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.

Figure 1

Functional metagenomic platform for discovery of novel functional genetic elements from diverse environmental microbiomes. Shown is a schematic detailing the key steps required for selecting functional genetic elements from diverse environments that confer a desired selective advantage to a microbial catalyst. Metagenomic DNA is directly extracted from arbitrary environmental samples without earlier culturing steps, purified, and transformed into a microbial host of interest. The entire library of putative functional genetic elements is subjected to a selection pressure (e.g. chemicals at inhibitory concentrations or recalcitrant substrates) that only allows survival of hosts containing functional genetic elements, which counteract the selection pressure (e.g. by allowing usage of the recalcitrant substrates or by conferring tolerance by intracellular or extracellular inactivation or efflux of the inhibitory compound). This scheme is ideally suited for discovery of novel functional genetic elements for biomass conversion to biofuels.

Figure 2

Sequence annotation and functional analysis of selected genetic elements improving biomass inhibitor tolerance in E. coli. (A, B) Improvements in inhibitor tolerance toward 2-furoic acid and syringaldehyde because of metagenomic inserts. Inhibitor concentrations resulting in 90% reductions in growth yield were determined for wild-type E. coli as 1.05 g/l for 2-furoic acid and 1.33 g/l for syringaldehyde. Improvements in E. coli growth yield at these concentrations because of metagenomic inserts were 6.9-fold for 2-furoic acid and 5.7-fold for syringaldehyde, showed here as the mean (and standard deviation) of triplicate readings after 24 h of growth. (C, D) The metagenomic inserts conferring tolerance to 2-furoic acid (mgFurAc) and syringaldehyde (mgSyrAld) were sequenced at 3 × coverage and annotated (Supplementary Tables II and III). Annotated genes for (C) mgFurAc and (D) mgSyrAld are shown as filled arrows, with the orientation denoting the relative direction of transcription based on an arbitrary sense strand. Transposon mutagenesis, followed by reselection of the tolerance phenotypes, was used to identify functional genetic elements in mgFurAc and mgSyrAld that contribute to the selected phenotypes (genes colored red and labeled) (Supplementary information). Vertical bars along the bottom of each sequence–position axis denote positions of transposon insertion in the loss-of-function study (black denotes no effect, red denotes loss-of-function).

Figure 3

Inhibitor tolerance phenotypes encoded by sub-cloned metagenomic genes for (A) 0.8 g/l 2-furoic acid, (B) 1.4 g/l syringaldehyde, and (C) mixtures of 2-furoic acid and syringaldehyde; 24 h kinetic growth curves are shown for E.coli clones harboring different combinations of sub-cloned metagenomic tolerance genes. Error bars represent standard deviation from triplicate kinetic readings. (A) For 2-furoic acid, the constructs containing the individual genes mgOrfX and mgRecA recapitulate only part of the tolerance phenotype; however, a bicistronic construct (mgRecA_mgOrfX) fully recapitulates the phenotype of the full-length selected fosmid (mgFurAc), showing that the two genes are necessary and sufficient for conferring the 2-furoic acid tolerance phenotype. (B) For syringaldehyde, the sub-cloned metagenomic UDP glucose-4-epimerase gene (mgUdpE) fully recapitulates the phenotype of the full-length selected fosmid (mgSyrAld). (C) A tri-cistronic construct (mgRecA_mgOrfX_mgUdpE) enables improved tolerance to mixtures of 2-furoic acid and syringaldehyde showing that the identified tolerance genes can be combined to generate multifunctional constructs.