Synthesis of cembratriene-ol and cembratriene-diol in yeast via the MVA pathway

Background Cembranoids are one kind of diterpenoids with multiple biological activities. The tobacco cembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis. Results We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20. Conclusions We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.

Synthesis of cembratriene-ol and cembratriene-diol in yeast via the MVA pathway

Background: Cembranoids are one kind of diterpenoids with multiple biological activities. The tobacco cembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis.Results: We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20.Conclusions: We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.

Modified Enzyme Substrates for the Detection of Bacteria: A Review

The ability to detect, identify and quantify bacteria is crucial in clinical diagnostics, environmental testing, food security settings and in microbiology research. Recently, the threat of multidrug-resistant bacterial pathogens pushed the global scientific community to develop fast, reliable, specific and affordable methods to detect bacterial species. The use of synthetically modified enzyme substrates is a convenient approach to detect bacteria in a specific, economic and rapid manner. The method is based on the use of specific enzyme substrates for a given bacterial marker enzyme, conjugated to a signalogenic moiety. Following enzymatic reaction, the signalophor is released from the synthetic substrate, generating a specific and measurable signal. Several types of signalophors have been described and are defined by the type of signal they generate, such as chromogenic, fluorogenic, luminogenic, electrogenic and redox. Signalophors are further subdivided into groups based on their solubility in water, which is key in defining their application on solid or liquid media for bacterial culturing. This comprehensive review describes synthetic enzyme substrates and their applications for bacterial detection, showing their mechanism of action and their synthetic routes.