Transcriptome Analysis Reveals a Promotion of Carotenoid Production by Copper Ions in Recombinant Saccharomyces cerevisiae

We previously constructed a Saccharomyces cerevisiae carotenoid producer BL03-D-4 which produced much more carotenoid in YPM (modified YPD) media than YPD media. In this study, the impacts of nutritional components on carotenoid accumulation of BL03-D-4 were investigated. When using YPM media, the carotenoid yield was increased 10-fold compared to using the YPD media. To elucidate the hidden mechanism, a transcriptome analysis was performed and showed that 464 genes changed significantly in YPM media. Furthermore, inspired by the differential gene expression analysis which indicated that ADY2, HES1, and CUP1 showed the most remarkable changes, we found that the improvement of carotenoid accumulation in YPM media was mainly due to the copper ions, since supplementation of 0.08 mM CuSO4 in YPD media could increase carotenoid yield 9.2-fold. Reverse engineering of target genes was performed and carotenoid yield could be increased 6.4-fold in YPD media through overexpression of ACE1. The present study revealed for the first time the prominent promotion of carotenoid yield by copper ions in engineered S. cerevisiae and provided a new target ACE1 for genetic engineering of S. cerevisiae for the bioproduction of carotenoids.


Introduction
Carotenoids are a large family of colored compounds naturally present in plants, algae, fungi, and bacteria. These compounds are known antioxidants that have a potentially beneficial effect on human health and may be used for nutrition, food, and cosmetic applications [1]. Currently, chemical synthesis and natural extraction from plants or algae are the major processes for carotenoid production in industry. However, chemical synthesis is very difficult due to the structural complexity of most carotenoids, and natural extraction based on solvent from plants and algae is confronted with a lot of challenges such as unpredictable feedstock availability, organic pollutants, and low yields [2]. Thus, the biosynthesis of certain carotenoids through homologous or heterologous microorganisms is becoming more and more attractive [3].
With the rapid development of metabolic engineering and synthetic biology, engineering microorganisms for the heterologous production of carotenoids has become a more sustainable process. Thus, there have been extensive studies to engineer microbial cell factories for the production of carotenoids [4]. The majority of these efforts were focused on the optimization of endogenous pathways, the introduction of heterologous genes to boost metabolic flux, and balancing cofactors [5]. Multiple strategies have been adopted to enhance the production of carotenoids in S. cerevisiae, such as boosting the metabolic flux towards the mevalonate (MVA) pathway through overexpression of truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase (tHMG1) [6], increasing the supply of precursors (acetyl-CoA) through downregulation of squalene synthase (Erg9) or overexpression of acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (ACS) [7,8]. Besides, strengthening the pathways such as the TCA cycle and pentose phosphate (PPP) pathway could increase the cofactor supply which carotenoid biosynthesis needs [9]. However, according to previous reports, little was demonstrated about the impact of nutritional components on carotenoids yield of recombinant strains, except one case that carotenoid production increased 7-fold using fructose as the carbon source in engineered E. coli [10].
Previously, we engineered a synthetic pathway for carotenoid biosynthesis (using lycopene as the model carotenoid) in S. cerevisiae and gained a higher production using the YPM media [11,12]. In this study, we aim to determine the impacts of nutritional components on the carotenoid yield of BL03-D-4 generated from our prior work, and then attempt to uncover the potential mechanisms of the promoted carotenoid production associated with supplementation of nutritional components by transcriptome analysis.

Strains and Cultivation
The original strain, engineered strains, and plasmids used in this study are listed in Table S1. Primers are provided in

Genetic Manipulation
CRISPR/Cpf1 system was applied for ADY2 and ACE1 deletion, and overexpression of HES1, ACE1, CUP1, and SOD1 by genomic integration. The pHCas9M-gRNA plasmid (Molecular Cloud: MC_0000739) was used for Cpf1-based genetic modifications by replacing the sgRNA and Cas9 on plasmid pHCas9M-gRNA with Cpf1 and crRNA from plasmid pCSN067 (Addgene#101748) [13]. The homologous arms of target genes under the yeast promoter Cit1 or TEF2 and native terminators were used as donor DNA. The edited plasmid including the guide RNA that targeted the chromosomal site was cotransformed into yeast cells with the donor DNA according to previous protocol [14]. Cells were screened on the YPD plate supplemented with 400 µg mL −1 G418. Positive colonies were confirmed by diagnostic PCR.
Growth was determined according to the OD 600 value and correlated to cell dry weight (CDW) by the CDW/OD 600 standard curve (y = 0.184x + 0.891 (x is OD 600 , y is CDW, R 2 = 0.992)).

Analytical Methods
Carotenoids were extracted using the method described in our previous work [11]. Briefly, strains cultures were centrifuged, washed, resuspended in 3 mol L −1 HCl boiling for 4 min, and cooled in an ice bath for 3 min. Then cell debris was washed twice, resuspended in acetone, ultrasonic extracted, and followed by centrifugation. The acetone supernatant was used for measuring total carotenoid by using a UV-VIS spectrometer (PerkinElmer Lambda 45, Waltham, MA, USA) at 470 nm. The extinction coefficient was adopted in acetone using an absorbance 1% 1 cm of 3450 [12]. Copper concentration was measured using the Copper (Cu) Colorimetric Assay Kit (Elabscience Biotechnology Co., Ltd., Wuhan, Hubei, China).

Transcriptome Analysis
Total RNA of different yeast cells was isolated after about 24 h cultivation using the HiPure Yeast RNA Kit (Magen, Guangzhou, Guangdong, China) according to the manufacturer's protocol. Transcriptome analysis was performed by Majorbio (Shanghai Majorbio Bio-pharm Technology Co., Ltd.) through RNA sequencing. Briefly, the RNA quality was determined by 2100 Bioanalyser (Agilent, Santa Clara, CA, USA) and quantified using the ND-2000 (NanoDrop Technologies, Tokyo, Japan). The RNA-seq transcriptome library was prepared following the TruSeqTM RNA sample preparation kit from Illumina (San Diego, CA, USA). Messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation, and "A" base addition according to Illumina's library construction protocol. All raw data for RNA-seq was deposited into NCBI (GEO accession number GSE164229). Transcriptome data analysis is provided in Table S3. The quantitative analyses of gene expression were performed using the Transcripts Per Million (TPM) method by RSEM for each sample. The data were analyzed on the free online platform of the Majorbio Cloud Platform (www.majorbio.com).

Quantitative PCR
The total RNA was extracted from each yeast strain cultivated in YPD2 media for 24 h using the above kit, referencing the manual of application. The RNA samples were reversely transcribed using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, Nanjing, Jiangsu, China). Quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) on a QuantStudio 6 Flex Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). The internal control gene ACT1 was chosen to normalize the different samples and the relative gene expression analysis was performed using the 2 −∆∆CT method.

Significant Promotion of Carotenoid Accumulation is Presented in a Modified YPD Medium
In our experimental experience, we found that the carotenoid yield in engineered S. cerevisiae was fluctuant using different lots of yeast extraction and the engineered strains could gain a stably higher carotenoid in the YPM media [11]. To clarify the impacts of nutritional components of the YPM medium on carotenoid yield, we compared the biomasses and carotenoid accumulations of BL03-D-4 growing on different YPD and YPM media. As illustrated in Figure 1A, obvious different growth and carotenoid accumulations were obtained when strains were cultured in different media. Both manufacturers (Oxoid and Angel) and lots of yeast extraction had significant effects on carotenoid accumulation indi-cating that there were different compounds in these different yeast extractions ( Figure S1). When cultured in the YPM media, the final carotenoid yield was 18.8 mg g −1 CDW, which was distinctly improved and over 10-fold higher compared to the yield of 1.85 mg g −1 CDW in the YPD2 medium ( Figure 1A). The maximum biomass reached 4.1 g L −1 of culture in the YPD2 media. When using YPM, the final cell mass could reach 7.0 g L −1 , which was 1.7-fold of that in the YPD2 media. The time-courses of carotenoid fermentation in YPD2 and YPM media were also measured ( Figure 1B); the growth curves and yield curves represented obvious differences. These results indicated that some substances in the YPM medium could promote cell growth and carotenoid biosynthesis. However, the reason behind this manner has not yet been known to necessitate further studies. For the sake of gaining a deeper understanding of the mechanisms, comprehensive analyses such as gene expression can be executed.

Transcriptome Analysis Reveals Multiple Differentially Expressed Genes
In this study, we investigated and analyzed the comprehensive transcriptome data of BL03-D-4 in YPD2 and YPM media. Strains typically reach a steady state level of transcription in the stationary phases [11]. Thus, gene expression patterns of BL03-D-4 in stationary phases (about 24 h) cultured with these two different media were compared using transcriptome sequencing. The results of RNA-seq data analysis are supplemented in Table S3 and showed that 464 genes changed their expression levels significantly (p < 0.05; |Log2FC| ≥ 1). Among aforesaid genes, 203 genes were upregulated and 261 were downregulated compared to using the YPD media.
KEGG pathway analysis was performed to search genes that might play key roles in the promotion of carotenoid accumulation. KEGG pathway analysis showed that genes in the glyoxylate and dicarboxylate metabolism, steroid biosynthesis, fatty acid degradation, glycolysis/gluconeogenesis, and PPP pathways changed significantly (Figure 2). These results were not surprising since the glycolysis and PPP pathways could provide energy for cell growth and cofactors for carotenoid biosynthesis [15]. Facilitation of steroid and fatty acid for carotenoid accumulation has been well studied previously [16], and interestingly these pathways were also shown to be clustered in this study. In particular, as shown in Figure 2C, most genes in the ergosterol biosynthetic pathway (ERG) containing ERG1, ERG11, ERG25, ERG26, ERG27, ERG6, ERG2, ERG3, ERG5, and ERG4 were significantly upregulated in the YPM media. This gene expression pattern of the ergosterol biosynthetic pathway was similar to a previous study which indicated that deletion of the acetate transporter gene ADY2 could improve the tolerance of S. cerevisiae against multiple stresses including acetic acid, ethanol, and hydrogen peroxide stresses [17]. The cell membrane of microorganisms contained a variety of sterols including ergosterol, fecosterol, and zymosterol [18]. Ergosterol played a key role in maintaining yeast membranes stabilization and was involved in carotenoid accumulation [16]. Besides, ergosterol biosynthesis showed facilitation for growth under ethanol stress [19]. Therefore, optimization of the sterol composition was an effective method of regulating membrane lipid homeostasis through changing the content of particular sterols and manipulating the sterol categories present [20,21]. Furthermore, sterol content has been reformed through overexpression of Erg3, Erg2, and Erg5 in sterol biosynthetic pathways [22]. However, based on the literature, the main factors that regulate ERG gene expression are the levels of unsaturated fatty acids, oxygen, and osmotic stress, and ERG gene expression is coordinated by sterol regulatory element (SRE)-binding proteins Upc2 and Ecm22, the heme-binding protein Hap1, and the repressor factors Rox1 and Mot3 [23]. We were interested in the significantly changed genes since these genes would provide indirect clues of the mechanisms that caused different carotenoid yields in different media. Among dramatically upregulated genes involved in steroid biosynthesis, HES1 encoded a protein that was implicated in the regulation of ergosterol biosynthesis, and it was one of the sterol transporters which transported sterol from the endoplasmic reticulum (ER) to the mitochondria [24]. On the other hand, acetate transporter gene ADY2, which was responsive for zinc ions, was downregulated 8-fold. Besides, the CUP1-2 gene, which encoded copper metallothionein, was upregulated about 94-fold. It is therefore of interest to study whether supplementation of zinc or/and copper ions can improve the carotenoid yield of BL03-D-4.

Copper Ions Play a Key Role in the Carotenoid Accumulation of BL03-D-4
To find out the key factor of the promotion of carotenoid accumulation, YPD media supplemented with different chemicals were used for carotenoid fermentation. As shown in Figure 3A, salt (10 g L −1 KH 2 PO 4 , 2.5 g L −1 MgSO 4 , 3.5 g L −1 K 2 SO 4 , 0.25 g L −1 Na 2 SO 4 ) and TMS (trace metal solution) had a promoting effect on carotenoid production. Better results were observed in the YPD2 medium supplemented with zinc (0.08 mM) and copper ions (0.08 mM). Especially, supplementation of copper ions could account for 90% of the improvement, and even gain lower biomass compared to using the YPM media. Furthermore, different concentrations of copper presented different promotions, while a very high concentration of copper was toxic to cells ( Figure 3C). It is worth mentioning that simultaneous supplementation of zinc and copper ions could increase the carotenoid yield by 9.7-fold to 19.8 mg g −1 CDW which exceeded the promotion of using the YPM media. It was likely that copper ions also influenced carotenoid biosynthesis by upregulating genes in the sterol biosynthetic pathway since there were no other significantly changed genes in any pathway like ergosterol ( Figure 2C). Figure 3 shows that those up to 0.08 mM copper-added media displayed good growth and carotenoid yield of BL03-D-4, while those with up to 1.6 mM copper in the media caused side effects. These findings were quite similar to the results of a previous study in microalgae which showed that the addition of a suitable concentration of copper in the media stimulated microalgal growth, as well as lutein content [25]. We also measured the copper concentration of different yeast extractions and found that there were significant differences in copper concentration of these yeast extractions ( Figure 3B). This result added to the growing body of evidence that copper ions are responsible for the promotion of carotenoid production. To verify whether the facilitation of copper ions was general, DM media and another carotenoid producer SC106 were used. As shown in Figure 3, Figure S1 (shake-flask fermentations), and Figure S1B,C (spot tests), copper ions could also facilitate carotenoid accumulation in DM media, although the carotenoid yield and cell masses were very low. Interestingly, the carotenoid yield of SC106 was increased 3.2-fold from xylose-glucose mixtures in YPD2 media supplemented with 0.08 mM copper ions.

ACE1 Overexpression Remarkably Improves Carotenoid Accumulation of BL03-D-4
To determine whether the selected targets were conducive to the promotion of carotenoid accumulation, gene deletion and gene overexpression were conducted in BL03-D-4, followed by the analysis of growth and carotenoid yield in YPD2 media. A single-gene knockout mutant of ADY2 and ACE1 were constructed using the CRISPR/Cpf1 system. The HES1, ACE1, CUP1, and SOD1-overexpressed strains were obtained, respectively, through integrating the overexpression cassettes in the 308a site [26]. As shown in Figure 4, the deletion of ADY2 and overexpression of HES1 did not increase the carotenoid accumulation. Interestingly, ACE1 which is the activating factor of CUP1 overexpressed strain MO3 can accumulate 6.4-fold higher carotenoid than the control strain, and this amount of increase accounts for over 60% of the carotenoid yield when using YPM media. However, no significant difference was observed in overexpression of ACE1 under the control of a stronger promoter TEF2. Since the facilitation partially accounts for the carotenoid promotion, these results prompted the speculation that the promotion of carotenoid accumulation might be regulated by a multi-gene which was responsible for zinc and copper ions. We also overexpressed the CUP1 gene; however, no facilitation was observed (Figure 4). We sequenced the genome of BL03-D-4 and the alterations in the copy numbers of the CUP genes were not observed. The fastq DNA-seq data were deposited in the Genome Sequence Archive (GSA) server at the BIG Data Center in Beijing Institute of Genomics (GSA accession No. CRA003704). It is worth mentioning that ACE1 also activates in response to copper other genes such as CRS5, SOD1, and FET3/FTR1 [27]. We have quantified the amount of these genes in MO3 using qPCR, and SOD1 represented remarkable upregulation. Furthermore, SOD1 was also overexpressed, and carotenoid yield increased 2.6-fold. This may provide clues to uncover the molecular mechanism for carotenoid promotion. The dataset generated during the current study are available in Table S4. To the best of our knowledge, our study demonstrated for the first time that the addition of copper and/or zinc ions had a significant promotion of carotenoid accumulation in S. cerevisiae, and the simultaneous supplementation of zinc and copper ions had a synergetic effect on carotenoid accumulation.

Supplementary Materials:
The following are available online at https://www.mdpi.com/2076-260 7/9/2/233/s1, Figure S1: Shake-flask fermentations and normalized spot tests, Table S1: Strains and plasmids used in this study, Table S2: Primers used in this study, Table S3: RNA-seq data analysis, Table S4: Raw data used in this study. Data Availability Statement: All raw data for RNA-seq were deposited into NCBI (GEO accession number GSE164229) and fastq DNA-seq data of BL03-D-4 were deposited in the Genome Sequence Archive (GSA) server at the BIG Data Center in Beijing Institute of Genomics (GSA accession No. CRA003704).

Conflicts of Interest:
The authors declare no conflict of interest.