High‐Throughput Screening for Terpene‐Synthase‐Cyclization Activity and Directed Evolution of a Terpene Synthase

Lauchli, Ryan; Rabe, Kersten S.; Kalbarczyk, Karolina Z.; Tata, Amulya; Heel, Thomas; Kitto, Rebekah Z.; Arnold, Frances H.

doi:10.1002/anie.201301362

Cited by 84 publications

(78 citation statements)

References 44 publications

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“…Since switching the BIS promoter from GAL10 to GAL1 increased bisabolene production 2-fold (comparing the pESC strain to the pRS strain), it seemed a reasonable assumption that BIS is a limiting factor in the yeast bisabolene biosynthetic pathway. This is in agreement with unpublished data on bisabolene production in yeast together with findings that, due to their relatively slow kinetics, terpene synthases are commonly found to be the limiting enzymes in terpene biosynthetic pathways (21)(22)(23).…”

Section: Resultssupporting

confidence: 92%

“…There is, however, a scarcity of tools available for high-throughput screening or selection of improved strains. An FPP analog which releases methanol upon enzyme-mediated cyclization has been used as a synthetic substrate to successfully increase the thermostability of a sesquiterpene synthase (22). This approach reduces processing time for screening a mutant library but still requires lysis and assay of each mutant on an individual basis.…”

Section: Discussionmentioning

confidence: 99%

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Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae

Kirby

Nishimoto

Chow

et al. 2014

Appl Environ Microbiol

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dTo facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. ␣-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in bisabolene titers, although mutants were found which made a mixture of ␣-bisabolene and ␤-farnesene, another candidate biofuel. Based on evidence that the 3= end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.

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Section: Resultssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae

Kirby

Nishimoto

Chow

et al. 2014

Appl Environ Microbiol

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“…Hence, metabolic engineering of terpene biosynthetic pathways has received a formidable interest [3] for the generation of valuable polycyclic and multichiral natural products that would be challenging to synthesize by traditional chemical methodologies [4]. Only recently the potential of terpene cyclases to generate complexity beyond nature's current capabilities started to become realized [5][6][7][8]. Harnessing the catalytic machineries of terpene synthases for novel chemistries could in principle be based on either of the two natural mechanisms for the generation of a reactive carbocationic species prone to further chemistries.…”

Section: Introductionmentioning

confidence: 99%

Squalene-hopene cyclases—evolution, dynamics and catalytic scope

Syrén

Henche

Eichler

et al. 2016

Current Opinion in Structural Biology

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“…To this end, Lauchli et al [22] demonstrated that TPS could be a target for directed evolution by using a surrogate substrate for FPP. They synthesized vinyl ether containing isoprenyl diphosphate, which is similar to FPP.…”

Section: Introductionmentioning

confidence: 99%

A High-Throughput Colorimetric Screening Assay for Terpene Synthase Activity Based on Substrate Consumption

et al. 2014

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Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

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High‐Throughput Screening for Terpene‐Synthase‐Cyclization Activity and Directed Evolution of a Terpene Synthase

Cited by 84 publications

References 44 publications

Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae

Use of Nonionic Surfactants for Improvement of Terpene Production in Saccharomyces cerevisiae

Squalene-hopene cyclases—evolution, dynamics and catalytic scope

A High-Throughput Colorimetric Screening Assay for Terpene Synthase Activity Based on Substrate Consumption

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