title: A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion.
abstract: Sustainable biofuel alternatives to fossil fuel energy are hampered by recalcitrance and toxicity of biomass substrates to microbial biocatalysts. To address this issue, we present a culture-independent functional metagenomic platform for mining Nature's vast enzymatic reservoir and show its relevance to biomass conversion. We performed functional selections on 4.7 Gb of metagenomic fosmid libraries and show that genetic elements conferring tolerance toward seven important biomass inhibitors can be identified. We select two metagenomic fosmids that improve the growth of Escherichia coli by 5.7- and 6.9-fold in the presence of inhibitory concentrations of syringaldehyde and 2-furoic acid, respectively, and identify the individual genes responsible for these tolerance phenotypes. Finally, we combine the individual genes to create a three-gene construct that confers tolerance to mixtures of these important biomass inhibitors. This platform presents a route for expanding the repertoire of genetic elements available to synthetic biology and provides a starting point for efforts to engineer robust strains for biofuel generation.
authors.affiliation: Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. sommer@genetics.med.harvard.edu
authors.forename: Morten O A
authors.initials: MO
authors.lastname: Sommer
authors.forename: George M
authors.initials: GM
authors.lastname: Church
authors.forename: Gautam
authors.initials: G
authors.lastname: Dantas
mesh_terms: D001547:Benzaldehydes / Q000378:metabolism; D056804:Biofuels; D018533:Biomass; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005663:Furans / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D056186:Metagenomics / Q000379:methods*; D010641:Phenotype; D049490:Systems Biology / Q000379:methods*
chemical_list: D001547:Benzaldehydes; D056804:Biofuels; D005663:Furans; C069665:syringaldehyde; C060089:2-furoic acid
references.citation: Science. 2008 Feb 29;319(5867):1238-40
references.pmid: 18258860
references.citation: J Biotechnol. 1998 Sep 17;64(1):53-62
references.pmid: 9823658
references.citation: Nat Genet. 2006 Dec;38(12):1406-12
references.pmid: 17086184
references.citation: Nat Rev Microbiol. 2005 Jun;3(6):470-8
references.pmid: 15931165
references.citation: Appl Microbiol Biotechnol. 2004 Nov;66(1):10-26
references.pmid: 15300416
references.citation: J Ind Microbiol Biotechnol. 1998 Feb;20(2):132-8
references.pmid: 9611822
references.citation: Appl Microbiol Biotechnol. 2006 Nov;73(1):27-36
references.pmid: 17028874
references.citation: Appl Environ Microbiol. 2005 Dec;71(12):8249-56
references.pmid: 16332810
references.citation: Science. 1994 Apr 15;264(5157):375-82
references.pmid: 8153624
references.citation: BMC Genomics. 2008 Feb 08;9:75
references.pmid: 18261238
references.citation: Appl Environ Microbiol. 2000 Jun;66(6):2541-7
references.pmid: 10831436
references.citation: Nature. 2007 Nov 22;450(7169):560-5
references.pmid: 18033299
references.citation: Biotechnol Bioeng. 2000 Jun 5;68(5):524-30
references.pmid: 10797238
references.citation: Science. 2006 Dec 8;314(5805):1598-600
references.pmid: 17158327
references.citation: Science. 2008 Feb 29;319(5867):1235-8
references.pmid: 18258862
references.citation: Annu Rev Microbiol. 1987;41:465-505
references.pmid: 3318677
references.citation: Appl Microbiol Biotechnol. 2003 Dec;63(3):258-66
references.pmid: 13680206
references.citation: Science. 2009 Aug 28;325(5944):1128-1131
references.pmid: 19713526
references.citation: Biotechnol Bioeng. 1999 Oct 5;65(1):24-33
references.pmid: 10440668
references.citation: Sci Am. 2006 Jun;294(6):44-51
references.pmid: 16711359
references.citation: Nature. 2006 Apr 13;440(7086):940-3
references.pmid: 16612385
references.citation: Biotechnol Bioeng. 1999;66(4):203-10
references.pmid: 10578090
references.citation: Appl Environ Microbiol. 2005 Oct;71(10):6335-44
references.pmid: 16204555
references.citation: Environ Microbiol. 2004 Sep;6(9):981-9
references.pmid: 15305923
references.citation: Science. 2007 Feb 9;315(5813):804-7
references.pmid: 17289988
references.citation: J Mol Biol. 1990 Oct 5;215(3):403-10
references.pmid: 2231712
references.citation: Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
references.pmid: 9092630
references.citation: J Am Chem Soc. 2004 Oct 20;126(41):13247-54
references.pmid: 15479078
references.citation: Science. 2006 Jan 27;311(5760):506-8
references.pmid: 16439656