High oil-yielding transgenic microalgae could be a promising source for biodiesel production. However, the biotechnological processes based on transgenic microalgae are still in infancy. In manipulation of genet­ically modified algae for high oil content, acetyl-CoA carboxylase (ACCase) was first isolated from the diatom Cyclotella cryptica by Roessler (1990), and then success­fully transformed into the diatoms C. cryptica and Navicula saprophila (Dunahay et al., 1995,1996; Sheehan et al., 1998). A plasmid was constructed that contained acc1 gene driven by the cauliflower mosaic virus 35S ribosomal gene promoter (CaMV35S) and the selectable marker nptII from Escherichia coli. Introduction of plasmids into the diatoms was mediated by micropro­jectile bombardment. The acc1 was overexpressed with the enzyme activity enhanced by threefold. These exper­iments demonstrated that ACCase could be transformed efficiently into microalgae, although no significant increase in lipid accumulation was observed in the transgenic diatoms (Dunahay et al., 1995, 1996). Recently, diacylglycerol acyltransferases (DGATs) homologous genes have been identified in the genome of Chlamydomonas reinhardtii and were overexpressed in the same microalga (Russa et al., 2012). This resulted in an enhanced mRNA expression level of DGAT genes, but did not boost the intracellular triacylglycerol (TAG) synthesis. Thus, till date, there is no success story with respect to lipid overproduction in microalgae using the genetic engineering approach.

Extensive studies have also been carried out on enhancement of lipid production using genetic engineer­ing approaches in different bacterial and plant species, which may provide valuable background for future studies with microalgae. Some of these studies are summarized in Table 11.2. The cytosolic ACCase from Arabidopsis sp. was overexpressed in Brassica napus (rapeseed) plastids. The fatty acid content of the

recombinant was 6% higher than that of the control (Roesler et al., 1997). In prokaryotes like E. coli, overex­pression of four ACCase subunits resulted in sixfold rise in the rate of fatty acid synthesis (Davis et al.,

2000) , confirming that the ACCase-catalyzed committing step was indeed the rate-limiting step for fatty acid biosynthesis in this strain. Nevertheless, Klaus et al.

(2004) achieved an increase in fatty acid synthesis and a more than fivefold rise in the amount of TAG in Sola — num tuberosum (potato) by overexpressing the ACCase from Arabidopsis in the amyloplasts of potato tubers.

Transformation of rape seed with a putative sn-2-acyl- transferase gene from Saccharomyces cerevisiae was car­ried out by Zou et al. (1997), leading to overexpression of seed lysophosphatidate acid acyl-transferase (LPAT) activity. This enzyme is involved in TAG formation and its overexpression led to profound rise in oil content from 8% to 48% on seed dry weight basis. However, it was cautioned that the steady state level of diacyl — glycerol could be perturbed by an increase in LPAT activity in the developing seeds. Transformations of S. cerevisiae with the Arabidopsis DGAT were performed
by Bouvier-Nave et al. (2000). About 600-fold rise in DGAT activity in the transformed S. cerevisiae was observed, which led to a ninefold increase in TAG accumulation. DGAT gene has also been overexpressed in the plant Arabidopsis and it was shown that the oil content was enhanced in correlation with the DGAT activity (Jako et al., 2001). All these results suggest that the reaction catalyzed by ACCase, LPAT and DGAT are important rate-limiting steps in lipid biosynthesis.

A few enzymes that are not directly involved in lipid metabolism have also been demonstrated to influence the rate of lipid accumulation. For instance, it was observed by Lin et al. (2006) that by overexpressing the acs gene in E. coli, the acetyl-CoA synthase activity was increased by ninefold, leading to a significant increase in the assimilation of acetate from the medium, which can contribute to lipid biosynthesis. The genes coding for malic enzyme from Mucor circinelloides (mal — EMt) and from Mortierella alpina (malEMc), respectively, were overexpressed in M. circinelloides which led to a

2.5- fold increase in lipid accumulation (Zhang et al.,

2007) . Lu et al. (2008) reported a 20-fold enhancement
of fatty acid productivity of E. coli by combining four targeted genotypic changes: deletion of the fadD gene encoding the first enzyme in fatty acid degradation, overexpression of the genes encoding the endogenous ACCase, and overexpression of both an endogenous thioesterase (TE) as well as a heterologous plant TE. Overexpression of wril gene from B. napus in transgenic Arabidopsis thaliana resulted into 40% increased seed oil content (Liu et al., 2010). Zhang et al. (2011) studied the effects of the overexpression of different acyl-ACP TE genes from Diploknema butyracea, Ricinus communis and J. curcas on free fatty acid contents of E. coli. The strain carrying the acyl-ACP TE gene from D. butyracea produced approximately 0.2 g/l of free fatty acid while the strains carrying acyl-ACP TE genes from R. commu­nis and J. curcas produced the free fatty acid at a high level of more than 2.0 g/l.