Tuning Geraniol Biosynthesis via a Novel Decane-Responsive Promoter in Candida glycerinogenes

Abstract

Geraniol is a rose-scented monoterpene with significant commercial and industrial value in medicine, condiments, cosmetics, and bioenergy. Here, we first targeted geraniol as a reporter metabolite and explored the suitability and potential of Candida glycerinogenes as a heterologous host for monoterpenoid production. Subsequently, dual-pathway engineering was employed to improve the production of geraniol with a geraniol titer of 858.4 mg/L. We then applied a synthetic hybrid promoter approach to develop a decane-responsive hybrid promoter based on the native promoter P GAP derived from C. glycerinogenes itself. The hybrid promoter was able to be induced by n-decane with 3.6 times higher transcriptional intensity than the natural promoter P GAP . In particular, the hybrid promoter effectively reduces the conflict between cell growth and product formation in the production of geraniol. Ultimately, 1194.6 mg/L geraniol was obtained at the shake flask level. The strong and tunable decane-responsive hybrid promoter developed in this study provides an important tool for fine regulation of toxic terpenoid production in cells.

EngineeringY. lipolyticafor the biosynthesis of geraniol

“…Recently, Zhao et al engineered the non-conventional yeast Candida glycerinogens using dual pathway engineering with MVA pathway and the isopentenol utilization pathway, and by dynamic regulation using an inducible promoter (Zhao et al, 2022). Shake-flask titers using solely glucose remained under 500 mg/L.…”

Metabolic Engineering of Candida glycerinogenes for Sustainable Production of Geraniol

“…Quantification and identification were performed by using a mass spectrometer (GC–MS-Trace1310-ISQ LT, Thermo Fisher Scientific) equipped with an HP-5 ms capillary column (30 m × 0.25 mm × 0.25 μm). The sample analysis procedure followed the method described previously . Briefly, the oven temperature was initially held at 50 °C for 3 min, sequentially increased at a rate of 20 °C min –1 to 270 °C, and held for 6 min.…”

“…The DNA manipulation followed the previously described methods involving the construction of single or multiple gene expression cassettes and gene editing using the transient CRISPR-Cas9 method. , Gene overexpression strains were constructed by transforming the corresponding integrative plasmids using the LiAC/SS carrier DNA/PEG method at 5.8S rDNA gene sites. The integrative plasmids were digested with the restriction enzyme Sac I or Xho I.…”

“…The transcription factor Upc2 achieves transcriptional activation of ERG genes by specifically binding to ergosterol regulatory elements (SREs, TATACGA) on the native promoter of ERG genes. 14,16 To optimize the performance of the Upc2-mediated ergosterol feedback system, the upstream of the promoter P ERG2 was fused with different numbers of SREs, which are recognized specifically by the transcription factor Upc2. Then, the fluorescence intensity of eGFP was monitored under the control of the hybrid promoters P ERG2(SREs*1) , P ERG2(SREs*2) , P ERG2(SREs*3) , P ERG2(SREs*4) , and P ERG2(SREs*5) (Figure 3a).…”

“…In a previous study, we employed a dual pathway engineering in C. glycerinogenes to obtain 858.4 mg L –1 of geraniol under the exogenous addition of 40 mM prenol . However, prenol as a potential gasoline alternative and a precursor of commodity chemicals was added to the medium to not only increase the fermentation cost but also not in line with the green and sustainable concept.…”