Improving CBCA synthase activity through rational protein design

Thomas, Fabian; Kayser, Oliver

doi:10.1016/j.jbiotec.2023.01.004

“…For Cs CBCAS, the variant C244W‐M165K was chosen. [ 65 ] The Cs CBDAS sequence was used without any modification. Multicopy integration of CBCAS revealed strain yCS19, which produced detectable amounts of CBCA‐C5 at pH 6.7.…”

Section: Resultsmentioning

confidence: 99%

Engineering cannabinoid production in Saccharomyces cerevisiae

Schmidt,

Aras,

Kayser

2024

Biotechnology Journal

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Phytocannabinoids are natural products with highly interesting pharmacological properties mainly produced by plants. The production of cannabinoids in a heterologous host system has gained interest in recent years as a promising alternative to production from plant material. However, the systems reported so far do not achieve industrially relevant titers, highlighting the need for alternative systems. Here, we show the production of the cannabinoids cannabigerolic acid and cannabigerol from glucose and hexanoic acid in a heterologous yeast system using the aromatic prenyltransferase NphB from Streptomyces sp. strain CL190. The production was significantly increased by introducing a fusion protein consisting of ERG20WW and NphB. Furthermore, we improved the production of the precursor olivetolic acid to a titer of 56 mg L−1. The implementation of the cannabinoid synthase genes enabled the production of Δ9‐tetrahydrocannabinolic acid, cannabidiolic acid as well as cannabichromenic acid, where the heterologous biosynthesis of cannabichromenic acid in a yeast system was demonstrated for the first time. In addition, we found that the product spectrum of the cannabinoid synthases localized to the vacuoles of the yeast cells was highly dependent on extracellular pH, allowing for easy manipulation. Finally, using a fed‐batch approach, we showed cannabigerolic acid and olivetolic acid titers of up to 18.2 mg L−1 and 117 mg L−1, respectively.

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Modifications of Prenyl Side Chains in Natural Product Biosynthesis

Wang,

Yang,

Abe

2024

Angewandte Chemie

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In recent years, there has been a growing interest in understanding the enzymatic machinery responsible for the modifications of prenyl side chains and elucidating their roles in natural product biosynthesis. This interest stems from the pivotal role such modifications play in shaping the structural and functional diversity of natural products, as well as from their potential applications to synthetic biology and drug discovery. In addition to contributing to the diversity and complexity of natural products, unique modifications of prenyl side chains are represented by several novel biosynthetic mechanisms. Representative unique examples of epoxidation, dehydrogenation, oxidation of methyl groups to carboxyl groups, unusual C‐C bond cleavage and oxidative cyclization are summarized and discussed. By revealing the intriguing chemistry and enzymology behind these transformations, this comprehensive and comparative review will guide future efforts in the discovery, characterization and application of modifications of prenyl side chains in natural product biosynthesis.

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Modifications of Prenyl Side Chains in Natural Product Biosynthesis

Wang,

Yang,

Abe

2024

Angew Chem Int Ed

0

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In recent years, there has been a growing interest in understanding the enzymatic machinery responsible for the modifications of prenyl side chains and elucidating their roles in natural product biosynthesis. This interest stems from the pivotal role such modifications play in shaping the structural and functional diversity of natural products, as well as from their potential applications to synthetic biology and drug discovery. In addition to contributing to the diversity and complexity of natural products, unique modifications of prenyl side chains are represented by several novel biosynthetic mechanisms. Representative unique examples of epoxidation, dehydrogenation, oxidation of methyl groups to carboxyl groups, unusual C‐C bond cleavage and oxidative cyclization are summarized and discussed. By revealing the intriguing chemistry and enzymology behind these transformations, this comprehensive and comparative review will guide future efforts in the discovery, characterization and application of modifications of prenyl side chains in natural product biosynthesis.

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Improving CBCA synthase activity through rational protein design

Cited by 6 publications

References 26 publications

Engineering cannabinoid production in Saccharomyces cerevisiae

Engineering cannabinoid production in Saccharomyces cerevisiae

Modifications of Prenyl Side Chains in Natural Product Biosynthesis

Modifications of Prenyl Side Chains in Natural Product Biosynthesis

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