TY - JOUR A1 - He, Hai A1 - Noor, Elad A1 - Ramos-Parra, Perla A. A1 - García-Valencia, Liliana E. A1 - Patterson, Jenelle A. A1 - Díaz de la Garza, Rocío I. A1 - Hanson, Andrew D. A1 - Bar-Even, Arren T1 - In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate JF - Metabolites N2 - 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. KW - one-carbon metabolism KW - spontaneous reaction KW - auxotrophy KW - serine cycle KW - phenotypic phase plane Y1 - 2019 U6 - https://doi.org/10.3390/metabo10020065 SN - 2218-1989 VL - 10 IS - 65 PB - MDPI CY - Basel ER - TY - JOUR A1 - He, Hai A1 - Höper, Rune A1 - Dodenhöft, Moritz A1 - Marlière, Philippe A1 - Bar-Even, Arren T1 - An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli JF - Metabolic Engineering N2 - 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. KW - Pathway design KW - Promiscuous enzymes KW - Formaldehyde assimilation KW - Serine cycle KW - Growth selection Y1 - 2020 U6 - https://doi.org/10.1016/j.ymben.2020.03.002 SN - 1096-7176 SN - 1096-7184 VL - 60 SP - 1 EP - 13 PB - Elsevier CY - Amsterdam [u.a.] ER - TY - JOUR A1 - Patterson, Jenelle A. A1 - He, Hai A1 - Folz, Jacob S. A1 - Li, Qiang A1 - Wilson, Mark A. A1 - Fiehn, Oliver A1 - Bruner, Steven D. A1 - Bar-Even, Arren A1 - Hanson, Andrew D. T1 - Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli JF - Biochemical Journal N2 - Formaldehyde (HCHO) is a reactive carbonyl compound that formylates and cross-links proteins, DNA, and small molecules. It is of specific concern as a toxic intermediate in the design of engineered pathways involving methanol oxidation or formate reduction. The interest in engineering these pathways is not, however, matched by engineering-relevant information on precisely why HCHO is toxic or on what damage-control mechanisms cells deploy to manage HCHO toxicity. The only well-defined mechanism for managing HCHO toxicity is formaldehyde dehydrogenase-mediated oxidation to formate, which is counterproductive if HCHO is a desired pathway intermediate. We therefore sought alternative HCHO damage-control mechanisms via comparative genomic analysis. This analysis associated homologs of the Escherichia coli pepP gene with HCHO-related one-carbon metabolism. Furthermore, deleting pepP increased the sensitivity of E. coli to supplied HCHO but not other carbonyl compounds. PepP is a proline aminopeptidase that cleaves peptides of the general formula X-Pro-Y, yielding X + Pro-Y. HCHO is known to react spontaneously with cysteine to form the close proline analog thioproline (thiazolidine-4-carboxylate), which is incorporated into proteins and hence into proteolytic peptides. We therefore hypothesized that certain thioproline-containing peptides are toxic and that PepP cleaves these aberrant peptides. Supporting this hypothesis, PepP cleaved the model peptide Ala-thioproline-Ala as efficiently as Ala-Pro-Ala in vitro and in vivo, and deleting pepP increased sensitivity to supplied thioproline. Our data thus (i) provide biochemical genetic evidence that thioproline formation contributes substantially to HCHO toxicity and (ii) make PepP a candidate damage-control enzyme for engineered pathways having HCHO as an intermediate. KW - formaldehyde KW - thiazolidine-4-carboxylic acid KW - thioproline KW - Xaa-Pro aminopeptidase Y1 - 2020 U6 - https://doi.org/10.1042/BCJ20200198 SN - 1470-8728 SN - 0006-2936 VL - 477 IS - 9 SP - 1745 EP - 1757 PB - Portland Press CY - London ER - TY - JOUR A1 - He, Hai A1 - Edlich-Muth, Christian A1 - Lindner, Steffen N. A1 - Bar-Even, Arren T1 - Ribulose Monophosphate Shunt Provides Nearly All Biomass and Energy Required for Growth of E. coli JF - ACS Synthetic Biology N2 - 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. KW - ribulose monophosphate cycle KW - methylotrophy KW - metabolic engineering KW - growth selection KW - carbon labeling KW - flux modeling KW - formaldehyde assimilation Y1 - 2018 U6 - https://doi.org/10.1021/acssynbio.8b00093 SN - 2161-5063 VL - 7 IS - 6 SP - 1601 EP - 1611 PB - ACS CY - Washington, DC ER -