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

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.


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 play important roles in the metabolic networks, due to various biocatalytic activities such as oxidation, epoxidation, hydroxylation, and demethylation (Stavropoulou et al., 2018). More importantly, 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). As a thoroughly studied model organism, Saccharomyces cerevisiae has advantages of clear genetic background, easy cultivation, and post-translational processing capability (Schuler and Werck-Reichhart 2003). Most importantly, the inner membrane systems of S. cerevisiae allow the functional anchoring of P450s and cytochrome P450 reductases (CPRs) (Hausjell et al., 2018). Therefore, S. cerevisiae is often selected as a preferred host for functional expression of P450s and accordingly biosynthesis of natural products.
Although a variety of P450s have been successfully expressed in S. cerevisiae, low expression level and activity have become the biggest challenge for fundamental and biotechnological application studies of P450s (Jiang et al., 2021). Accordingly, different strategies have been implemented to increase the expression level and/or activity of P450s in S. cerevisiae, including N-terminal truncation, protein molecular modification of P450s through protein engineering, and co-expression with CPRs (Jiang et al., 2021). Unfortunately, the effects of these engineering strategies are often varied case by case. In other words, it is of great demand to develop a generally applicable strategy to improve the functional expression of a wide range of P450s in S. cerevisiae.
In plants, there are many kinds of P450s, interacting with a variety of substrates to participate in the synthesis and degradation of alkaloids, terpenes, flavonoids, fatty acids, plant hormones, and signal molecules (Bolwell et al., 1994). Thus, plants have evolved a complex gene regulation system to control the expression and folding of P450s (Yang et al., 2016). It is speculated that the introduction of key genes involved in the plant regulatory network may have positive effects on the functional expression of a series of P450s in yeast.
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). Firstly, an A. thaliana cDNA library was overexpressed in S. cerevisiae, which was combined with biosensor-based highthroughput screening ( Figure 1A) to identify target genes that can improve the functional expression of a P450 (CYP76AD1) in yeast. Then, the synergistic interactions and molecular mechanisms of these newly identified targets were further explored. Finally, these plant genes were overexpressed to increase the activity of another P450 protein (encoded by CYP736A167) involved in the hydroxylation of α-santalene to Z-α-santalol. The present study promises the establishment of a platform yeast strain for functional expression of a wide variety of P450s.

Strains and Plasmids
The yeast biosensor strain yJS1256 for high throughput screening was kindly provided by Prof. Dueber from the University of California at Berkeley (DeLoache et al., 2015). The A. thaliana cDNA library under the control of ADH1 promoter and ADH1 terminator in plasmid pGADT7-AD was kindly provided by Prof. Zeng Tao from Zhejiang University. To facilitate the construction of the A. thaliana cDNA overexpression library in S. cerevisiae, a helper plasmid pRS416-TEF1p was constructed by cloning TEF1 promoter, upstream and downstream homology arms of the cDNA library, as well as EcoRI and BamHI restriction sites into pRS416. Then pRS416-TEF1p was digested by EcoRI and BamHI and cotransformed with the PCR amplified cDNA library fragments with homology arms into yJS1256 for in vivo assembly ( Figure 1B). The identified plasmids from the A. thaliana cDNA library were isolated using a ZymoPrep Yeast Plasmid Miniprep Kit (Zymo Research, Irvine, CA) and transformed into Escherichia coli DH5α for plasmid amplification. The Z-α-santalol producing S. cerevisiae strain was constructed by integrating four copies of the α-santalene synthase (SAS) gene (Dong et al., 2020) and one copy of CYP736A167 and CPR2 from Santalum album (XII5 locus) into the genome of BY4741 (MATa his△1 leu2△0 met15△0 ura3△0), via CRISPR-Cas9-mediated genome editing technology (Lian et al., 2017;Lian et al., 2019). In addition, the mevalonate (MVA) pathway genes were overexpressed to enhance the precursor supply for Z-α-santalol biosynthesis, with tHMG1-ERG8-ERG13-ERG20-ERG12 overexpression cassettes and EGR10-MVD1-IDI1-tHMG1 overexpression cassettes integrated into X4 and XI3 loci, The betaxanthin biosynthetic pathway starting from L-tyrosine, including a P450 enzyme (CYP76AD1, shown in red) and an L-DOPA dioxygenase (DOD). CYP76AD1 has been determined to be rate-limiting for betaxanthin production, indicating that the production of betaxanthin (yellow color or fluorescence) can function as a biosensor for the expression level of CYP76AD1. (B) Workflow of the genome-scale engineering experiments. The A. thaliana cDNA overexpression library was introduced into the betaxanthin-producing yeast (yJS1256, biosensor strain) via in vivo homologous recombination. Clones with obvious color changes were selected and inoculated into SCD-URA medium in 96 deep-well plates and the fluorescence intensities were measured using a microplate reader. Then the isolated plasmids were re-transformed into the biosensor strain and the corresponding genes were identified by DNA sequencing.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org December 2021 | Volume 9 | Article 764851 respectively. Q5 polymerase, T4 DNA ligase, and all restriction enzymes used were purchased from New England Biolabs (Ipswich, MA). All chemicals were bought from Sigma (Sigma Aldrich, St. Louis, MO) unless otherwise stated.

High-Throughput Screening and Fluorescence Intensity Measurement
The A. thaliana cDNA overexpression library was constructed by the in vivo DNA assembly method in S. cerevisiae. After transformation, 10 6 independent clones were observed on SCD-URA agar plates, indicating at least a 50-fold coverage of the A. thaliana cDNA library. For biosensor-based high-throughput screening, 76 clones with the highest yellow color intensities were selected from SCD-URA agar plates and inoculated into 1 ml SCD-URA medium in a 96 deep-well plate. The yeast strains were pre-cultured for 2 days and then inoculated into fresh SCD-URA medium with an initial OD 600 of 0.1. Mid-log phase yeast cells were collected and diluted 2-fold in ddH 2 O for measuring betaxanthin fluorescence intensity at 498-533 nm using a Tecan microplate reader. The fluorescence intensity (relative fluorescence units; RFU) was normalized to cell density that was determined by the same microplate reader.

Z-α-Santalol Production and Quantification
Z-α-Santalol producing strains were pre-cultured in SCD-URA medium for 2 days, inoculated into 50 ml fresh medium in 250 ml shaker flasks with an initial OD 600 of 0.1, and cultured at 30°C and 250 rpm for 5 days. Then 2 ml yeast cells were collected by centrifuge at 12,000×g for 2 min and resuspended in 700 μL ethyl acetate to be disrupted by bead milling. The cell lysate was centrifuged for 10 min and the supernatant was filtered for GCMS (SHIMADZU, Japan) analysis on a DB-5MS column. 2 μL of each sample was injected with a 20:1 split mode at 280°C. The initial column temperature was 40°C and kept for 3 min. Subsequently, the temperature was increased to 130°C at a rate of 10°C/min, followed by to 180°C at a rate of 2°C/min and to 300°C at a rate of 50°C/min, and finally kept at 300°C for 10 min. The production of α-santalene and Z-α-santalol was quantified using the standard curve method. The α-santalene to Z-α-santalol conversion rate was calculated as

Transcriptomic Analysis
Yeast cells in biological triplicates were grown at 30°C overnight in 5 ml of SCD medium for 2 days and inoculated into 50 ml SCD medium in 250 ml shaker flasks with an initial OD 600 of 0.1. Then cells were harvested in the early stationary phase by centrifugation at 4,000 g for 15 min at 4°C, with the total RNA extracted and sequenced (RNA-Seq) by Shanghai Majorbio Bio-pharm Technology Co., Ltd. RNA-Seq data manipulation and differential gene expression profiling were performed on the free online Majorbio Cloud Platform (www.majorbio.com) with default settings. The RNA-Seq data are available from the NCBI Sequence Read Archive, with an accession number PRJNA760804.

RESULTS
Biosensor-Based High Throughput Screening of A. thaliana cDNA Overexpression Library in S. cerevisiae A major challenge for P450 engineering is the lack of a high throughput screening method. The betaxanthin-producing yeast strain yJS1256 developed by the Dueber group (DeLoache et al., 2015) was employed as a biosensor for high-throughput screening of yeast strains with improved functional expression of P450s. The biosensor is composed of a P450 enzyme mutant (CYP76AD1 W13L−F309L ) from Beta vulgaris and an L-DOPA (dihydroxyphenylalanine) dioxygenase (DOD) from Mirabilis jalapa, which can catalyze the conversion of L-tyrosine to produce betaxanthin ( Figure 1A). In addition, two transporter genes, encoding Qdr2p and Yor1p, were deleted to accumulate betaxanthin intracellularly (Savitskaya et al., 2019). With the introduction of an additional copy of CYP76AD1 W13L−F309L , the production of betaxanthin was significantly improved, indicating that betaxanthin biosynthesis in yeast was limited by CYP76AD1 (Supplementary Figure S1), which was consistent with the previous report (DeLoache et al., 2015). Considering the color and fluorescence characteristics of betaxanthin and rate-limiting of CYP76AD1 in betaxanthin biosynthesis, a biosensor-based high throughput screening method was established to isolate yeast mutants with improved production of betaxanthin ( Figure 1B). Then the A. thaliana cDNA library was cloned into the single-copy plasmid pRS416 and overexpressed in S. cerevisiae. After yeast transformation, 20 clones were randomly selected for diagnostic PCR verification to investigate the diversity of the A. thaliana cDNA library in S. cerevisiae (Supplementary Figure S2A). Clones with obvious color changes were selected and inoculated into SCD-URA medium to measure the change in fluorescence intensities (Supplementary Figure S2B). Compared with the control strain containing empty plasmid, the fluorescence intensities of 25 clones were increased to varying degrees. The plasmids from these clones were extracted and sequenced to identify the candidate genes which could improve the production of betaxanthin in yeast.

Verification of the Candidate Genes for Improved Functional Expression of CYP76AD1
After the extraction of plasmids from the isolated clones, they were re-transformed into the biosensor strain yJS1256 to verify the ability of these genes to improve the functional expression of Frontiers in Bioengineering and Biotechnology | www.frontiersin.org December 2021 | Volume 9 | Article 764851 CYP76AD1 and accordingly the production of betaxanthin in yeast. Three plasmids, A3, A6, and E9 harboring the genes of A. thaliana glycine-rich RNA-binding protein (AtGRP7), membrane steroid binding protein 1 (AtMSBP1), and A. thaliana CO-like four protein (AtCOL4), showed 1.32-, 1.86-, and 1.10-fold improvement in betaxanthin production, respectively (Figures 2A,B). AtGRP7 (A3) participates in the negative feedback loop of circadian rhythm regulation and pre-mRNA splicing and plays an important role in a complex network of transcripts in A. thaliana (Koster et al., 2014;Streitner et al., 2010). AtMSBP1 (A6), an ER-located protein, demonstrates steroid-binding activity in vitro and is involved in the inhibition of elongation and brassinosteroid signaling (Shi et al., 2011). More importantly, MSBP1 and its homologue MSBP2 can form homomers and heteromers on the ER membrane, which interact with three monolignin P450 enzymes to form MSBP-P450 protein complexes, thereby improving the stability and activity of P450s as well as the production of lignin (Gou et al., 2018). AtCOL4 (E9), a putative novel transcription factor, has transcriptional activation activity and is an important regulator of plant tolerance to abiotic stress (Min et al., 2015). Afterwards, the synergistic or additive interactions among AtGRP7, AtMSBP1, and AtCOL4 were further investigated. Considering the stability of heterologous genes, these gene expression cassettes were integrated into XII5, X4, and XI3 loci of the yeast genome for combinatorial optimization ( Figure 2B). When AtGPR7 (A3) or AtCOL4 (E9) was expressed together with MSBP1 (A6), synergistic and/or additive effects on the production of betaxanthin were observed, representing 2.21-and 2.18-fold higher than the control strain, respectively. In contrast, the effect of simultaneous expression of AtGPR7 (A3) and AtCOL4 (E9) was not obvious. The highest production of betaxanthin was achieved in the strain with these three genes (A3-A6-E9) being expressed simultaneously, representing a 2.36-fold improvement over the control strain. These results indicated that there might be synergistic interactions between AtMSBP1 and AtGRP7, as well as AtMSBP1 and AtCOL4, but not AtGRP7 and AtCOL4.

Effects of the A. thaliana Target Gene Overexpression on the Synthesis of Z-α-Santalol in S. cerevisiae
To further demonstrate the general applicability of AtGRP7, AtMSBP1, and AtCOL4 from A. thaliana in improving the functional expression of P450s, these genes were overexpressed in a Z-α-santalol producing yeast strain and their effects on the functional expression of CYP736A167, whose gene product catalyzes the hydroxylation of α-santalene to produce Z-α-santalol, was investigated ( Figure 3A). Based on the previously constructed α-santalene producing strain (Dong et al., 2020), CYP736A167 and CPR2 were further integrated into the yeast genome to produce Z-αsantalol. To increase the accumulation of α-santalene, the substrate of CYP736A167 and the precursor of Z-α-santalol biosynthesis, genes involved in MVA pathway were overexpressed, including tHMG1, ERG8, ERG10, ERG12, ERG13, ERG20, IDI1, and MVD1. As shown in Supplementary Figures S3, S4, α-santalene was accumulated to a relatively high level, indicating that the production of Z-α-santalol was limited by the low activity of CYP736A167. The introduction of AtGRP7 (A3), AtMSBP1 (A6), and AtCOL4 (E9) all demonstrated positive effects on the hydroxylation of α-santalene to Z-α-santalol, with the conversion rate increased for 1.89-, 1.71-, and 1.73-fold, respectively. What's more, when these three genes (A3-A6-E9) were overexpressed simultaneously (integrated into XI2, XII2, and XI3 loci of the yeast genome), the conversion rate was the highest, representing a 2.97-fold improvement when compared with the control strain ( Figure 3B). Surprisingly, the titer of Z-α-santalol was not increased as significantly as the α-santalene to Z-α-santalol conversion rate (Supplementary Figure S5). To figure out the possible reasons, the expression level of MVA pathway genes were profiled. As shown in Supplementary Figure S6, all the MVA pathway genes except for ERG10 were down-regulated to different degrees. In other words, the advantage of overexpressing AtGRP7 (A3), AtMSBP1 (A6), and AtCOL4 (E9) in improving the functional expression of CYP736A167 should be combined with other metabolic engineering strategies to enhance the MVA pathway fluxes, which has been well established in yeast.

DISCUSSION
In recent years, there is a growing interest in establishing yeast as cell factories for the production of plant natural products. Considering the significance in natural product biosynthesis as well as the low activity of P450s, various strategies have been attempted to address the challenges in functional expression of P450s in yeast. In contrast to previous strategies, such as N-terminal truncation and protein molecular engineering, the establishment of platform strains, especially those with ER expansion, has attracted more attention (Jiang et al., 2021). As most eukaryotic P450s and CPRs are membrane-bound proteins and anchored to the ER outer membranes, ER expansion has been determined to be a generally applicable strategy to improve the functional expression of P450s Emmerstorfer et al. found that the overexpression of ICE2 could increase the stability and activity of P450/CPR (Emmerstorfer et al., 2015). DNA repair and recombination gene RAD52 was also reported to improve the functional expression level of P450s (Wriessnegger et al., 2016). The overexpression of IN O 2 and the deletion of PAH1 and OPI1, which had been found to enlarge the ER, could also improve the functional expression of P450s (Schuck et al., 2009;Arendt et al., 2017;Kim et al., 2019). Therefore, the identification of novel genetic engineering targets is a promising Considering the complex regulatory machinery in P450 expression and folding, genome-scale engineering has been proved as an effective strategy to overcome our limited knowledge and identify new engineering targets (Lian et al., 2018;Lian et al., 2019). Among various genome-scale engineering strategies, cDNA overexpression library is the simplest and most commonly employed, with the advantages of high-level expression and the introduction of heterologous genes to enable improved and even novel phenotypes of interests. Baumann et al. successfully screened two new targets that could increase the titer of octanoic acid by overexpressing the yeast cDNA library in the octanoic acid-producing S. cerevisiae (Baumann et al., 2021). Similarly, Shi et al. overexpressed the Yarrowia lipolytica cDNA library and identified key targets that could improve the production of fatty acids in S. cerevisiae (Shi et al., 2016). Encouraged by these successful examples, the present study screened the A. thaliana cDNA library to identify the key genes that were closely related to functional expression of P450s. Using the biosensor-based high throughput screening method, three target genes from A. thaliana (AtGRP7, AtMSBP1, and AtCOL4) were identified and proved to be effective in improving the functional expression of CYP76AD1 and CYP736A167, whose gene products had been determined to be ratelimiting for the production of betaxanthin and Z-α-santalol, respectively. The positive role of these genes in improving the functional expression of P450s with different origins (CYP76AD1 from B. vulgaris and CYP736A167 from S. album) indicated the potential of general applicability in functional expression of a wide variety of P450s.
In conclusion, an A. thaliana cDNA library was successfully expressed in yeast, and three target genes AtGRP7, AtMSBP1, and AtCOL4 from A. thaliana were identified to be effective in improving the functional expression of CYP76AD1 and accordingly the production of betaxanthin. More importantly, these target genes could also effectively increase the activity of CYP736A167, catalyzing the hydroxylation of α-santalene to produce Z-α-santalol, whose conversion rate was increased for 2.97-fold when these three genes were overexpressed simultaneously. The target genes identified in the present study promise the construction of a platform yeast strain for functional expression of P450s and accordingly production of natural products.

DATA AVAILABILITY STATEMENT
The raw reads of the NGS data were deposited into the NCBI Sequence Read Archive (SRA) database (accession number: PRJNA760804).

AUTHOR CONTRIBUTIONS
LJ and JL designed the study, analyzed the data, and wrote the paper. LJ, CD, and TL conducted the experiments. All authors revised and approved the manuscript.