Functional mining of novel terpene synthases from metagenomes

Abstract

Background: Terpenes are one of the most diverse and abundant classes of natural biomolecules, collectively enabling a variety of therapeutic, energy, and cosmetic applications. Recent genomics investigations have predicted a large untapped reservoir of bacterial terpene synthases residing in the genomes of uncultivated organisms living in the soil, indicating a vast array of putative terpenoids waiting to be discovered.

Results: We aimed to develop a high-throughput functional metagenomic screening system for identifying novel terpene synthases from bacterial metagenomes by relieving the toxicity of terpene biosynthesis precursors to the Escherichia coli host. The precursor toxicity was achieved using an inducible operon encoding the prenyl pyrophosphate synthetic pathway and supplementation of the mevalonate precursor. Host strain and screening procedures were finely optimized to minimize false positives arising from spontaneous mutations, which avoid the precursor toxicity. Our functional metagenomic screening of human fecal metagenomes yielded a novel β-farnesene synthase, which does not show amino acid sequence similarity to known β-farnesene synthases. Engineered S. cerevisiae expressing the screened β-farnesene synthase produced 120 mg/L β-farnesene from glucose (2.86 mg/g glucose) with a productivity of 0.721 g/L∙h.

Conclusions: A unique functional metagenomic screening procedure was established for screening terpene synthases from metagenomic libraries. This research proves the potential of functional metagenomics as a sequence-independent avenue for isolating targeted enzymes from uncultivated organisms in various environmental habitats.

Keywords: Functional metagenomics; Prenyl pyrophosphate; Terpene synthase; β-Farnesene.

Fig. 1

Schematic illustration of the precursor toxicity-based functional metagenomic screening system

Fig. 2

Construction and optimization of the precursor toxicity-based functional metagenomic screening system. a Structure of pA5c–MBIS plasmid overexpressing the prenyl pyrophosphate precursors synthetic (MBIS) pathway under the control of IPTG-inducible P_lacUV5. A terpene synthase mitigates the toxicity of excessive prenyl pyrophosphate precursors (red) by converting them into non-toxic terpene molecules. b Gap between growth inhibitions on the negative (green) and positive (gray) controls was maximized by adjusting the concentration of supplemented mevalonate. The MBIS operon expression was induced by 0.5 mM IPTG. Upper panel, the timepoint the strain exhibited maximum specific growth rate (μ_max) with selected mevalonate concentration. Lower panel, maximum specific growth rate. c Growth profiles of negative and positive controls in the optimized screening medium containing 8 mM mevalonate

Fig. 3

Control of spontaneous mutagenesis during the precursor toxicity-based screening. a, b Survival rates of DH10B- and LowMut-based positive a and negative b controls on the screening plate with 1 mM IPTG and a variation of mevalonate concentration. Survival rate was the ratio of colony forming units (CFUs) on the selected mevalonate concentration to CFUs on 0 mM mevalonate. Pie graphs in b represent the location of mutagenesis in the MBIS operon of 4 randomly selected colonies from each condition. White, no mutation in the MBIS operon; olive, ERG12; cyan, ERG8 (see the color scheme of Fig. 1a). c Recovery of LowMut-based positive control strains from a simulated mixture on the screening plate with 1 mM IPTG and 8 mM mevalonate. d Scheme of the second screening process using fresh LowMut pA5c–MBIS strain to exclude false positives that evade the precursor toxicity via spontaneous mutations (red crosses)

Fig. 4

Characterization of TS10F1. a TS10F1 ORF was isolated from 3 different contigs screened from a human fecal metagenome library 40301 (Additional file 1: Fig. S2). TS10F1 showed 75% identity to a histidine phosphatase family protein of Prevotella pectinovora (sky blue, correct match; blue, functional equivalence; plum, not similar amino acid). b − d Gas chromatography–mass spectrometry (GC–MS) analysis of the TS10F1 in vivo test cultures. With IPTG induction of the MBIS pathway and supplemented mevalonate, BM_TS10F1 (blue) generated more β-farnesene and its derivatives than BM_AgBis (orange) and BM_Empty (green, no IPTG induction). e Confirmation after the purification of TS10F1 (estimated size is 21.54 KDa). M, protein marker. f GC–MS analysis of in vitro enzyme assay products at a 3 h timepoint and β-farnesene standard (top)

Fig. 5

Microbial production of β-farnesene from glucose via TS10F1. a Overview of biosynthesis of β-farnesene from glucose by SHE_TS10F1. Upper glycolysis was downregulated, and ZWF1 was overexpressed to shunt metabolic fluxes from glucose toward the pentose phosphate pathway to regenerate NADPH efficiently. ERG9 was also downregulated to maximize FPP availability for β-farnesene biosynthesis. ERG10 and tHMG1 were episomally overexpressed with TS10F1 to enhance the flux through the MVA pathway. b − d Comparison of culture profiles of SHE_TS10F1 and negative control SHE_Empty on glucose, including cell growth (b), glucose consumption (c), and production of major extracellular metabolites (d). e β-Farnesene and farnesoic acid production through the glucose culture of SHE_TS10F1. SHE_Empty did not produce both compounds at detectable levels