Combinatorial engineering of mevalonate pathway for improved amorpha‐4,11‐diene production in budding yeast

Abstract

Combinatorial genome integration of mevalonate pathway genes was performed with the aim of optimizing the metabolic flux for improved production of terpenoids in budding yeast. In the present study, we developed a novel δ-integration platform to achieve multiple genome integrations through modulating the concentration of antibiotics. By exploiting carotenoid biosynthesis as screening module, we successfully created a library of yeast colonies appeared with various intensities of orange color. As proof-of-concept that carotenoid overproducers could serve to boost the titer of other terpenoids, we further tested engineered strains for the production of amorpha-4,11-diene, an important precursor for antimalarial drug. However, we experienced some limitations of the carotenoid-based screening approach as it was only effective in detecting a small range of pathway activity improvement and further increasing mevalonate pathway activity led to a decreased orange color. By far, we were only able to obtain one mutant strain yielded more than 13-fold amorpha-4,11-diene over parental strains, which was approximately 64 mg/L of caryophyllene equivalents. Further qPCR studies confirmed that erg10, erg13, thmg1 and erg12 involved in mevalonate pathway were overexpressed in this mutant strain. We envision the current δ-integration platform would form the basis of a generalized technique for multiple gene integrations in yeast-a method that would be of significant interest to the metabolic engineering community.

Construction of yeast producing patchoulol by global metabolic engineering strategy

“…Also, by overexpressing ERG20 in the yeast S. cerevisiae , the production of cineole, a monoterpene, was successfully improved (Ignea et al, 2011). Yuan and Ching (2014) improved the production of β‐carotene and amorpha‐4,11‐diene by modifying the mevalonate pathway by δ‐integration. Then, the transcription amount of the gene related to the mevalonate pathway of the recombinant yeast was examined by quantitative PCR.…”

Upregulating the mevalonate pathway and repressing sterol synthesis in Saccharomyces cerevisiae enhances the production of triterpenes

“…HMGR ( HMG1 ) catalyzes the rate-limiting step of the pathway and is subject to strict feedback control, so we overexpressed a truncated form of the enzyme (tHMGR) which no longer responds to feedback inhibition by enzyme degradation due to a missing ubiquitination signal (DeBose-Boyd 2008). We also overexpressed HMGS ( ERG13 ) which promotes isoprenoid biosynthesis by supplying the substrate for HMGR (Yuan and Ching 2014). Finally, we knocked out the ROX1 gene, which encodes a negative regulator of the MVA pathway and late sterol biosynthesis (Henry et al 2002; Montañés et al 2011; Özaydin et al 2013; Jakočiūnas et al 2015).…”

“…Furthermore, the overexpression of ERG13 increases flux through the pathway, given that a 13-fold increase in amorpha-4,11-diene production was observed when ERG13 was overexpressed in combination with tHMGR , ERG10 , and ERG12 (Yuan and Ching 2014). We chose the ROX1 locus as an integration site for our overexpression cassette, thus knocking out this negative regulator of the MVA pathway and late sterol biosynthesis (Henry et al 2002; Montañés et al 2011; Özaydin et al 2013; Jakočiūnas et al 2015).…”

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae

“…To compare the production of amorphadiene by engineered yeast strains on glucose and xylose, engineered yeasts were cultured in 50 mL of synthetic complete (SC) medium (MP Biomedicals, Santa Ana, CA) following previous studies (Paradise et al, ; Yuan and Ching, , ). The medium was buffered by 50 mM potassium hydrogen phthalate buffer at pH 5.5, and 40 g/L of substrates were added.…”