Biology subjects

Schann, K.

Publications and source records attributed to Schann, K..

4 recordsLinked to original sources

bioRxiv2024

The Serine Shunt enables formate conversion to formaldehyde in vivo

Microbial valorization of CO2-derived substrates has emerged as a promising approach to address climate change and resource scarcity. Formate, which can be efficiently produced from CO2, shows great potential as a sustainable feedstock for biotechnological production. However, the scope of formate assimilation pathways is restricted by the limited number of natural formate-assimilating enzymes. To overcome this limitation, several new-to-nature routes for formate assimilation based on its reduction to formaldehyde have been proposed, but they suffer from low catalytic efficiencies and cannot yet support bacterial growth. Here, we propose the Serine Shunt as a novel formate reduction route and demonstrate its activity in vivo. In this pathway, formate is attached to glycine to form serine, which is subsequently cleaved into formaldehyde and glycine, thereby effectively converting formate to formaldehyde. Unlike other formate reduction routes, the Serine Shunt mainly utilizes natural reactions with favorable enzyme kinetics, while requiring the same amount of ATP and NAD(P)H as the most efficient new-to-nature route. We implemented the Serine Shunt in engineered E. coli strains using a step-wise approach by dividing the pathway into metabolic modules. After validating the individual module activities, we demonstrated the in vivo activity of the complete Serine Shunt by measuring intracellular formaldehyde production with a GFP sensor and coupling its activity to cell growth. Our results indicate that the Serine Shunt could be applied as a novel formate reduction route in methylotrophic hosts relevant for biotechnology.

bioengineering↗

bioRxiv2023

Design, construction and optimization of formaldehyde growth biosensors with broad application in Biotechnology

Formaldehyde is a key metabolite in natural and synthetic one-carbon metabolism as well as an important environmental toxin with high toxicity at low concentrations. To engineer efficient formaldehyde producing enzymes and to detect formaldehyde in industrial or environmental samples, it is important to establish highly sensitive, easy to use and affordable formaldehyde detection methods. Here, we transformed the workhorse bacterium Escherichia coli into biosensors that can detect a broad range of formaldehyde concentrations. Based on natural and promiscuous formaldehyde assimilation enzymes, we designed and engineered three different E. coli strains that depend on formaldehyde assimilation for cellular growth. After in depth characterization of these biosensors, we show that the formaldehyde sensitivity can be improved through adaptive laboratory evolution or modification of metabolic branch points. The metabolic engineering strategy presented in this work allowed the creation of E. coli biosensors that can detect formaldehyde in a concentration range from [~]30 M to [~]13 mM. Using the most sensitive strain, we benchmarked the in vivo activities of different, widely used NAD-dependent methanol dehydrogenases, the rate-limiting enzyme in synthetic methylotrophy. We also show that the strains can grow upon external addition of formaldehyde indicating their potential use for applications beyond enzyme engineering. The formaldehyde biosensors developed in this study are fully genomic and can be used as plug and play devices for screening large enzyme libraries. Thus, they have the potential to greatly advance enzyme engineering and might even be used for environmental monitoring or analysis of industrial probes. Highlights- Conversion of E. coli into three different formaldehyde growth biosensors - Biosensors are fully genomic and grow robustly when formaldehyde is present - Biosensors can detect formaldehyde concentrations ranging from [~]30 M to [~]13 mM - Benchmarking of biotechnological relevant methanol dehydrogenases reveals potential of biosensors for enzyme engineering - Biosensors grow upon direct addition of formaldehyde indicating potential use in environmental or industrial settings

bioengineering↗

bioRxiv2023

Engineering the biological conversion of formate into crotonate in Cupriavidus necator

To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO2 and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacterium C. necator as cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growing C. necator in minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacterium via a modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse {beta}-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 {+/-} 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical.

synthetic biology↗

bioRxiv2022

Synthetic carbon fixation via the autocatalytic serine threonine cycle

Atmospheric CO2 poses a major threat to life on Earth by causing global warming and climate change. On the other hand, it is the only carbon source that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO2 to reduced one-carbon (C1) molecules (e.g. formate) using renewable energy are improving fast. Driven by the idea of creating sustainable bioproduction platforms, natural and synthetic C1-utilization pathways are engineered into industrially relevant microbes. The realization of synthetic C1-assimilation cycles in living organisms is a promising but challenging endeavour. Here, we engineer the autocatalytic serine threonine cycle, a synthetic C1-assimilation route in Escherichia coli. Our stepwise engineering approach in tailored selection strains combined with adaptive laboratory evolution experiments enabled the organism to grow on formate. The synthetic strain uses formate as the sole carbon and energy source and is capable of growing at ambient CO2 concentrations, demonstrating the feasibility of establishing synthetic C1-assimilation cycles over laboratory timescales.

synthetic biology↗