Biosynthesis of cannabigerol and cannabigerolic acid: the gateways to further cannabinoid production.

Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway of Cannabis sativa generates cannabigerolic acid (CBGA), the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analog of this compound, cannabigerol (CBG), represents an alternate gateway into the cannabinoid space as a substrate either to non-canonical cannabinoid synthase homologs or to synthetic chemical reactions. Herein, we describe the identification and repurposing of aromatic prenyltransferase (AtaPT), which when coupled with native enzymes of C. sativa can form an Escherichia coli production system for CBGA in cell lysates and CBG in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics toward CBGA production for subsequent use in a proof-of-concept lysate system. For the first time, we show a synthetic biology platform for CBG biosynthesis in E. coli cells by employing AtaPT under an optimized microbial system. Our results have therefore set the foundation for sustainable production of well-researched and rarer cannabinoids in an E. coli chassis. Graphical Abstract.

Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in type III polyketide synthases.

In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three-step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 â†’ C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 â†’ C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an 'aldol switch' is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable 'aldol switch' consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue-specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high-resolution structural analysis of multiple enzyme homologues with classical site-directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. ENZYMES: TKS, EC 2.3.1.206; OAC, EC 4.4.1.26; chalcone synthase, EC 2.3.1.74; stilbene synthase, EC 2.3.1.95; 2-PS, EC 2.3.1.-. ACCESSION NUMBERS: The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number 6GW3.

The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803.

Synthetic metabolism allows new metabolic capabilities to be introduced into strains for biotechnology applications. Such engineered metabolic pathways are unlikely to function optimally as initially designed and native metabolism may not efficiently support the introduced pathway without further intervention. To develop our understanding of optimal metabolic engineering strategies, a two-enzyme ethanol pathway consisting of pyruvate decarboxylase and acetaldehyde reductase was introduced into Synechocystis sp. PCC 6803. We characteriseda new set of ribosome binding site sequences in Synechocystis sp. PCC 6803 providing a range of translation strengths for different genes under test. The effect of ribosome-bindingsite sequence, operon design and modifications to native metabolism on pathway flux was analysed by HPLC. The accumulation of all introduced proteins was also quantified using selected reaction monitoring mass spectrometry. Pathway productivity was more strongly dependent on the accumulation of pyruvate decarboxylase than acetaldehyde reductase. In fact, abolishment of reductase over-expression resulted in the greatest ethanol productivity, most likely because strains harbouringsingle-gene constructs accumulated more pyruvate decarboxylase than strains carrying any of the multi-gene constructs. Overall, several lessons were learned. Firstly, the expression level of the first gene in anyoperon influenced the expression level of subsequent genes, demonstrating that translational coupling can also occur in cyanobacteria. Longer operons resulted in lower protein abundance for proximally-encoded cistrons. And, implementation of metabolic engineering strategies that have previously been shown to enhance the growth or yield of pyruvate dependent products, through co-expression with pyruvate kinase and/or fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, indicated that other factors had greater control over growth and metabolic flux under the tested conditions.