**Isolation and Analysis of the** *Cppsy* **Gene and Promoter from** *Chlorella protothecoides* **CS-41**

**Meiya Li 1,2,†, Yan Cui 1,†, Zhibing Gan 1, Chunlei Shi 1,\* and Xianming Shi 1**


Academic Editor: Graziano **Riccioni**

*Received: 25 June 2015 / Accepted: 9 September 2015 / Published:* 

**Abstract:** Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the *psy* gene from *Chlorella protothecoides* CS-41, designated *Cppsy*, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid\_Biosyn\_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg2+ binding sites. The *Cppsy* gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the 

*Cppsy* expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in *C. protothecoides*. 

**Keywords:** *Chlorella protothecoides* CS-41; phytoene synthase; *Cppsy*; promoter 

## **1. Introduction**

Lutein is one of more than 750 known naturally occurring carotenoids, and is synthesized by 

higher plants, bacteria, fungi, and algae. Based on its molecular structure (containing oxygen), it belongs to the xanthophyll family, one of the two major carotenoid families. In the plant kingdom, lutein provides photoprotection by scavenging singlet oxygen and peroxyl radicals [1], and its bright yellow color helps plants achieve effective cross pollination. Humans cannot synthesize lutein themselves, yet it is essential for the human body. Lutein is the predominant carotenoid in the infant brain [2], and is supplemented in newborn babies in the first hours of life. Lutein can increase biological antioxidant potential and reduce the plasma concentration of total hydroperoxides. It also reduces free radical-induced damage [3]. Lutein is the main carotenoid in the human retina; hence, it has been used as a therapeutic agent for the prevention of age-related macular degeneration [4,5]. Epidemiologic data suggest that lutein plays an active role in delaying chronic diseases [6], stimulating the immune response [7], and hampering the development of cataracts and atherosclerosis [8,9]. A recent study showed that a lutein-based dye used during chromovitrectomy in humans could improve the identification and removal of the vitreous, internal limiting membrane and the epiretinal membrane [10]. 

As lutein has many functions, it has become increasingly important to find and create more sources of lutein production. In recent years, algae have received a great deal of attention in the production of carotenoids and proteins. Previous studies in our laboratory showed that heterotrophically cultivated *Chlorella protothecoides* CS-41 can produce considerable amounts of lutein [11]. Furthermore, optimization of the cultivation conditions, medium composition, and extraction techniques can improve lutein yields [12,13]. However, to date, there are no reports of the enhancement of lutein production by this alga using genetic modification, although genetic engineering technologies have become increasingly popular in the field of carotenoid production. The first step is to determine the genes involved in lutein biosynthesis—information that is essential for genetic modification. 

It has been found that phytoene synthase (PSY) is the rate-limiting enzyme in the carotenoid biosynthesis pathway in photosynthetic organisms [14–16]. In many  cases, the rate of lutein formation through the carotenoid biosynthetic pathway appears to be controlled by PSY, which catalyzes the 

head-to-head condensation of two geranylgeranyl diphosphate molecules to yield phytoene—the first committed reaction in carotenogenesis. Since PSY plays a key role in the first step of carotenogenesis, it has unsurprisingly been chosen for genetic engineering studies of carotenoid production. 

PSY has been extensively studied in bacteria and higher plants, but its study in algae is still in its infancy. For unicellular green algae, the *psy* gene has previously been investigated in *Chlamydomonas reinhardtii* [17], *Duanliella bardawil* [18], and *Haematococcus pluvialis* [19]. In *C. reinhardtii*, deletion of the *psy* gene resulted in a white phenotype [20]. For *Haematococcus*, *psy* was shown to be 

up-regulated under stress conditions of high light and low nutrient availability [21]. Overexpression of exogenous *psy* from *D. salina* [22] or *C. zofingiensis* [23] in *C. reinhardtii* has been shown to increase the lutein content to over 1.25 and 2.2-fold, respectively. 

As an efficient lutein-production alga, *C. protothecoides* CS-41 has high potential for application in the commercial production of lutein; however, its lutein biosynthesis pathway has not been well studied. Our research group previously cloned other key enzyme genes in the lutein biosynthesis pathway of this alga, such as the phytoene desaturase (*pds*) (GenBank accession No. FJ968162) [24], zetacarotene desaturase (*zds*) (GenBank accession No. GU269622) [25], and lycopene-<sup>Ή</sup>cyclase (*lyce*) (GenBank accession No. FJ752528) genes. The *psy* gene is essential for determining the complete lutein biosynthesis pathway in this alga. Therefore, in this study, the *psy* gene from the unicellular microalga *C. protothecoides* CS-41 and its promoter were isolated and analyzed. This study provides an important theoretical basis for the genetic modification of lutein biosynthesis in *C. protothecoides* CS-41, including gene sequences, expression promoter candidates, and possible regulatory environmental factors for gene expression. 

## **2. Results and Discussion**
