol in a chemical process step, which is then converted into biomass for the feed industry or acid derivatives for the chemical industry with the help of Methylorubrum extorquens strains.

The focus of the work of the DECHEMA-Forschungsinstitut is on characterizing and optimizing the robustness of the methylotrophic bacterium against process fluctuations. In particular, we are looking for mutants that can tolerate longer “hunger” phases better than previously available strains and are therefore also suitable for unsteady process conditions with possible methanol peaks and hunger phases. With the aim of coupling methanol synthesis and biotechnological implementation without laborious purification steps, the direct utilization of the methanol / water mixture is also analyzed and possible inhibiting components identified. Moreover, we aim at improving the growth rate and the medium and will test the developed processes on a laboratory scale.

Chiramet – Chiral building blocks produced from the biomass conversion product methanol

Chiral substances play an important role in the manufacture of fine chemicals, especially pharmaceuticals. The Chiramet project aims at the further development of possibilities for the biotechnological synthesis of enantiopure chemicals from methanol. With this biomass conversion product as starting material, an important contribution is made to the biologization of chemistry. Due to its availability from petrochemical and renewable resources, methanol is a very flexible raw material that, in the medium term, can very likely also be produced from residual and waste streams that are otherwise hardly usable, such as urban waste. Here, the microorganism Methylorubrum extorquens comes into play, which can grow with methanol as the sole carbon source. This allows to manufacture the products from methanol, which is easy to handle and toxic to most other microorganisms. In order to enable the biotechnological synthesis of various chiral substances from methanol, intermediates of a primary metabolic pathway are secreted by heterologously expressed thioesterases. Together with another academic partner and two companies, appropriate enzymes are identified and optimized. Further optimizations in the metabolism of the bacterium proposed by in silico models are carried out.  Production processes are carried out on a laboratory scale.

Production of ethylmalonyl-CoA pathway derived dicarboxylic acids mesaconic and (2S)-methylsuccinic acid from methanol by Methylobacterium extorquens

There is a high need for sustainably producible chemical building blocks being applicable e.g. as monomers for novel bioplastics. The ethylmalonyl-CoA pathway (EMCP) harbors several CoA-esters such as ethylmalonyl-, methylsuccinyl- or mesaconyl-CoA whose free dicarboxylic acid derivatives potentially present promising synthons for chemical industry. The EMCP in Methylobacterium extorquens offers the possibility to produce these new building blocks directly from the cheap and non-food competing C-source methanol.

Suitable thioesterases for production of different EMCP-derived dicarboxylic acids were identified by in vitro assays and their expression levels in M. extorquens optimized. Productivities of different strains were compared and increased by different metabolic engineering and medium optimization strategies. A current focus of this project is the identification of acid reuptake and utilization mechanisms. Altogether we aim to establish the biotechnological production of unconventional dicarboxylic acids from methanol with high efficiency.

Engineering Methylobacterium extorquens for production of the sesquiterpenoid α-humulene

Over the last 10 to 15 years, metabolic engineering of microbes has become a versatile tool for high-level de novo synthesis of terpenoids, with the sesquiterpenoids armopha-1,4-diene, farnesene and artemisinic acid as prime examples. However, almost all cell factory approaches towards terpenoids to date have been based on sugar as the raw material, which is mainly used as a food resource and subject to high price volatilities.

This project focuses on the de novo synthesis of the sesquiterpenoid α-humulene from the abundantly available non-food carbon source methanol by metabolically engineered Methylobacterium extorquens. We want to make use of the intermediate acetoacetyl-CoA, which is part of the central metabolism of the organism but also the starting metabolite of the terpene-producing mevalonate pathway. Expression of this pathway together with prenyltransferase and a plant α-humulene synthase resulted in production of this model terpenoid. The use of a tightly controlled expression system and fine tuning of the translation rates of specific mRNAs allowed the production of 1.6g/L humulene in a bioreactor.

This project demonstrates the potential of M. extorquens as a future platform strain for the production of high-value terpenoids from the alternative carbon source methanol.