Display options
Cite
Wang Y, Nguyen N, Lee SH, et al. Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates. Biotechnol Bioeng. 2021;119(1):315-320doi: 10.1002/bit.27956.
Wang, Y., Nguyen, N., Lee, S. H., Wang, Q., May, J. A., Gonzalez, R., & Cirino, P. C. (2022). Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates. Biotechnology and bioengineering, 119(1), 315-320. https://doi.org/10.1002/bit.27956
Wang, Yixi, et al. "Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates." Biotechnology and bioengineering vol. 119,1 (2022): 315-320. doi: https://doi.org/10.1002/bit.27956
Wang Y, Nguyen N, Lee SH, Wang Q, May JA, Gonzalez R, Cirino PC. Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates. Biotechnol Bioeng. 2022 Jan;119(1):315-320. doi: 10.1002/bit.27956. Epub 2021 Oct 16. PMID: 34633065.
Copy Download .nbib Format: NLM
Share it on

Biotechnol Bioeng. 2022 Jan;119(1):315-320. doi: 10.1002/bit.27956. Epub 2021 Oct 16.

Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates.

Biotechnology and bioengineering

Yixi Wang, Nam Nguyen, Seung H Lee, Qinxuan Wang, Jeremy A May, Ramon Gonzalez, Patrick C Cirino

Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, USA.
  2. Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, Florida, USA.
  3. Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas, USA.
  4. Department of Chemistry, University of Houston, Houston, Texas, USA.

PMID: 34633065 DOI: 10.1002/bit.27956

Abstract

In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a subterminal carbon, producing an alkylsuccinate. Alkylsuccinate synthases are complex, multi-subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkylsuccinate synthases have been identified, primarily from nitrate- and sulfate-reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host. Here, we describe the functional expression of the (1-methylalkyl)succinate synthase (Mas) system from Azoarcus sp. strain HxN1 in recombinant Escherichia coli. Mass spectrometry confirms anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane. Maximum production of (1-methylpentyl)succinate is observed when masC, masD, masE, masB, and masG are all present on the expression plasmid; omitting masC reduces production by 66% while omitting any other gene eliminates production. Meanwhile, deleting iscR (encoding the repressor of the E. coli iron-sulfur cluster operon) improves product titer, as does performing the biotransformation at reduced temperature (18°C), both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this enzyme system.

© 2021 Wiley Periodicals LLC.

Keywords: alkane activation; alkylsuccinate; fumarate addition; glycyl radical enzyme; iron-sulfur cluster

References

  1. Akhtar, M. K., & Jones, P. R. (2008). Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3). Applied Microbiology and Biotechnology, 78(5), 853-862. https://doi.org/10.1007/s00253-008-1377-6 - PubMed
  2. Bali, A. P., Lennox-Hvenekilde, D., Myling-Petersen, N., Buerger, J., Salomonsen, B., Gronenberg, L. S., Sommer, M. O. A., & Genee, H. J. (2020). Improved biotin, thiamine, and lipoic acid biosynthesis by engineering the global regulator IscR. Metabolic Engineering, 60, 97-109. https://doi.org/10.1016/j.ymben.2020.03.005 - PubMed
  3. Callaghan, A. V., Gieg, L. M., Kropp, K. G., Suflita, J. M., & Young, L. Y. (2006). Comparison of mechanisms of alkane metabolism under sulfate-reducing conditions among two bacterial isolates and a bacterial consortium. Applied and Environmental Microbiology, 72(6), 4274-4282. https://doi.org/10.1128/AEM.02896-05 - PubMed
  4. Callaghan, A. V., Tierney, M., Phelps, C. D., & Young, L. Y. (2009). Anaerobic biodegradation of n-hexadecane by a nitrate-reducing consortium. Applied and Environmental Microbiology, 75(5), 1339-1344. https://doi.org/10.1128/AEM.02491-08 - PubMed
  5. Davidova, I. A., Gieg, L. M., Nanny, M., Kropp, K. G., & Suflita, J. M. (2005). Stable isotopic studies of n-alkane metabolism by a sulfate-reducing bacterial enrichment culture. Applied and Environmental Microbiology, 71(12), 8174-8182. https://doi.org/10.1128/Aem.71.12.8174-8182.2005 - PubMed
  6. Ehrenreich, P., Behrends, A., Harder, J., & Widdel, F. (2000). Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria. Archives of Microbiology, 173(1), 58-64. https://doi.org/10.1007/s002030050008 - PubMed
  7. Grundmann, O., Behrends, A., Rabus, R., Amann, J., Halder, T., Heider, J., & Widdel, F. (2008). Genes encoding the candidate enzyme for anaerobic activation of n-alkanes in the denitrifying bacterium, strain HxN1. Environmental Microbiology, 10(2), 376-385. https://doi.org/10.1111/j.1462-2920.2007.01458.x - PubMed
  8. Haynes, C. A., & Gonzalez, R. (2014). Rethinking biological activation of methane and conversion to liquid fuels. Nature Chemical Biology, 10(5), 331-339. https://doi.org/10.1038/nchembio.1509 - PubMed
  9. Heider, J., Szaleniec, M., Martins, B. M., Seyhan, D., Buckel, W., & Golding, B. T. (2016). Structure and function of benzylsuccinate synthase and related fumarate-adding glycyl radical enzymes. Journal of Molecular Microbiology and Biotechnology, 26(1-3), 29-44. https://doi.org/10.1159/000441656 - PubMed
  10. Jarling, R., Kuhner, S., Janke, E. B., Gruner, A., Drozdowska, M., Golding, B. T., Rabus, R., & Wilkes, H. (2015). Versatile transformations of hydrocarbons in anaerobic bacteria: Substrate ranges and regio- and stereo-chemistry of activation reactions. Frontiers in Microbiology, 6, 880. https://doi.org/10.3389/fmicb.2015.00880 - PubMed
  11. Kniemeyer, O., Musat, F., Sievert, S. M., Knittel, K., Wilkes, H., Blumenberg, M., Michaelis, W., Classen, A., Bolm, C., Joye, S. B., & Widdel, F. (2007). Anaerobic oxidation of short-chain hydrocarbons by marine sulphate-reducing bacteria. Nature, 449(7164), 898-901. https://doi.org/10.1038/nature06200 - PubMed
  12. Kuppers, J., Mitschke, N., Heyen, S., Rabus, R., Wilkes, H., & Christoffers, J. (2020). Metabolites of the anaerobic degradation of n-hexane by denitrifying betaproteobacterium strain HxN1. ChemBioChem, 21(3), 373-380. https://doi.org/10.1002/cbic.201900375 - PubMed
  13. Leuthner, B., Leutwein, C., Schulz, H., Horth, P., Haehnel, W., Schiltz, E., Schagger, H., & Heider, J. (1998). Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: A new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Molecular Microbiology, 28(3), 615-628. https://doi.org/10.1046/j.1365-2958.1998.00826.x - PubMed
  14. Prince, R. C., Amande, T. J., & McGenity, T. J. (2018). Prokaryotic hydrocarbon degraders. In T. McGenity (Ed.), Taxonomy, genomics and ecophysiology of hydrocarbon-degrading microbes (pp. 1-41). Springer. https://doi.org/10.1007/978-3-319-60053-6_15-1 - PubMed
  15. Rabus, R., Wilkes, H., Behrends, A., Armstroff, A., Fischer, T., Pierik, A. J., & Widdel, F. (2001). Anaerobic initial reaction of n-alkanes in a denitrifying bacterium: Evidence for (1-methylpentyl)succinate as initial product and for involvement of an organic radical in n-hexane metabolism. Journal of Bacteriology, 183(5), 1707-1715. https://doi.org/10.1128/JB.183.5.1707-1715.2001 - PubMed
  16. Salii, I., Szaleniec, M., Zein, A. A., Seyhan, D., Sekula, A., Schuhle, K., Kaplieva-Dudek, I., Linne, U., Meckenstock, R. U., & Heider, J. (2021). Determinants for substrate recognition in the glycyl radical enzyme benzylsuccinate synthase revealed by targeted mutagenesis. ACS Catalysis, 11(6), 3361-3370. https://doi.org/10.1021/acscatal.0c04954 - PubMed
  17. Schwartz, C. J., Giel, J. L., Patschkowski, T., Luther, C., Ruzicka, F. J., Beinert, H., & Kiley, P. J. (2001). IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proceedings of the National Academy of Sciences of the United States of America, 98(26), 14895-14900. https://doi.org/10.1073/pnas.251550898 - PubMed
  18. Shisler, K. A., & Broderick, J. B. (2014). Glycyl radical activating enzymes: Structure, mechanism, and substrate interactions. Archives of Biochemistry and Biophysics, 546, 64-71. https://doi.org/10.1016/j.abb.2014.01.020 - PubMed
  19. Toth, C. R. A., & Gieg, L. M. (2017). Time course-dependent methanogenic crude oil biodegradation: Dynamics of fumarate addition metabolites, biodegradative genes, and microbial community composition. Frontiers in Microbiology, 8, 2610. https://doi.org/10.3389/fmicb.2017.02610 - PubMed
  20. Wilkes, H., Kuhner, S., Bolm, C., Fischer, T., Classen, A., Widdel, F., & Rabus, R. (2003). Formation of n-alkane- and cycloalkane-derived organic acids during anaerobic growth of a denitrifying bacterium with crude oil. Organic Geochemistry, 34(9), 1313-1323. https://doi.org/10.1016/S0146-6380(03)00099-8 - PubMed

Publication Types

Grant support