Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast

Thomas, Fabian; Schmidt, Christina; Kayser, Oliver

doi:10.1007/s00253-020-10798-3

Cited by 23 publications

(19 citation statements)

References 59 publications

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“…With the availability of the precursors, an aromatic prenyltransferase named geranylpyrophosphate:olivetolate geranyltransferase (GOT) is responsible to convert GPP and OA into cannabigerolic acid (CBGA) [13], the central precursor for phytocannabinoids biosynthesis. This enzyme was detected in 1998 and is assumed to be an integral membrane protein, although some activity was found in soluble fractions [65,66].…”

Section: Biosynthesis Of Phytocannabinoidsmentioning

confidence: 99%

“…Improvement and redesign of metabolic pathways toward the product is the main strategy to enhance higher concentrations of cannabinoids. In fact, metabolic bottlenecks for the biosynthesis of ∆ 9 -THCA have been recently analyzed in silico and reported [66] for an engineered S. cerevisiae strain. The kinetics of reactions toward cannabinoids were modeled using MATLAB ® (version 9.4, The Mathworks, Inc., Natick, MA, USA) with the SimBiology extension [97], in which ∆ 9 -THCA was produced from glucose instead of galactose-a much-appreciated upgrade since galactose is up to 100-fold more expensive than glucose.…”

Section: Metabolic Engineering In Silicomentioning

confidence: 99%

“…The first challenge lies in acetyl-CoA, the committed precursor for mevalonate and olivetolic acid pathways, responsible for the GPP and OA pool, respectively. Thomas et al [66] suggested the replacement of acetaldehyde dehydrogenase (ADH) as well as acetyl-CoA synthetase with aldehyde dehydrogenase acylating (ADA) from Dickeya zeae, an optimization that grants higher specific activity, demands less energy, and prevents acetate formation. Moreover, the ethanol generated by aerobic cultivation on glucose can be converted back into acetaldehyde with the addition of ADH2 under specific promoter control.…”

Section: Metabolic Engineering In Silicomentioning

confidence: 99%

“…OA feeding is also not recommended due to its high cost, low absorbance by the yeast, and chemical instability. Moreover, the CBGA production is a limiting step toward the optimization of the process as shown by Thomas et al [66] in a sensitivity analysis. The membrane-bound enzyme CBGAS from Cannabis sativa L. was replaced by the soluble prenyltransferase NphB present in Streptomyces spp., especially due to a CBGA-specific variant recently reported [99].…”

Section: Metabolic Engineering In Silicomentioning

confidence: 99%

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Converting Sugars into Cannabinoids—The State-of-the-Art of Heterologous Production in Microorganisms

et al. 2022

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The legal cannabis market worldwide is facing new challenges regarding innovation in the production of cannabinoid-based drugs. The usual cannabinoid production involves growing Cannabis sativa L. outdoor or in dedicated indoor growing facilities, followed by isolation and purification steps. This process is limited by the growth cycles of the plant, where the cannabinoid content can deeply vary from each harvest. A game change approach that does not involve growing a single plant has gained the attention of the industry: cannabinoids fermentation. From recombinant yeasts and bacteria, researchers are able to reproduce the biosynthetic pathway to generate cannabinoids, such as (-)-∆9-tetrahydrocannabinol (∆9-THC), cannabidiol (CBD), and (-)-∆9-tetrahydrocannabivarin (∆9-THCV). This approach avoids pesticides, and natural resources such as water, land, and energy are reduced. Compared to growing cannabis, fermentation is a much faster process, although its limitation regarding the phytochemical broad range of molecules naturally present in cannabis. So far, there is not a consolidated process for this brand-new approach, being an emerging and promising concept for countries in which cultivation of Cannabis sativa L. is illegal. This survey discusses the techniques and microorganisms already established to accomplish the task and those yet in seeing for the future, exploring upsides and limitations about metabolic pathways, toxicity, and downstream recovery of cannabinoids throughout heterologous production. Therapeutic potential applications of cannabinoids and in silico methodology toward optimization of metabolic pathways are also explored. Moreover, conceptual downstream analysis is proposed to illustrate the recovery and purification of cannabinoids through the fermentation process, and a patent landscape is presented to provide the state-of-the-art of the transfer of knowledge from the scientific sphere to the industrial application.

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Section: Biosynthesis Of Phytocannabinoidsmentioning

confidence: 99%

Section: Metabolic Engineering In Silicomentioning

confidence: 99%

Section: Metabolic Engineering In Silicomentioning

confidence: 99%

Section: Metabolic Engineering In Silicomentioning

confidence: 99%

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Converting Sugars into Cannabinoids—The State-of-the-Art of Heterologous Production in Microorganisms

et al. 2022

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“…Engineered S. cerevisiae was successfully used to produce a high level of natural products from other sources, for example, artemisinic acid up to 100 mg/L [71], tetrahydrocannabinolic acid up to 8.0 mg/L, cannabidiolic acid, and tetrahydrocannabivarinic acid up to 4.8 mg/L [72]. Engineered S. cerevisiae also produced some natural products from Streptomyces with high titers, such as gamma aminobutyric acid up to 62.6 g/L [73] and cannabigerolic acid up to 299.8 mg/L [74].…”

Section: Other Hostsmentioning

confidence: 99%

Recent Advances in the Heterologous Biosynthesis of Natural Products from Streptomyces

et al. 2021

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Streptomyces is a significant source of natural products that are used as therapeutic antibiotics, anticancer and antitumor agents, pesticides, and dyes. Recently, with the advances in metabolite analysis, many new secondary metabolites have been characterized. Moreover, genome mining approaches demonstrate that many silent and cryptic biosynthetic gene clusters (BGCs) and many secondary metabolites are produced in very low amounts under laboratory conditions. One strain many compounds (OSMAC), overexpression/deletion of regulatory genes, ribosome engineering, and promoter replacement have been utilized to activate or enhance the production titer of target compounds. Hence, the heterologous expression of BGCs by transferring to a suitable production platform has been successfully employed for the detection, characterization, and yield quantity production of many secondary metabolites. In this review, we introduce the systematic approach for the heterologous production of secondary metabolites from Streptomyces in Streptomyces and other hosts, the genome analysis tools, the host selection, and the development of genetic control elements for heterologous expression and the production of secondary metabolites.

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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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Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast

Cited by 23 publications

References 59 publications

Converting Sugars into Cannabinoids—The State-of-the-Art of Heterologous Production in Microorganisms

Converting Sugars into Cannabinoids—The State-of-the-Art of Heterologous Production in Microorganisms

Recent Advances in the Heterologous Biosynthesis of Natural Products from Streptomyces

Engineering cannabinoid production in Saccharomyces cerevisiae

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