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

Lihong Jiang,Zeng Tao,Tengfei Liu,Handing Wang,Jiazhang Lian,Chang Dong,Yi Shi,Yan Liang

doi:10.3389/fbioe.2021.764851

Lihong Jiang, Zeng Tao + Show 6 more

Open Access

https://doi.org/10.3389/fbioe.2021.764851

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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.

Highlights

Cytochrome P450 enzymes (P450s), first discovered in the early 1960s, are a superfamily of heme-thiolate proteins widely existing in animals, plants, and microorganisms (Elfaki et al, 2018; Guengerich et al, 2016)
The present study aims to identify key genes that can improve the functional expression of P450s in S. cerevisiae through screening a genome-scale cDNA library from Arabidopsis thaliana, a model plant widely used in plant genetic, cellular, developmental, and molecular biology researches (Hayashi and Nishimura 2006)
The biosensor is composed of a P450 enzyme mutant (CYP76AD1W13L−F309L) from Beta vulgaris and an L-DOPA dioxygenase (DOD) from Mirabilis jalapa, which can catalyze the conversion of L-tyrosine to produce betaxanthin (Figure 1A)

Summary

Introduction

Cytochrome P450 enzymes (P450s), first discovered in the early 1960s, are a superfamily of heme-thiolate proteins widely existing in animals, plants, and microorganisms (Elfaki et al, 2018; Guengerich et al, 2016). P450s are involved in the biosynthesis of many natural products, such as opioids (Galanie et al, 2015), artemisinic acid (Paddon et al, 2013), and glycyrrhetinic acid (Zhu et al, 2018). The Improving P450 Expression in Yeast inner membrane systems of S. cerevisiae allow the functional anchoring of P450s and cytochrome P450 reductases (CPRs) (Hausjell et al, 2018). S. cerevisiae is often selected as a preferred host for functional expression of P450s and biosynthesis of natural products

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