@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{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}
}