Reconstruction of the Biosynthetic Pathway of Santalols under Control of the GAL Regulatory System in Yeast

Abstract

Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/ steroid biosynthesis to sesquiterpenoid production, expression of ERG9 (encoding yeast squalene synthase) was depressed by replacing its innate promotor with P HXT1 and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of GAL promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of GAL promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L Z-α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from Santalum album) and WL19 expressing SanSyn (α-santalene synthase from Clausena lansium) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.

Rationally engineering santalene synthase to readjust the component ratio of sandalwood oil

“…As mentioned above, Z - α -santalol and Z - β -santalol are the most important components which contribute to the odor and bioactivity of sandalwood oil, and used for quality control in ISO 3518:2002. However, SaSSy produces much exo - α -bergamotene, resulting in the large ratios of exo - α -bergamotene and Z - exo - α -bergamotol in the fermentation oil, which are close to those of α -santalene and Z - α -santalol and even larger than those of β -santalene and Z - β -santalol 22 . In this study, we sought to elevate the ratios of Z - α -santalol and Z - β -santalol through engineering santalene synthase to attenuate the production of exo - α -bergamotene and Z - exo - α -bergamotol.…”

“…CYP736A167 exhibits the approximately equal substrate preference to α -santalene, β -santalene, epi - β -santalene and exo - α -bergamotene, and hence the component profile of sandalwood oil is shaped mainly by SaSSy 22 . As mentioned above, Z - α -santalol and Z - β -santalol are the most important components which contribute to the odor and bioactivity of sandalwood oil, and used for quality control in ISO 3518:2002.…”

“…The metabolic engineering efforts have led to construction of α -santalene-producing microbial platforms 18 – 21 . Recently, we built an engineered Saccharomyces cerevisiae (yeast) which can produce both santalenes and santalols, with 35.7% Z - α -santalol and 17.8% Z - β -santalol which is very close to the standard of ISO 3518:2002 22 .

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Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast

“…As more than 25 expression cassettes under the control of GAL promoters have been integrated, the transcription machinery might not be sufficient to drive the expression of all ajmalicine biosynthetic pathway genes (Zha et al, 2020). Therefore, the transcription activator of the yeast GAL regulatory system (GAL4) was overexpressed in AJM6, resulting in the construction of AJM7.…”

“…In addition, the disruption of ARI1 and ADH6 was found to benefit cis-trans-nepetalactol production in yeast. Herein, AJM6 was constructed by inserting SAM2 expression cassette into the ARI1 locus, followed by integrating another copy of SLS2 and STR expression As more than 25 expression cassettes under the control of GAL promoters have been integrated, the transcription machinery might not be sufficient to drive the expression of all ajmalicine biosynthetic pathway genes (Zha et al, 2020). Therefore, the transcription activator of the yeast GAL regulatory system (GAL4) was overexpressed in AJM6, resulting in the construction of AJM7.…”