Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity

Abstract

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6,051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.

FIG. 1.

Conversion of DMAPP to isoprene by IspS.

FIG. 2.

Enrichment for ADS-expressing cells [DH10B(pMBIS)(pADS); black] over the empty vector control [DH10B-K1(pMBIS)(pTrc99A); white] in a mixed culture over 8.5 h. Cultures were inoculated at 0.5% with a 1:1 mixture of DH10B-K1(pMBIS)(pTrc99A) and DH10B(pMBIS)(pADS).

FIG. 3.

Enrichment for and against ADS-expressing clones [DH10B-K1(pMBIS)(pADS)] in a 1:10,000 initial mixture with GFP-expressing clones [DH10B(pMBIS)(pTrcGFP)]. Cultures were tested in the presence (squares) and absence (diamonds) of 10 mM mevalonate.

FIG. 4.

YhfR expression is sufficient to overcome IPP/DMAPP toxicity. Growth of DH10B(pMBIS) with either pYhfR (A) or pTrc99A (B) in 96-well plates at various mevalonate concentrations: 0 mM (dark blue), 5 mM (purple), 10 mM (light blue), 15 mM (red), and 20 mM (yellow).

FIG. 5.

Low-level NudF expression is sufficient to overcome IPP/DMAPP toxicity. Growth of DH10B(pMBIS) with either pC9b (A) or pTrc99A (B) in 96-well plates at various mevalonate concentrations: 0 mM (blue), 5 mM (purple), 15 mM (red), and 20 mM (yellow).

FIG. 6.

Crude lysate assays with pYhfR (A) or pTrc99A (B) lysates with a DMAPP concentration of 0 (black) or 50 (gray) mM. The peak at a retention time of 4.9 min is 2-methyl-3-buten-1-ol.

FIG. 7.

Growth (open symbols) and isopentenol production (solid symbols) of E. coli DH1 expressing NudF (squares) or the pTrc99A control (circles).