Engineering improved bio-jet fuel tolerance in Escherichia coli using a transgenic library from the hydrocarbon-degrader Marinobacter aquaeolei

Biotechnology for Biofuels

Timothy A. Tomko¹  Mary J. Dunlop¹ 

[1] School of Engineering, University of Vermont, 33 Colchester Ave, Burlington 05405, VT, USA

关键词: Transgenic;    Genomic library;    Monoterpene;    Pinene;    YceI;    Marinobacter aquaeolei;    Biofuel tolerance;   

Others  :  1229688
DOI  :  10.1186/s13068-015-0347-3

 received in 2015-06-08, accepted in 2015-09-22,  发布年份 2015

【 摘 要 】

Background

Recent metabolic engineering efforts have generated microorganisms that can produce biofuels, including bio-jet fuels, however these fuels are often toxic to cells, limiting production yields. There are natural examples of microorganisms that have evolved mechanisms for tolerating hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters.

Results

Using genomic DNA from the hydrocarbon-degrading microbe Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy with a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance.

Conclusions

The genomes of hydrocarbon-tolerant microbes represent a rich resource for tolerance engineering. Using a transgenic library, we were able to identify a single gene that improves E. coli’s tolerance to the bio-jet fuel precursor pinene.

【 授权许可】

   
2015 Tomko and Dunlop.

【 图 表 】

Fig.1.

Fig.2.

Fig.3.

Fig.1.

Fig.2.

Fig.3.

【 参考文献 】

• [1]Fortman JL, Chhabra S, Mukhopadhyay A, Chou H, Lee TS, Steen E, et al.: Biofuel alternatives to ethanol: pumping the microbial well. Trends Biotechnol 2008, 26(7):375-381.
• [2]Lee SK, Chou H, Ham TS, Lee TS, Keasling JD: Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 2008, 19(6):556-563.
• [3]Fischer CR, Klein-Marcuschamer D, Stephanopoulos G: Selection and optimization of microbial hosts for biofuels production. Metab Eng 2008, 10(6):295-304.
• [4]Camilli R, Reddy CM, Yoerger DR, Van Mooy BAS, Jakuba MV, Kinsey JC, et al.: Tracking hydrocarbon plume transport and biodegradation at Deepwater Horizon. Science 2010, 330(6001):201-204.
• [5]Rojas A, Duque E, Mosqueda G, Golden G, Hurtado A, Ramos JL, et al.: Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E. J Bacteriol 2001, 183(13):3967-3973.
• [6]Huu NB, Denner EB, Ha DT, Wanner G, Stan-Lotter H: Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oil-producing well. Int J Syst Bacteriol 1999, 49(Pt 2):367-375.
• [7]Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD, et al.: Engineering microbial biofuel tolerance and export using efflux pumps. Mol Syst Biol 2011, 7:487.
• [8]Schneiker S, dos Santos VAPM, Bartels D, Bekel T, Brecht M, Buhrmester J, et al.: Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis. Nat Biotechnol 2006, 24(8):997-1004.
• [9]Hazen TC, Dubinsky EA, DeSantis TZ, Andersen GL, Piceno YM, Singh N, et al.: Deep-sea oil plume enriches indigenous oil-degrading bacteria. Sci NY. 2010, 330(6001):204-208.
• [10]Schobert HH. The chemistry of hydrocarbon fuels. Butterworth-Heinemann; 2013.
• [11]Brennan TCR, Turner CD, Krömer JO, Nielsen LK: Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae. Biotechnol Bioeng 2012, 109(10):2513-2522.
• [12]Harvey BG, Wright ME, Quintana RL: High-density renewable fuels based on the selective dimerization of pinenes. Energy Fuels 2010, 24(1):267-273.
• [13]Chuck CJ, Donnelly J: The compatibility of potential bioderived fuels with Jet A-1 aviation kerosene. Appl Energy 2014, 118:83-91.
• [14]Sarria S, Wong B, García Martín H, Keasling JD, Peralta-Yahya P: Microbial synthesis of pinene. ACS Synth Biol 2014, 3(7):466-475.
• [15]Alonso-Gutierrez J, Chan R, Batth TS, Adams PD, Keasling JD, Petzold CJ, et al.: Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metab Eng 2013, 19:33-41.
• [16]Dunlop MJ: Engineering microbes for tolerance to next-generation biofuels. Biotechnol Biofuels 2011, 4:32. BioMed Central Full Text
• [17]Ramos JL, Duque E, Gallegos M-T, Godoy P, Ramos-Gonzalez MI, Rojas A, et al.: Mechanisms of solvent tolerance in gram-negative bacteria. Annu Rev Microbiol 2002, 56:743-768.
• [18]Nicolaou SA, Gaida SM, Papoutsakis ET: A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation. Metab Eng 2010, 12(4):307-331.
• [19]Peabody GL, Winkler J, Kao KC: Tools for developing tolerance to toxic chemicals in microbial systems and perspectives on moving the field forward and into the industrial setting. Curr Opin Chem Eng 2014, 6:9-17.
• [20]Doshi R, Nguyen T, Chang G: Transporter-mediated biofuel secretion. Proc Natl Acad Sci USA 2013, 110(19):7642-7647.
• [21]Foo JL, Leong SSJ: Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules. Biotechnol Biofuels 2013, 6(1):81. BioMed Central Full Text
• [22]Reyes LH, Almario MP, Kao KC: Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli. PLoS One 2011, 6(3):e17678.
• [23]Fiocco D, Capozzi V, Goffin P, Hols P, Spano G: Improved adaptation to heat, cold, and solvent tolerance in Lactobacillus plantarum. Appl Microbiol Biotechnol 2007, 77(4):909-915.
• [24]Alsaker KV, Paredes C, Papoutsakis ET: Metabolite stress and tolerance in the production of biofuels and chemicals: Gene-expression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum. Biotechnol Bioeng 2010, 105(6):1131-1147.
• [25]Weber H, Polen T, Heuveling J, Wendisch VF, Hengge R: Genome-wide analysis of the general stress response network in Escherichia coli: S-Dependent genes, promoters, and sigma factor selectivity. J Bacteriol 2005, 187(5):1591-1603.
• [26]Woodruff LBA, Pandhal J, Ow SY, Karimpour-Fard A, Weiss SJ, Wright PC, et al.: Genome-scale identification and characterization of ethanol tolerance genes in Escherichia coli. Metab Eng 2013, 15(C):124-133.
• [27]Hong M-E, Lee K-S, Yu BJ, Sung Y-J, Park SM, Koo HM, et al.: Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering. J Biotechnol 2010, 149(1–2):52-59.
• [28]Nicolaou SA, Gaida SM, Papoutsakis ET: Exploring the combinatorial genomic space in Escherichia coli for ethanol tolerance. Biotechnol J 2012, 7(11):1337-1345.
• [29]Zingaro KA, Nicolaou SA, Yuan Y, Papoutsakis ET: Exploring the heterologous genomic space for building, stepwise, complex, multicomponent tolerance to toxic chemicals. ACS Synth Biol. 2014, 3(7):476-486.
• [30]Ruegg TL, Kim E-M, Simmons BA, Keasling JD, Singer SW, Lee TS, et al.: An auto-inducible mechanism for ionic liquid resistance in microbial biofuel production. Nat Commun. 2014, 5:3490.
• [31]Foo JL, Jensen HM, Dahl RH, George K, Keasling JD, Lee TS, et al.: Improving microbial biogasoline production in Escherichia coli using tolerance engineering. MBio. 2014, 5(6):e01932.
• [32]Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G: Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 2006, 314(5805):1565-1568.
• [33]Atsumi S, Wu T-Y, Machado IMP, Huang W-C, Chen P-Y, Pellegrini M, et al.: Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli. Mol Syst Biol 2010, 6:449.
• [34]Clarke L, Carbon J: A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976, 9(1):91-99.
• [35]Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, et al.: BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 2011, 5(1):12. BioMed Central Full Text
• [36]Handa N, Terada T, Doi-Katayama Y, Hirota H, Tame JRH, Park S-Y, et al.: Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8. Protein Sci 2005, 14(4):1004-1010.
• [37]Sisinni L, Cendron L, Favaro G, Zanotti G: Helicobacter pylori acidic stress response factor HP1286 is a YceI homolog with new binding specificity. FEBS J 2010, 277(8):1896-1905.
• [38]Stancik LM, Stancik DM, Schmidt B: pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli. J Bacteriol 2002, 184(15):4246-4258.
• [39]Turner WJ, Dunlop MJ: Trade-offs in improving biofuel tolerance using combinations of efflux pumps. ACS Synth Biol 2014.
• [40]Wargo MJ, Szwergold BS, Hogan DA: Identification of two gene clusters and a transcriptional regulator required for Pseudomonas aeruginosa glycine betaine catabolism. J Bacteriol 2008, 190(8):2690-2699.
• [41]Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6(5):343-345.