Catnip can be "brewed" through bioengineering to achieve a large-scale production capacity of 153 mg/L
At present, the most widely used and most effective mosquito repellent in the world is DEET, but because some mosquito populations have developed resistance to DEET, it is urgent to find a suitable alternative.
Previous studies have shown that catnip, which makes cats "hey", has a powerful repellent effect on insects (especially mosquitoes), and has been recognized as a natural substitute for traditional synthetic chemical insecticides (DEET).
The key ingredient in catnip to repel mosquitoes is nepetalactone, which is a key ingredient for mosquito repellent.
Nepetalactone has a special effect on approximately 75% of cats. The cat's response to this substance is related to genes, and the location of perception is the olfactory epithelium. For humans, nepetalactone has many milder effects and can be used as weak hypnotics, anticonvulsants, antipyretics and antibacterial drugs.
However, due to the insufficient efficiency and yield of nepetalactone directly extracted from catnip, it cannot be applied to large-scale production.
For many years, scientists have been committed to using the eukaryotic host Saccharomyces cerevisiae to synthesize nepetalactone. At present, they have realized the use of yeast-based whole-cell biocatalysis to produce semi-biosynthetic nepetalactone from low-cost precursors through a four-step enzymatic method. , The subsequent factorial design and response surface method optimization method increased the nepetalactone titer by 5.8 times to 153 mg/L.
However, the realization of this technology was not achieved overnight, and many detours were also taken.
The Vincent Martin team from Concordia University produced 11.4mg/L of precursor geraniol based on the yeast strain at the time in 2016. After the introduction of G10H, the strain successfully produced 5.3mg/L of 10- Hydroxygeraniol, but after expressing 10HGO and ISY, GC-MS could not detect geraniol, 10-hydroxygeraniol, 10-oxogeraniol or nepetalactone, but citronella Obvious peaks of alcohol and 10-hydroxycitronellol fractionated products. Later, it was discovered that ISY is a hybrid enzyme that transfers carbon source flux to the split product.
Subsequently, the John M. Billingsley team from the Department of Chemical and Biomolecular Engineering at the University of California tried to use NEPS1 cyclase/oxidase to produce nepetalactone precursors from the provided substrate 8-hydroxygeraniol, and 8-hydroxyl Geraniol can be easily synthesized using geranyl acetate, thereby improving the economic feasibility of the biological process.
In this study, NEPS1 was used to produce semi-biosynthetic nepetalactone in Saccharomyces cerevisiae using whole-cell biocatalysis, and the biotransformation process parameters were optimized one factor at a time and multivariate; the prediction of the implementation of the RSM method resulted in product drops. Degree increased by 5.8 times.
Strategies for the production of nepetalactone in yeast. The production of nepetalactone requires the expression of NAD+-dependent geraniol oxidoreductase (GOR) and NAD(P)H-dependent iridoid synthase (ISY) (Figure A). Previously expressing GOR and ISY in wild-type yeast strains can clarify the termination of the two metabolic pathways, and now the downstream enzyme NEPS1 can provide nepetalactone through stereoselective cyclization and subsequent oxidation (Figure A). NEPS1 oxidizes the unstable hemiacetal in nepetalactone to lactone, provides a more stable final product, is not easy to open or epimerize, and promotes metabolite analysis and purification of biocatalyst products. The MLS1 promoter and SPG5 terminator were selected to coordinate gene expression. They have roughly the same induction and expression profiles as the promoters and terminator that drive the expression of GOR and ISY. The expression cassette was cloned into GOR and GOR using the yeast homologous recombination method. Downstream of ISY, the plasmid pJB133 (Figure B) was produced and transformed into yeast strain YJB051. The standard single fed-batch biotransformation was carried out. Through GC-MS, the yield of nepetalactone was 26mg/L, corresponding to the yield. It is 0.08 (practical/theoretical),
but they still need to study the characteristics of nepetalactone as a cat attractant, in case we carry this molecule, although it has achieved the effect of repelling mosquitoes, but facing the cat chasing you situation.