Frank Sonntag

Methylobacterium extorquens : methylotrophy and biotechnological applications

Methylotrophy is the ability to use reduced one-carbon compounds, such as methanol, as a single source of carbon and energy. Methanol is, due to its availability and potential for production from renewable resources, a valuable feedstock for biotechnology. Nature offers a variety of methylotrophic microorganisms that differ in their metabolism and represent resources for engineering of value-added products from methanol. The most extensively studied methylotroph is the Alphaproteobacterium Methylobacterium extorquens. Over the past five decades, the metabolism of M. extorquens has been investigated physiologically, biochemically, and more recently, using complementary omics technologies such as transcriptomics, proteomics, metabolomics, and fluxomics. These approaches, together with a genome-scale metabolic model, facilitate system-wide studies and the development of rational strategies for the successful generation of desired products from methanol. This review summarizes the knowledge of methylotrophy in M. extorquens, as well as the available tools and biotechnological applications.

Polycistronic expression of a β-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to β-ionone production

The flavour and fragrance compound β-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for β-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three β-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of β-carotene. To enable β-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the β-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, β-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound.

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.

Effect of Linoleic Acids on the Release of β-Carotene from Carotenoid-Producing Saccharomyces cerevisiae into Sunflower Oil

In situ extraction is important for highly productive and cost-efficient processes in industrial biotechnology, but it is difficult to establish for intracellularly accumulating carotenoids like β-carotene. In this study, the organic solvent used in aqueous-organic two-phase media exerted a strong effect on the release of β-carotene from recombinant yeast cells. The carotenoid-synthesizing Saccharomyces cerevisiae strain YB/I/E was cultivated in two-liquid-phase media with 20% dodecane or 20% sunflower oil. Up to 0.6 µg/ml β-carotene was released into sunflower oil, but less than 0.1 µg/ml into dodecane, although biocompatibility and solubility of β-carotene is appropriate for both solvents. Addition of linoleic acid, the main component of sunflower oil, to the dodecane phase increased the amount of β-carotene released, indicating that linoleic acid is the component responsible for the β-carotene release into sunflower oil. These findings demonstrate that the effect of the organic solvent should be taken into consideration for further research on in situ extraction of carotenoids.

Methanol Utilisation Technologies

Oil and gas are raw materials—the availability of which is prognosticated to run short in the near future. The peak oil discussion is an example generally perceived as proof of this development to come.