Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

Henard, Calvin A.; Smith, Holly K.; Guarnieri, Michael T.

doi:10.1016/j.ymben.2017.03.007

Title: Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

Journal Article · Sun Apr 02 00:00:00 EDT 2017 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2017.03.007· OSTI ID:1355139

Henard, Calvin A. ^[1]; Smith, Holly K. ^[1]; Guarnieri, Michael T. ^[1]

National Renewable Energy Lab. (NREL), Golden, CO (United States)

Microbial conversion of methane to high-value bio-based chemicals and materials offers a path to mitigate GHG emissions and valorize this abundant-yet -underutilized carbon source. In addition to fermentation optimization strategies, rational methanotrophic bacterial strain engineering offers a means to reach industrially relevant titers, carbon yields, and productivities of target products. The phosphoketolase pathway functions in heterofermentative bacteria where carbon flux through two sugar catabolic pathways to mixed acids (lactic acid and acetic acid) increases cellular ATP production. Importantly, this pathway also serves as an alternative route to produce acetyl-CoA that bypasses the CO₂ lost through pyruvate decarboxylation in the Embden-Meyerhof-Parnas pathway. Thus, the phosphoketolase pathway can be leveraged for carbon efficient biocatalysis to acetyl-CoA-derived intermediates and products. Here, we show that the industrially promising methane biocatalyst, Methylomicrobium buryatense, encodes two phosphoketolase isoforms that are expressed in methanol- and methane-grown cells. Overexpression of the PktB isoform led to a 2-fold increase in intracellular acetyl-CoA concentration, and a 2.6-fold yield enhancement from methane to microbial biomass and lipids compared to wild-type, increasing the potential for methanotroph lipid-based fuel production. Off-gas analysis and metabolite profiling indicated that global metabolic rearrangements, including significant increases in post-translational protein acetylation and gene expression of the tetrahydromethanopterin-linked pathway, along with decreases in several excreted products, coincided with the superior biomass and lipid yield observed in the engineered strain. Further, these data suggest that phosphoketolase may play a key regulatory role in methanotrophic bacterial metabolism. As a result, given that acetyl-CoA is a key intermediate in several biosynthetic pathways, phosphoketolase overexpression offers a viable strategy to enhance the economics of an array of biological methane conversion processes.

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Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office (BETO)

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1355139

Alternate ID(s):: OSTI ID: 1397076

Report Number(s):: NREL/JA-5100-67217

Journal Information:: Metabolic Engineering, Vol. 41, Issue C; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 54 works

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References (34)

Anthropogenic emissions of methane in the United States Miller, S. M.; Wofsy, S. C.; Michalak, A. M. Proceedings of the National Academy of Sciences, Vol. 110, Issue 50 https://doi.org/10.1073/pnas.1314392110	journal	November 2013
Phosphoketolase, a Neglected Enzyme of Microbial Carbohydrate Metabolism Rohr, Lukas M.; Teuber, Michael; Meile, Leo CHIMIA International Journal for Chemistry, Vol. 56, Issue 6 https://doi.org/10.2533/000942902777680379	journal	June 2002
Metabolic engineering in methanotrophic bacteria Kalyuzhnaya, Marina G.; Puri, Aaron W.; Lidstrom, Mary E. Metabolic Engineering, Vol. 29 https://doi.org/10.1016/j.ymben.2015.03.010	journal	May 2015
Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae Sonderegger, Marco; Schümperli, Michael; Sauer, Uwe Applied and Environmental Microbiology, Vol. 70, Issue 5 https://doi.org/10.1128/AEM.70.5.2892-2897.2004	journal	May 2004
Bioconversion of natural gas to liquid fuel: Opportunities and challenges Fei, Qiang; Guarnieri, Michael T.; Tao, Ling Biotechnology Advances, Vol. 32, Issue 3 https://doi.org/10.1016/j.biotechadv.2014.03.011	journal	May 2014
Bioconversion of methane to lactate by an obligate methanotrophic bacterium Henard, Calvin A.; Smith, Holly; Dowe, Nancy Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep21585	journal	February 2016
Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria Xiong, Wei; Lee, Tai-Chi; Rommelfanger, Sarah Nature Plants, Vol. 2, Issue 1, Article No. 15187 https://doi.org/10.1038/nplants.2015.187	journal	December 2015
Electroporation-Based Genetic Manipulation in Type I Methanotrophs Yan, Xin; Chu, Frances; Puri, Aaron W. Applied and Environmental Microbiology, Vol. 82, Issue 7 https://doi.org/10.1128/AEM.03724-15	journal	January 2016
Bioreactor performance parameters for an industrially-promising methanotroph Methylomicrobium buryatense 5GB1 Gilman, Alexey; Laurens, Lieve M.; Puri, Aaron W. Microbial Cell Factories, Vol. 14, Issue 1 https://doi.org/10.1186/s12934-015-0372-8	journal	November 2015
Arabinose is metabolized via a phosphoketolase pathway in Clostridium acetobutylicum ATCC 824 Servinsky, M. D.; Germane, K. L.; Liu, S. Journal of Industrial Microbiology & Biotechnology, Vol. 39, Issue 12 https://doi.org/10.1007/s10295-012-1186-x	journal	August 2012
Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway Kocharin, Kanokarn; Siewers, Verena; Nielsen, Jens Biotechnology and Bioengineering, Vol. 110, Issue 8 https://doi.org/10.1002/bit.24888	journal	March 2013
Methanotrophic bacteria. Hanson, R. S.; Hanson, T. E. Microbiological reviews, Vol. 60, Issue 2 https://doi.org/10.1128/MR.60.2.439-471.1996	journal	January 1996
Highly efficient methane biocatalysis revealed in a methanotrophic bacterium Kalyuzhnaya, M. G.; Yang, S.; Rozova, O. N. Nature Communications, Vol. 4, Issue 1 https://doi.org/10.1038/ncomms3785	journal	December 2013
Search for Methanotrophic Producers of Exopolysaccharides Malashenko, Yu. R.; Pirog, T. P.; Romanovskaya, V. A. Applied Biochemistry and Microbiology, Vol. 37, Issue 6, p. 599-602 https://doi.org/10.1023/A:1012307202011	journal	November 2001
Bacterial protein acetylation: the dawning of a new age: Bacterial protein acetylation Hu, Linda I.; Lima, Bruno P.; Wolfe, Alan J. Molecular Microbiology, Vol. 77, Issue 1 https://doi.org/10.1111/j.1365-2958.2010.07204.x	journal	May 2010
Post-translational modifications as key regulators of bacterial metabolic fluxes Pisithkul, Tippapha; Patel, Nishaben M.; Amador-Noguez, Daniel Current Opinion in Microbiology, Vol. 24 https://doi.org/10.1016/j.mib.2014.12.006	journal	April 2015
Phosphoketolase pathway engineering for carbon-efficient biocatalysis Henard, Calvin Andrew; Freed, Emily Frances; Guarnieri, Michael Thomas Current Opinion in Biotechnology, Vol. 36 https://doi.org/10.1016/j.copbio.2015.08.018	journal	December 2015
Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae Bergman, Alexandra; Siewers, Verena; Nielsen, Jens AMB Express, Vol. 6, Issue 1 https://doi.org/10.1186/s13568-016-0290-0	journal	November 2016
Envisioning the Bioconversion of Methane to Liquid Fuels Conrado, R. J.; Gonzalez, R. Science, Vol. 343, Issue 6171 https://doi.org/10.1126/science.1246929	journal	February 2014
Synthetic non-oxidative glycolysis enables complete carbon conservation Bogorad, Igor W.; Lin, Tzu-Shyang; Liao, James C. Nature, Vol. 502, Issue 7473 https://doi.org/10.1038/nature12575	journal	September 2013
Studies of the Production of Fungal Polyketides in Aspergillus nidulans by Using Systems Biology Tools Panagiotou, Gianni; Andersen, Mikael R.; Grotkjaer, Thomas Applied and Environmental Microbiology, Vol. 75, Issue 7 https://doi.org/10.1128/AEM.01461-08	journal	January 2009
Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway de Jong, Bouke Wim; Shi, Shuobo; Siewers, Verena Microbial Cell Factories, Vol. 13, Issue 1 https://doi.org/10.1186/1475-2859-13-39	journal	January 2014
Rewriting yeast central carbon metabolism for industrial isoprenoid production Meadows, Adam L.; Hawkins, Kristy M.; Tsegaye, Yoseph Nature, Vol. 537, Issue 7622 https://doi.org/10.1038/nature19769	journal	September 2016
Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath) in a single cell protein production process Bothe, Harald; Jensen, K. Møller; A., Mergel Applied Microbiology and Biotechnology, Vol. 59, Issue 1 https://doi.org/10.1007/s00253-002-0964-1	journal	June 2002
Methane as a Resource: Can the Methanotrophs Add Value? Strong, P. J.; Xie, S.; Clarke, W. P. Environmental Science & Technology, Vol. 49, Issue 7 https://doi.org/10.1021/es504242n	journal	March 2015
Cofactor engineering of intracellular CoA/acetyl-CoA and its effect on metabolic flux redistribution in Escherichia coli Vadali, R. Metabolic Engineering, Vol. 6, Issue 2 https://doi.org/10.1016/j.ymben.2004.02.001	journal	April 2004
Contribution of anthropogenic and natural sources to atmospheric methane variability Bousquet, P.; Ciais, P.; Miller, J. B. Nature, Vol. 443, Issue 7110 https://doi.org/10.1038/nature05132	journal	September 2006
Acetate kinase-an enzyme of the postulated phosphoketolase pathway in Methylomicrobium alcaliphilum 20Z Rozova, Olga N.; Khmelenina, Valentina N.; Gavletdinova, Juliya Z. Antonie van Leeuwenhoek, Vol. 108, Issue 4 https://doi.org/10.1007/s10482-015-0549-5	journal	August 2015
Aerobic Methylotrophic Prokaryotes Chistoserdova, Ludmila; Lidstrom, Mary E. The Prokaryotes https://doi.org/10.1007/978-3-642-30141-4_68	book	January 2013
Exometabolome analysis reveals hypoxia at the up-scaling of a Saccharomyces cerevisiae high-cell density fed-batch biopharmaceutical process Fu, Zhibiao; Verderame, Thomas D.; Leighton, Julie M. Microbial Cell Factories, Vol. 13, Issue 1 https://doi.org/10.1186/1475-2859-13-32	journal	January 2014
Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1) de la Torre, Andrea; Metivier, Aisha; Chu, Frances Microbial Cell Factories, Vol. 14, Issue 1 https://doi.org/10.1186/s12934-015-0377-3	journal	November 2015
Innovative metabolic pathway design for efficient l-glutamate production by suppressing CO2 emission Chinen, Akito; Kozlov, Yuri I.; Hara, Yoshihiko Journal of Bioscience and Bioengineering, Vol. 103, Issue 3 https://doi.org/10.1263/jbb.103.262	journal	March 2007
Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense Puri, Aaron W.; Owen, Sarah; Chu, Frances Applied and Environmental Microbiology, Vol. 81, Issue 5 https://doi.org/10.1128/AEM.03795-14	journal	December 2014
Building carbon–carbon bonds using a biocatalytic methanol condensation cycle Bogorad, Igor W.; Chen, Chang-Ting; Theisen, Matthew K. Proceedings of the National Academy of Sciences, Vol. 111, Issue 45 https://doi.org/10.1073/pnas.1413470111	journal	October 2014

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