It was reported that overexpression

It was reported that overexpression of the rat ACL gene in tobacco increased the total ACL activity by 4 fold, which increased the production of fatty acids by 16% [8]. Recently, a 1.7-fold increase of fatty acids and 1.9-fold increase of triacylglycerol (TAG) was observed in Aspergillus oryzae with an ACL-enhanced thiostrepton receptor [9]. Also, overexpression of ACL from Mus musculus in Yarrowia lipolytica enhanced the lipid accumulation from 7.3% to 23.1% of cell dry weight, which suggested that ACL is an important acetyl-CoA producer and responsible for lipid accumulation in oleaginous yeast Y. lipolytica[10]. Animal ACL has only one subunit with a molecular mass of 110±120kDa that are encoded by a single gene [6], [11]. In contrast, ACLs of most bacteria, fungi and plants are thought to be composed of two different subunits, ACL1 and ACL2 (or ACLA and ACLB; for distinction the small subunit is abbreviated to ACLA or ACL1, and large subunit is abbreviated to ACLB or ACL2) [2], [7], [12], [13]. The two ACL subunits are encoded by different genes whose deduced two polypeptides display significant homology to the N- and C-terminal parts of animal ACL polypeptides, respectively. Very recently, the large subunit of algal ACL sequence has been discovered from the glaucophyte alga Cyanophora paradoxa, which shows 64–68% amino-acid sequence identity with the ACLB from plants [14]. To our knowledge, few algal ACL genes have been cloned. The chlorophyte Dunaliella is highly salt tolerant, simple to cultivate and not easy to be polluted, which make it possible to be large-scale outdoor cultivation. D. tertiolecta was reported to contain high lipid content (up to 57.02% per dry cell weight), which made it a potential biodiesel feedstock [15]. In this study, cDNAs of the small and large subunits of ACL (DtACLA and DtACLB) and their genomic sequences were isolated from Dunaliella tertiolecta using RT-PCR, RACEs and genomic walking techniques. Subsequently, bioinformatics tools were employed to analyze the deduced proteins, conserved domains, and phylogenetic analysis of the obtained DtACLA and DtACLB. Heterologous expression of DtACLA and DtACLB and their transcription levels in response to nitrogen deficiency were analyzed.
Materials and methods
Results
Discussion ACL genes are present in eukaryotes, including fungi, plants, protists, and animals, as well as prokaryotes, like a green sulfur bacterium. Mammalian ACL is a homotetramer with a subunit of about 1100 amino acids. The green alga, Chlorella vulgaris, photosynthetic protists, including C. reinhardtii and the glaucophyte, Cyanophora paradoxa[14], have a similar heteromeric structure. Ascomycota, including Aspergillus and Sordaria macrospora, contain ACLA and ACLB two separate genes. However, in the GenBank database, it is indicated that S. cerevisiae does not contain these genes, while acetyl-CoA is generated by acetyl-CoA synthetase in S. cerevisiae[23]. As for prokaryote, bacteria ACL has been reported from the photosynthetic green sulfur bacterium Chlorobium limicola[12]. In this study, genes encoding the two distinct subunits (ACLA and ACLB) from D. tertiolecta were isolated. The heteromeric organization of ACL in D. tertiolecta is common to other algae, green plants, fungi, and bacteria. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that an evolutionary fusion of the ACLA and ACLB genes probably occurred [24]. Based on the conserved domains of ACL subunits, SCS subunits and CS (Fig. 5A and B) and phylogenetic analysis (Fig. 6), the small subunit ACLA may share the common evolutionary origin with SCSβ, and the large subunit ACLB may have a common origin from the fusion and divergence of SCSα and CS. Fusions of genes have been found in genes encoding different subunits of one enzyme or enzymes, like the TPI-GAPDH fusion gene of the glycolytic pathway in dinoflagellates and oomycetes [25], [26], the bifunctional ICL-MS gene of the glyoxylate cycle in metazoan [27], and the TPS/TPP for the trehalose biosynthesis in eukaryotes [28]. While using pET32a as the expression vector, E. coli BL21 (DE3) as the host strain, the recombinant DtACLA or DtACLB seemed to be expressed mainly in the form of insoluble fraction (Fig. 7). The activity of DtACLA and DtACLB will be studied further, when we explore the suitable induction system for expressions of DtACLA and DtACLB.