Development of an autotrophic fermentation technique for the production of fatty acids using an engineered Ralstonia eutropha cell factory
Massive emission of CO2 into atmosphere from consumption of carbon deposit is causing climate change. Researchers have applied metabolic engineering and synthetic biology techniques for improving CO2 fixation efficiency in many species. One solution might be the utilization of autotrophic bacteria, which have great potential to be engineered into microbial cell factories for CO2 fixation and the production of chemicals, independent of fossil resources. In this work, several pathways of Ralstonia eutropha H16 were modulated by manipulation of heterologous and endogenous genes related to fatty acid synthesis. The resulting strain B2(pCT, pFP) was able to produce 124.48 mg/g (cell dry weight) free fatty acids with fructose as carbon source, a fourfold increase over the parent strain H16. To develop a truly autotrophic fermentation technique with H2, CO2 and O2 as substrates, we assembled a relatively safe, continuous, lab-scale gas fermentation system using micro-fermentation tanks, H2 supplied by a hydrogen generator, and keeping the H2 to O2 ratio at 7:1. The system was equipped with a H2 gas alarm, rid of heat sources and placed into a fume hood to further improve the safety. With this system, the best strain B2(pCT, pFP) produced 60.64 mg free fatty acids per g biomass within 48 h, growing in minimal medium supplemented with 9 × 103 mL/L/h hydrogen gas. Thus, an autotrophic fermentation technique to produce fatty acids was successfully established, which might inspire further research on autotrophic gas fermentation with a safe, lab-scale setup, and provides an alternative solution for environmental and energy problems.
KeywordsAutotrophic fermentation Fatty acid synthesis Ralstonia eutropha
This research was financially supported by the Key Research Program of the Chinese Academy of Science (KFZD-SW-215, ZDRW-ZS-2016-3), National Natural Science Foundation of China (31522002, 31770105).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interests.
- 2.Quadrelli Elsje Alessandra, Centi Gabriele, Duplan Jean-Luc, Perathoner Siglinda, Quadrelli EA, Centi G, Duplan JL, Perathoner S (2011) Carbon dioxide recycling: emerging large-scale technologies with industrial potential. Chemsuschem 4:1194–1215. https://doi.org/10.1002/cssc.201100473 CrossRefGoogle Scholar
- 7.Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Pötter M, Schwartz E, Strittmatter A, Voß I, Gottschalk G, Steinbüchel A, Friedrich B, Bowien B (2006) Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nat Biotechnol 24:1257–1262. https://doi.org/10.1038/nbt1244 CrossRefGoogle Scholar
- 8.Schwartz E, Henne A, Cramm R, Eitinger T, Friedrich B, Gottschalk G (2003) Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H2-based Lithoautotrophy and Anaerobiosis. J Mol Biol 332:369–383. https://doi.org/10.1016/s0022-2836(03)00894-5 CrossRefGoogle Scholar
- 14.Bi Changhao, Peter Su, Müller Jana, Yeh Yi-Chun, Chhabra Swapnil R, Beller Harry R, Singer Steven W, Hillson Nathan J (2013) Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production. Microb Cell Fact 12:1–10. https://doi.org/10.1186/1475-2859-12-107 CrossRefGoogle Scholar
- 15.Tanaka Kenji, Ishizaki Ayaaki, Kanamaru Toshihisa, Kawano Takeharu (1994) Production of poly(d-3-hydroxybutyrate) from CO2, H2, and O2 by high cell density autotrophic cultivation of Alcaligenes eutrophus. Biotechnol Bioengineering 45:268–275. https://doi.org/10.1002/bit.260450312 CrossRefGoogle Scholar
- 20.Tung T, Hoang RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86. https://doi.org/10.1016/s0378-1119(98)00130-9 CrossRefGoogle Scholar
- 24.Zaigao Tan JMY, Chowdhury Anupam, Burdick Kaitlin, Jarboe Laura R, Maranas Costas D, Shanks Jacqueline V (2018) Engineering of E. coli inherent fatty acid biosynthesis capacity to increase octanoic acid production. Biotechnol Biofuels 11:1. https://doi.org/10.1186/s13068-018-1078-z CrossRefGoogle Scholar
- 28.Toshikazu Sugimoto TT, Tanaka Kenji, Ishizaki Ayaaki (1999) Control of acetic acid concentration by ph-stat continuous substrate feeding in heterotrophic culture phase of two-stage cultivation of Alcaligenes eutrophus for production of P(3HB) from CO2, H2, and O2 under non-explosive conditions. Biotechnol Bioeng 62:625–631. https://doi.org/10.1002/(SICI)1097-0290(19990320)62:6%3c625:AID-BIT1%3e3.0.CO;2-D CrossRefGoogle Scholar
- 32.Schwartz E, Voigt B, Zuhlke D, Pohlmann A, Lenz O, Albrecht D, Schwarze A, Kohlmann Y, Krause C, Hecker M, Friedrich B (2009) A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16. Proteomics 9:5132–5142. https://doi.org/10.1002/pmic.200900333 CrossRefGoogle Scholar