Biology subjects

Wenk, S.

Publications and source records attributed to Wenk, S..

5 recordsLinked to original sources

bioRxiv2025

Evolution-assisted engineering of formate assimilation via the formyl phosphate route in Escherichia coli

The transition towards a sustainable bioeconomy requires the use of alternative feedstocks, with CO2-derived formate emerging as a promising candidate for industrial biotechnology. Despite its beneficial characteristics as a feedstock, microbial assimilation of formate is limited by the inefficiency of naturally evolved formate-fixing pathways. To overcome this limitation, synthetic formate reduction cascades could enable formate assimilation via formaldehyde, a key intermediate of several existing one carbon assimilation pathways. Recently, the formyl phosphate route, combining ATP-dependent activation of formate to formyl phosphate, followed by its reduction to formaldehyde, was developed through enzyme engineering and characterized in vitro. In this work, we successfully established the formyl phosphate route in vivo by developing a selection strategy that couples formate reduction to growth in a threonine/methionine auxotrophic Escherichia coli. Through adaptive laboratory evolution, we achieved formate-dependent growth via this novel pathway. Evolved strains were capable of growing robustly with formate concentrations between 20 mM and 100 mM with glucose in the co-feed. Genomic and proteomic analyses together with activity assays uncovered that formate activation was limiting in vivo. This discovery guided the rational engineering of a strain capable of efficient formate assimilation through the formyl phosphate route. By demonstrating that novel enzyme activities can link formate reduction to cell growth, our study shows how synthetic metabolic routes can be functionally established inside the cell, paving the way for the engineering of more complex synthetic pathways.

bioengineering↗

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↗

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↗

bioRxiv2022

Paving the way for synthetic C1- metabolism in Pseudomonas putida through the reductive glycine pathway

One-carbon (C1) compounds such as methanol, formate, and CO2 are alternative, sustainable microbial feedstocks for the biobased production of chemicals and fuels. In this study, we engineered the carbon metabolism of the industrially important bacterium Pseudomonas putida to assimilate these three substrates through the reductive glycine pathway. First, we demonstrated the functionality of the C1-assimilation module by coupling the growth of auxotrophic strains to formate assimilation. Next, we extended the module from formate to methanol using both NAD and PQQ - dependent methanol dehydrogenases. Finally, we demonstrated CO2-dependent growth through CO2 reduction to formate by the native formate dehydrogenase, which required short-term evolution to rebalance the cellular NADH/NAD+ ratio. This research paves the way to engineer P. putida towards growth on formate, methanol, and CO2 as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell factory.

synthetic biology↗