@phdthesis{Wenk2020, author = {Wenk, Sebastian}, title = {Engineering formatotrophic growth in Escherichia coli}, school = {Universit{\"a}t Potsdam}, pages = {V, 107}, year = {2020}, abstract = {To meet the demands of a growing world population while reducing carbon dioxide (CO2) emissions, it is necessary to capture CO2 and convert it into value-added compounds. In recent years, metabolic engineering of microbes has gained strong momentum as a strategy for the production of valuable chemicals. As common microbial feedstocks like glucose directly compete with human consumption, the one carbon (C1) compound formate was suggested as an alternative feedstock. Formate can be easily produced by various means including electrochemical reduction of CO2 and could serve as a feedstock for microbial production, hence presenting a novel entry point for CO2 to the biosphere and a storage option for excess electricity. Compared to the gaseous molecule CO2, formate is a highly soluble compound that can be easily handled and stored. It can serve as a carbon and energy source for natural formatotrophs, but these microbes are difficult to cultivate and engineer. In this work, I present the results of several projects that aim to establish efficient formatotrophic growth of E. coli - which cannot naturally grow on formate - via synthetic formate assimilation pathways. In the first study, I establish a workflow for growth-coupled metabolic engineering of E. coli. I demonstrate this approach by presenting an engineering scheme for the PFL-threonine cycle, a synthetic pathway for anaerobic formate assimilation in E. coli. The described methods are intended to create a standardized toolbox for engineers that aim to establish novel metabolic routes in E. coli and related organisms. The second chapter presents a study on the catalytic efficiency of C1-oxidizing enzymes in vivo. As formatotrophic growth requires generation of both energy and biomass from formate, the engineered E. coli strains need to be equipped with a highly efficient formate dehydrogenase, which provides reduction equivalents and ATP for formate assimilation. I engineered a strain that cannot generate reducing power and energy for cellular growth, when fed on acetate. Under this condition, the strain depends on the introduction of an enzymatic system for NADH regeneration, which could further produce ATP via oxidative phosphorylation. I show that the strain presents a valuable testing platform for C1-oxidizing enzymes by testing different NAD-dependent formate and methanol dehydrogenases in the energy auxotroph strain. Using this platform, several candidate enzymes with high in vivo activity, were identified and characterized as potential energy-generating systems for synthetic formatotrophic or methylotrophic growth in E. coli.   In the third chapter, I present the establishment of the serine threonine cycle (STC) - a synthetic formate assimilation pathway - in E. coli. In this pathway, formate is assimilated via formate tetrahydrofolate ligase (FtfL) from Methylobacterium extorquens (M. extorquens). The carbon from formate is attached to glycine to produce serine, which is converted into pyruvate entering central metabolism. Via the natural threonine synthesis and cleavage route, glycine is regenerated and acetyl-CoA is produced as the pathway product. I engineered several selection strains that depend on different STC modules for growth and determined key enzymes that enable high flux through threonine synthesis and cleavage. I could show that expression of an auxiliary formate dehydrogenase was required to achieve growth via threonine synthesis and cleavage on pyruvate. By overexpressing most of the pathway enzymes from the genome, and applying adaptive laboratory evolution, growth on glycine and formate was achieved, indicating the activity of the complete cycle. The fourth chapter shows the establishment of the reductive glycine pathway (rGP) - a short, linear formate assimilation route - in E. coli. As in the STC, formate is assimilated via M. extorquens FtfL. The C1 from formate is condensed with CO2 via the reverse reaction of the glycine cleavage system to produce glycine. Another carbon from formate is attached to glycine to form serine, which is assimilated into central metabolism via pyruvate. The engineered E. coli strain, expressing most of the pathway genes from the genome, can grow via the rGP with formate or methanol as a sole carbon and energy source.}, language = {en} } @article{HeEdlichMuthLindneretal.2018, author = {He, Hai and Edlich-Muth, Christian and Lindner, Steffen N. and Bar-Even, Arren}, title = {Ribulose Monophosphate Shunt Provides Nearly All Biomass and Energy Required for Growth of E. coli}, series = {ACS Synthetic Biology}, volume = {7}, journal = {ACS Synthetic Biology}, number = {6}, publisher = {ACS}, address = {Washington, DC}, issn = {2161-5063}, doi = {10.1021/acssynbio.8b00093}, pages = {1601 -- 1611}, year = {2018}, abstract = {The ribulose monophosphate (RuMP) cycle is a highly efficient route for the assimilation of reduced one-carbon compounds. Despite considerable research, the RuMP cycle has not been fully implemented in model biotechnological organisms such as Escherichia coli, mainly since the heterologous establishment of the pathway requires addressing multiple challenges: sufficient formaldehyde production, efficient formaldehyde assimilation, and sufficient regeneration of the formaldehyde acceptor, ribulose 5-phosphate. Here, by efficiently producing formaldehyde from sarcosine oxidation and ribulose 5-phosphate from exogenous xylose, we set aside two of these concerns, allowing us to focus on the particular challenge of establishing efficient formaldehyde assimilation via the RuMP shunt, the linear variant of the RuMP cycle. We have generated deletion strains whose growth depends, to different extents, on the activity of the RuMP shunt, thus incrementally increasing the selection pressure for the activity of the synthetic pathway. Our final strain depends on the activity of the RuMP shunt for providing the cell with almost all biomass and energy needs, presenting an absolute coupling between growth and activity of key RuMP cycle components. This study shows the value of a stepwise problem solving approach when establishing a difficult but promising pathway, and is a strong basis for future engineering, selection, and evolution of model organisms for growth via the RuMP cycle.}, language = {en} } @phdthesis{He2019, author = {He, Hai}, title = {Exploring and engineering formaldehyde assimilation}, doi = {10.25932/publishup-47386}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-473867}, school = {Universit{\"a}t Potsdam}, pages = {vi, 105}, year = {2019}, abstract = {Increasing concerns regarding the environmental impact of our chemical production have shifted attention towards possibilities for sustainable biotechnology. One-carbon (C1) compounds, including methane, methanol, formate and CO, are promising feedstocks for future bioindustry. CO2 is another interesting feedstock, as it can also be transformed using renewable energy to other C1 feedstocks for use. While formaldehyde is not suitable as a feedstock due to its high toxicity, it is a central intermediate in the process of C1 assimilation. This thesis explores formaldehyde metabolism and aims to engineer formaldehyde assimilation in the model organism Escherichia coli for the future C1-based bioindustry. The first chapter of the thesis aims to establish growth of E. coli on formaldehyde via the most efficient naturally occurring route, the ribulose monophosphate pathway. Linear variants of the pathway were constructed in multiple-gene knockouts strains, coupling E. coli growth to the activities of the key enzymes of the pathway. Formaldehyde-dependent growth was achieved in rationally designed strains. In the final strain, the synthetic pathway provides the cell with almost all biomass and energy requirements. In the second chapter, taking advantage of the unique feature of its reactivity, formaldehyde assimilation via condensation with glycine and pyruvate by two promiscuous aldolases was explored. Facilitated by these two reactions, the newly designed homoserine cycle is expected to support higher yields of a wide array of products than its counterparts. By dividing the pathway into segments and coupling them to the growth of dedicated strains, all pathway reactions were demonstrated to be sufficiently active. The work paves a way for future implementation of a highly efficient route for C1 feedstocks into commodity chemicals. In the third chapter, the in vivo rate of the spontaneous formaldehyde tetrahydrofolate condensation to methylene-tetrahydrofolate was assessed in order to evaluate its applicability as a biotechnological process. Tested within an E. coli strain deleted in essential genes for native methylene-tetrahydrofolate biosynthesis, the reaction was shown to support the production of this essential intermediate. However, only low growth rates were observed and only at high formaldehyde concentrations. Computational analysis dependent on in vivo evidence from this strain deduced the slow rate of this spontaneous reaction, thus ruling out its substantial contribution to growth on C1 feedstocks. The reactivity of formaldehyde makes it highly toxic. In the last chapter, the formation of thioproline, the condensation product of cysteine and formaldehyde, was confirmed to contribute this toxicity effect. Xaa-Pro aminopeptidase (PepP), which genetically links with folate metabolism, was shown to hydrolyze thioproline-containing peptides. Deleting pepP increased strain sensitivity to formaldehyde, pointing towards the toxicity of thioproline-containing peptides and the importance of their removal. The characterization in this study could be useful in handling this toxic intermediate. Overall, this thesis identified challenges related to formaldehyde metabolism and provided novel solutions towards a future bioindustry based on sustainable C1 feedstocks in which formaldehyde serves as a key intermediate.}, language = {en} } @article{delaCruzMachensMesserschmidtetal.2019, author = {de la Cruz, Jorge Gonzalez and Machens, Fabian and Messerschmidt, Katrin and Bar-Even, Arren}, title = {Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast}, series = {ACS synthetic biology}, volume = {8}, journal = {ACS synthetic biology}, number = {5}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.8b00464}, pages = {911 -- 917}, year = {2019}, abstract = {One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO2 and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO2 in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.}, language = {en} }