Improved Functional Expression of Cytochrome P450s in Saccharomyces cerevisiae Through Screening a cDNA Library From Arabidopsis thaliana

Abstract

Cytochrome P450 enzymes (P450s) are a superfamily of heme-thiolate proteins widely existing in various organisms and play a key role in the metabolic network and secondary metabolism. However, the low expression levels and activities have become the biggest challenge for P450s studies. To improve the functional expression of P450s in Saccharomyces cerevisiae, an Arabidopsis thaliana cDNA library was expressed in the betaxanthin-producing yeast strain, which functioned as a biosensor for high throughput screening. Three new target genes AtGRP7, AtMSBP1, and AtCOL4 were identified to improve the functional expression of CYP76AD1 in yeast, with accordingly the accumulation of betaxanthin increased for 1.32-, 1.86-, and 1.10-fold, respectively. In addition, these three targets worked synergistically/additively to improve the production of betaxanthin, representing a total of 2.36-fold improvement when compared with the parent strain. More importantly, these genes were also determined to effectively increase the activity of another P450 enzyme (CYP736A167), catalyzing the hydroxylation of α-santalene to produce Z-α-santalol. Simultaneous overexpression of AtGRP7, AtMSBP1, and AtCOL4 increased α-santalene to Z-α-santalol conversion rate for more than 2.97-fold. The present study reported a novel strategy to improve the functional expression of P450s in S. cerevisiae and promises the construction of platform yeast strains for the production of natural products.

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

“…On the contrary, the S. cerevisiae strain engineered with four copies of SAS integrated into the genome and all the mevalonate pathway genes overexpressed was able to produce α-santalene with a titer of 77.4 ± 7.3 mg/L under the same fermentation conditions. In other words, STE-1 with a single copy of SAS showed higher α-santalene production than the extensively engineered S.…”

“…Moreover, STE-1 and STE-0 showed comparable growth profiles (Supporting Information, Figure S4), indicating that the overproduction of α-santalene did not result in cytotoxicity and metabolic burdens. On the contrary, the S. cerevisiae strain engineered with four copies of SAS integrated into the genome and all the mevalonate pathway genes overexpressed 31 was able to produce α-santalene with a titer of 77.4 ± 7.3 mg/L under the same fermentation conditions. In other words, STE-1 with a single copy of SAS showed higher αsantalene production than the extensively engineered S. cerevisiae, indicating the advantage of K. phaffii in expressing heterologous genes and accordingly the production of terpenoids.…”

“…phaffii was found to have the highest production of CYPs among several yeast species . In addition, several strategies to improve the functional expression of CYPs in yeast have been established. , Considering the high production of α-santalene and relatively high expression of CYPs, K. phaffii is expected to be the most appropriate microbial cell factory for the production of α-santalol.…”

“…The integration donor helper plasmids (containing the homology arms for genome integration and promoters and terminators for heterologous gene expression) and the corresponding sgRNA plasmids were constructed in our previous studies. 26 The αsantalene biosynthetic pathway genes, including tHMG1, IDI1, ERG20, and SAS, were amplified from the chromosome of the α-santaleneproducing S. cerevisiae strain constructed in our previous studies 31 and subsequently cloned into the restriction sites of the donor helper plasmids using digestion/ligation and/or Gibson assembly methods. All the plasmids used in this study are listed in the Supporting Information, Table S1.…”

“…The α-santalene biosynthetic pathway genes, including tHMG1 , IDI1 , ERG20 , and SAS , were amplified from the chromosome of the α-santalene-producing S. cerevisiae strain constructed in our previous studies and subsequently cloned into the restriction sites of the donor helper plasmids using digestion/ligation and/or Gibson assembly methods. All the plasmids used in this study are listed in the Supporting Information, Table S1.…”