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

[Vee]

Key points

• First time critical review of the heterologous process for recombinant THCA/CBDA production and critical review of bottlenecks and limitations for a bioengineered technical process

• Integrative approach of protein engineering, systems biotechnology, and biochemistry of yeast physiology and biosynthetic cannabinoid enzymes

• Comparison of NphB and CsPT aromatic prenyltransferases as rate-limiting catalytic steps towards cannabinoids in yeast as platform organisms

Introduction
Since the legalization of cannabis products for medicinal use, cannabinoids like tetrahydrocannabinol (THC) and cannabidiol (CBD) get attraction as for direct use or as potential drug candidates for various diseases. Besides isolation from plant material, the biotechnological production of cannabinoids like THC and CBD is an exciting alternative. Over the last years, the genetic blueprint of THC and CBD biosynthesis is understood, and genes have been functionally expressed in various microorganisms as documented in scientific papers. So far, no report has been communicated showing scaled up biosynthesis and how an industrial process must be designed to allow feasible economic production in a bioreactor. In this review, we take for the first time the endeavor to analyze basic biological parameters and to develop concepts and strategies for an engineered process to identify limitations, bottlenecks, and opportunities.


HERE:



Tetrahydrocannabinolic acid (THCA) biosynthesic pathway in C. sativa L.. A total of six enzymes (AAE1, GPPS, OLS, OAC, CBGAS, THCAS)
form THCA from isopentenyl diphosphate (IPP) and dimethylallylphosphate (DMAPP) synthesized in the MEP pathway,
as well as hexanoic acid provided by the fatty acid biosynthesis

Conclusion
A systems biotechnology approach for high yield heterologous THCA biosynthesis is still in its infancy and must be characterized by low titers. Today, the question is not anymore what genes and biocatalysts must be used; the driving force in bioengineering 2.0 is to make kg and not mg to be competitive with plants. After careful analysis of metabolic bottlenecks in the heterologous THCA production, we can identify as critical the following:

1. 1
   Insufficient hexanoic acid formation in the fatty acid biosynthesis
2. 2
   Low acetyl-CoA precursor delivery to the hexanoic acid biosynthesis and the mevalonate pathway
3. 3
   Limiting catalytic activity of NphB or CsPT and THCAS
4. 4
   Insufficient ATP and NADPH regeneration
5. 5
   Ethanol production by Crabtree effect

From our calculations and experimental data, it is obvious that hexanoic acid production is a limiting step for the complete cannabinoid biosynthesis. Here, future metabolic work must resolve this bottleneck towards the delivery of olivetolic acid. By calculation of yeast performance to deliver a sufficient amount of olivetolic acid, a serious bottleneck remains unsolved. Hexanoic acid is at low concentrations and cannot cover the demand for sufficient olivetolic acid production. Only 5 % of HA is converted to OA, while 90 % is olivetol in practice. If further modifications like glycosylation are necessary, a third of THCA is converted according to Elshahawi et al. (2015) and Gachon et al. (2005) following simple rules of glycosylation. Taking into account that no delivery of HA as precursor will satisfy the demand of olivetolic acid for prenylation with GPP, feeding with synthetic OA with all its consequences is only a second option.
A combination of the kinetics provided by Chen et al. (2012) and Förster et al. (2003) are used for the throughput calculation of glycolysis (Förster et al. 2003; Sheng and Feng 2015). The reactions of acetyl-CoA to isopentenyl pyrophosphate and dimethylallyl pyrophosphate were modeled with the data and kinetics of Smallbone et al. (2013) and the geranyl pyrophosphate synthase with the data of Ku et al. (2005).
THCA production by a genetically engineered S. cerevisiae strain was successfully modeled, and a bioprocess was in silico designed that allows prediction of industrial applications. Modeled pathways and obtained data showed clearly that the supply of GPP and olivetolic acid from the primary pathways is limiting the THCA biosynthesis in yeast. Especially low concentration of hexanoic acid is critical for high yield production of THCA. This can be solved but must be considered the top priority for any metabolic engineering of the yeast. We have not discussed other aspects of rational and smart metabolic engineering like transcription factors, import and export of substrate, and THCA as final products, but these must be validated as well in future modeling approaches. With all limitations, S cerevisiae is still the best platform organism we have to produce heterologously cannabinoids, but it is obvious that a perfect running yeast will not compete with C. sativa L. varieties known for THC concentrations of 20 % and more. Plant extraction will stay for a long time the first choice for THC delivery. But the cannabis biotechnology has its unique niche for the production of so-called minor or rare cannabinoids, which are present at very low concentrations of less than 0.5 % in dried flowers. Smart bioengineering will be an attractive alternative to the cannabis plant.

The Bottom Line:
In future the need for corporations to increase profitability it will be easier and very much cheaper to buy your THC/CBD from a lab in Europe derived from yeast than to use extracts from grown plants....as many of us have long suspected

 

[Deez. Nutz]

I guess this is where theory (kinetic modeling) and practice don't necessarily equate to a standardized theoretical outcome.

They didn't address why synthetic OA would not mediate the bottleneck in Biosynthetic pathways of thca production.

Ultimately whether or not this were to become economically readable would be consumer dependent, without a market they would obviously fail.

It would be far more interesting, with further generational testing for plant breeders to select for individual therapeutic cannabinoids, thus rendering synthetic production unnecessary.

 

[Highland Rogue]

very much cheaper to buy your THC/CBD from a lab in Europe derived from yeast than to use extracts from grown plants....as many of us have long suspected

Yeast?

So rather than smoking weed to get high, we're all gonna be puffing on a piece of bread !?
Those fuckers!

 

[Vee]

Above is the McDonald/Coke Cola, idea of selling THC type foods,

but I think we will always have the old schoolers with dirt and trees

selling into a market similar to today's wine/vinters market

 

[Oreguhnism]

Other cannibinoids, acetyls or any other type of chemical produced in situ from an environment, are they produced as well?

It reminds me of high thc dabs that are flavorless but will get you ultra high for ten minutes then fall, or a well grown random plant with unusual terpenes will make the "high" complex more nuanced aka trip weed.

It seems like a stab at capatalism without the expense of exposing it to the medical community.....cannabis plants also produce thc, lol....why grow something else with less?

 

[H.A.F.]

It seems like a stab at capatalism without the expense of exposing it to the medical community.....cannabis plants also produce thc, lol....why grow something else with less?

It sounds like capitalism doing what capitalism does. See a need - fill the need. THC et.al.
People want to get high
Costco can't sell weed
Whoever can market a legal "THC replacement" first makes bank.
No one that has access to actual weed will bother with it other than out of curiosity

 

[Deez. Nutz]

I can see it having a place in pharmaceuticals, such as in the production of pure thc for therapeutics, eg. Marinol. Not necessarily in the realm recreational use.