Coupled Electrochemical and Microbial Catalysis for the Production of Polymer Bricks.

Power-to-X technologies have the potential to pave the way towards a future resource-secure bioeconomy as they enable the exploitation of renewable resources and CO2 . Herein, the coupled electrocatalytic and microbial catalysis of the C5 -polymer precursors mesaconate and 2S-methylsuccinate from CO2 and electric energy by in situ coupling electrochemical and microbial catalysis at 1 L-scale was developed. In the first phase, 6.1±2.5 mm formate was produced by electrochemical CO2 reduction. In the second phase, formate served as the substrate for microbial catalysis by an engineered strain of Methylobacterium extorquens AM-1 producing 7±2 µm and 10±5 µm of mesaconate and 2S-methylsuccinate, respectively. The proof of concept showed an overall conversion efficiency of 0.2 % being 0.4 % of the theoretical maximum.

Towards electroenzymatic processes involving old yellow enzymes and mediated cofactor regeneration.

Old yellow enzymes are able to catalyze asymmetric C=C reductions. A mediated electroenzymatic process to regenerate the NADPH in combination with an old yellow enzyme was investigated. Due to the fact that the overall process was affected by a broad set of parameters, a design of experiments (DoE) approach was chosen to identify suitable process conditions. Process conditions with high productivities of up to 2.27 mM/h in combination with approximately 90% electron transfer efficiency were identified.

Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol.

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. In this study we present de novo synthesis of the sesquiterpenoid α-humulene from the abundantly available non-food carbon source methanol by metabolically engineered Methylobacterium extorquens AM1. Expression of α-humulene synthase from Zingiber zerumbet in combination with farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae led to concentrations of up to 18 mg/L α-humulene. Introduction of a prokaryotic mevalonate pathway from Myxococcus xanthus in combination with ribosome binding site optimization of α-humulene and FPP synthases increased product concentration 3-fold. This value was additionally raised by 30% using a carotenoid synthesis deficient mutant strain. Final product concentrations of up to 1.65 g/L were obtained in methanol limited fed-batch cultivations, which is the highest titer of de novo synthesized α-humulene reported to date. This study demonstrates the potential of M. extorquens as a future platform strain for the production of high-value terpenoids from the alternative carbon source methanol.