A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion.
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.
Sommer
Morten O A
MO
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. sommer@genetics.med.harvard.edu
Church
George M
GM
Dantas
Gautam
G
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*
D001547:Benzaldehydes; D056804:Biofuels; D005663:Furans; C069665:syringaldehyde; C060089:2-furoic acid
Science. 2008 Feb 29;319(5867):1238-40
18258860
J Biotechnol. 1998 Sep 17;64(1):53-62
9823658
Nat Genet. 2006 Dec;38(12):1406-12
17086184
Nat Rev Microbiol. 2005 Jun;3(6):470-8
15931165
Appl Microbiol Biotechnol. 2004 Nov;66(1):10-26
15300416
J Ind Microbiol Biotechnol. 1998 Feb;20(2):132-8
9611822
Appl Microbiol Biotechnol. 2006 Nov;73(1):27-36
17028874
Appl Environ Microbiol. 2005 Dec;71(12):8249-56
16332810
Science. 1994 Apr 15;264(5157):375-82
8153624
BMC Genomics. 2008 Feb 08;9:75
18261238
Appl Environ Microbiol. 2000 Jun;66(6):2541-7
10831436
Nature. 2007 Nov 22;450(7169):560-5
18033299
Biotechnol Bioeng. 2000 Jun 5;68(5):524-30
10797238
Science. 2006 Dec 8;314(5805):1598-600
17158327
Science. 2008 Feb 29;319(5867):1235-8
18258862
Annu Rev Microbiol. 1987;41:465-505
3318677
Appl Microbiol Biotechnol. 2003 Dec;63(3):258-66
13680206
Science. 2009 Aug 28;325(5944):1128-1131
19713526
Biotechnol Bioeng. 1999 Oct 5;65(1):24-33
10440668
Sci Am. 2006 Jun;294(6):44-51
16711359
Nature. 2006 Apr 13;440(7086):940-3
16612385
Biotechnol Bioeng. 1999;66(4):203-10
10578090
Appl Environ Microbiol. 2005 Oct;71(10):6335-44
16204555
Environ Microbiol. 2004 Sep;6(9):981-9
15305923
Science. 2007 Feb 9;315(5813):804-7
17289988
J Mol Biol. 1990 Oct 5;215(3):403-10
2231712
Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
9092630
J Am Chem Soc. 2004 Oct 20;126(41):13247-54
15479078
Science. 2006 Jan 27;311(5760):506-8
16439656