@article{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 = {Metabolic Engineering}, volume = {60}, journal = {Metabolic Engineering}, publisher = {Elsevier}, address = {Amsterdam [u.a.]}, issn = {1096-7176}, doi = {10.1016/j.ymben.2020.03.002}, 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{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} } @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{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 = {Metabolites}, volume = {10}, journal = {Metabolites}, number = {65}, publisher = {MDPI}, address = {Basel}, issn = {2218-1989}, doi = {10.3390/metabo10020065}, pages = {15}, 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} } @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} } @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} } @article{PattersonHeFolzetal.2020, author = {Patterson, Jenelle A. and He, Hai and Folz, Jacob S. and Li, Qiang and Wilson, Mark A. and Fiehn, Oliver and Bruner, Steven D. and Bar-Even, Arren and Hanson, Andrew D.}, title = {Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli}, series = {Biochemical Journal}, volume = {477}, journal = {Biochemical Journal}, number = {9}, publisher = {Portland Press}, address = {London}, issn = {1470-8728}, doi = {10.1042/BCJ20200198}, pages = {1745 -- 1757}, year = {2020}, abstract = {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.}, language = {en} }