Gene acquisition and plasmid construction

We used HarmOR10 gene which comes from helicoverpa armigera as a receptor for benzaldehyde detection. To test the receptor, we used FUS1 promoter from the yeast GPCR signaling pathway to link with the mCherry gene, forming a reporter gene fragment.

Due to the low GC ratio of the FUS1 promoter fragment, our PCR experiment failed many times. We redesigned the fragment by inserting three base pairs from the Fus1 gene at the start of the mCherry fragment, finally achieved successful amplification.

Gene acquisition and plasmid construction

To ensure the expression efficiency of HarmOR10, we used CRISPR-Cas9 system to knock out the yeast's own GPCR receptor Ste2 in the original strain BY4741-Cas9, and replaced the HarmOR10 gene at the position of the Ste2 gene.

Based on the strain named harm-1 in which we successfully integrate harmOR10 gene, we integrate Fus1p-mCherry gene on the verified yeast gene integration site int21 to construct harmOR10 receptor test strain T1.

We integrated fus1p-mCherry gene into the original strain BY4741-Cas9 to form a positive control strain.

Test of the strain

The fluorescence intensity and biomass of T1 strain was tested after cultivated in full nutrient SCD medium with a benzaldehyde concentration gradient for 24 hours

Optimization

Since no red fluorescence reaction positively correlated with benzaldehyde concentration was detected in strain T1, we process improvement of previous experiments: in the process of integrating HarmOR10 gene to the genome, we replace the original gene promoter by GAL promoter which has high efficiency, and use galactose in 24-hour fluorescence intensity test to induce the expression of harmOR10.

Source of the gene we use

We choose MLPLA(major latex protein-like protein A) and NEPS1(nepetalactone related short chain dehydrogenase 1) coming from the perennial herb Nepeta cataria to catalyze the conversion from the precursor to nepetalactol and the conversion from the nepetalactol to nepetalactone.

Original strain

The starting strain we used was the AJM-3 strain with a low production of nepetalactol.

Gene integration

Integrating the MLPLA gene on the validated yeast gene integration site int21 to construct a high-yielding-nepetalactol strain M1.
Integrating the NEPS1 gene on the int20 gene integration site to form a high-yielding- nepetalactone strain M1-N1

The fermentation experiments

In order to verify the products and yields of each strain during the experiment, we performed multiple rounds of fermentation experiments, the products were detected by GC-MS, the peak times of the nepetalactol and nepetalactone ester were 8.7 min and 9.4 min.

Standard curve

Different concentrations of nepetalactol and nepetalactone were used for GC-MS determination to establish a linear relationship between peak area and product concentration.

Optimization of fermentation time condition

The fermentation experiments of origin strain and M1 strain were carried out, and the concentrations of related products (nepetalactol or nepetalactone) were measured at 24h, 48h and 72h, respectively. It was found that there was no significant difference in the concentration of the three time points, so in the subsequent experiments, what’s more, most of the data measured at 24h were higher than those at the other two time points, so 24h fermentation was used as the sampling time point in the further experiments.

We performed multiple rounds of fermentation experiments on the original strain, M1 strain and M1-N1 strain, and the concentrations of related products in fermentation broth were measured at 24h of fermentation. We found that the integration of the MLPLA gene greatly increased nepetalactol production, and the integration of the NEPS1 gene convert almost all the nepetalactol to nepetalactone.

By-product analysis

From M1 and M1-N1 strains‘ fermentation experiment data, we can find that although the integration of NEPS1 gene resulted in complete conversion of the nepetalactol, nepetalactone yield is far lower than the nepetalactol yield. This indicates that the NEPS1 enzyme may produce by-products in the process of catalyzing the nepetalactol conversation. Thus, we scanned all products of fermentation extract and tried to analyze the possible by-products based on the map, providing the basis for the further modification of enzyme.

After comparing the two full-scan maps, we found that the M1-N1 strain have high peaks at around 10.7 min and 11.2 min, which is not found in the maps of M1 strain. We analyzed the two peaks and get the possible by-products below.

Means of improvement

Preliminary experiments showed that the catalytic efficiency of NEPS1 enzyme was high, and nepetalactol in M1-N1 strain were completely converted to nepetalactone, which could not achieve our purpose of regulating the ratio of nepetalactol and nepetalactone. Therefore, we planned to improve the yield of nepetalactol through metabolic engineering in further experiments. And the promoter GAL1,10 used by the NEPS1 gene should be replaced with a weaker, regulatable promoter, such as the CUP1 promoter.