@misc{HeHoeperDodenhoeftetal.2020,
  author    = {He, Hai and H{\"o}per, Rune and Dodenh{\"o}ft, Moritz and Marli{\`e}re, Philippe and Bar-Even, Arren},
  title     = {An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli},
  series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  journal   = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  number    = {997},
  issn      = {1866-8372},
  doi       = {10.25932/publishup-47645},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-476452},
  pages     = {1 -- 13},
  year      = {2020},
  abstract  = {Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform.},
  language  = {en}
}
@misc{HeNoorRamosParraetal.2020,
  author    = {He, Hai and Noor, Elad and Ramos-Parra, Perla A. and Garc{\´i}a-Valencia, Liliana E. and Patterson, Jenelle A. and D{\´i}az de la Garza, Roc{\´i}o I. and Hanson, Andrew D. and Bar-Even, Arren},
  title     = {In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate},
  series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  journal   = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  number    = {998},
  issn      = {1866-8372},
  doi       = {10.25932/publishup-47647},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-476472},
  pages     = {17},
  year      = {2020},
  abstract  = {Formaldehyde is a highly reactive compound that participates in multiple spontaneous reactions, but these are mostly deleterious and damage cellular components. In contrast, the spontaneous condensation of formaldehyde with tetrahydrofolate (THF) has been proposed to contribute to the assimilation of this intermediate during growth on C1 carbon sources such as methanol. However, the in vivo rate of this condensation reaction is unknown and its possible contribution to growth remains elusive. Here, we used microbial platforms to assess the rate of this condensation in the cellular environment. We constructed Escherichia coli strains lacking the enzymes that naturally produce 5,10-methylene-THF. These strains were able to grow on minimal medium only when equipped with a sarcosine (N-methyl-glycine) oxidation pathway that sustained a high cellular concentration of formaldehyde, which spontaneously reacts with THF to produce 5,10-methylene-THF. We used flux balance analysis to derive the rate of the spontaneous condensation from the observed growth rate. According to this, we calculated that a microorganism obtaining its entire biomass via the spontaneous condensation of formaldehyde with THF would have a doubling time of more than three weeks. Hence, this spontaneous reaction is unlikely to serve as an effective route for formaldehyde assimilation.},
  language  = {en}
}