Increasing the intracellular isoprenoid pool in Saccharomyces cerevisiae by structural fine-tuning of a bifunctional farnesyl diphosphate synthase

Wang

et al. 2022

J. Agric. Food Chem.

As a natural sesquiterpene compound with numerous biological activities, α-santalene has extensive applications in the cosmetic and pharmaceutical industries. Although several α-santalene-producing microbial strains have been constructed, low productivity still hampers large-scale fermentation. Herein, we present a case of engineered sesquiterpene biosynthesis where the insufficient downstream pathway capacity limited high-level α-santalene production in Escherichia coli. The initial strain was constructed, and it produced 6.4 mg/L α-santalene. To increase α-santalene biosynthesis, we amplified the flux toward farnesyl diphosphate (FPP) precursor by screening and choosing the right FPP synthase and reprogrammed the rate-limiting downstream pathway by generating mutations in santalene synthase (Clausena lansium; ClSS). Santalene synthase was engineered by site-directed mutagenesis, resulting in the improved soluble expression of ClSS and an α-santalene titer of 887.5 mg/L; the α-santalene titer reached 1078.8 mg/L after adding a fusion tag to ClSS. The most productive pathway, which included combining precursor flux amplification and mutant synthases, conferred an approximate 169-fold increase in α-santalene levels. Maximum titers of 1272 and 2916 mg/L were achieved under shake flask and fed-batch fermentation, respectively, and were among the highest levels reported using E. coli as the host.

Section: Resultscontrasting

confidence: 54%

Section: Resultsmentioning

confidence: 77%

Sesquiterpene Synthase Engineering and Targeted Engineering of α-Santalene Overproduction in Escherichia coli

Wang

et al. 2022

J. Agric. Food Chem.

“…Therefore, the initial strategy for engineering higher 1,8-cineole titers focused on optimizing and balancing expression levels of the two terminal enzymes: GPPS and 1,8-cineole synthase. Engineering a high monoterpene titer requires a balance between GPPS and terpene synthase activities that provides sufficient flux to accumulate the target product while avoiding growth-inhibitory levels of GPP [ 11 , 27 ]. Promoters for these synthases were selected from native R. toruloides genes.…”

Section: Resultsmentioning

confidence: 99%

Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

Kirby

Geiselman

Yaegashi

et al. 2021

Biotechnol Biofuels

Background Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

“…Engineering a high monoterpene titer requires a balance between GPPS and terpene synthase activities that provides su cient ux to accumulate the target product while avoiding growth-inhibitory levels of GPP [11,27]. Promoters for these synthases were selected from native R. toruloides genes.…”

Section: Transcriptomics and Proteomics Analysis Of α-Bisabolene Prodmentioning

confidence: 99%

Further Engineering of R. Toruloides for the Production of Terpenes From Lignocellulosic Biomass

Kirby¹,

Geiselman²,

Yaegashi

et al. 2020

Preprint

Background: Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results:This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2 L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer 4-fold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional 10-fold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions:This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.