*2.1. Enzymes for High-Value Added Lipids and Biodiesel Production*

Microalgae are known to accumulate large amounts of lipids [16], with triglycerides (TAGs) and poly-unsaturated fatty acids (PUFA) being the most studied from a biotechnological application standpoint, particularly for the production of biodiesel and nutraceuticals [9,16–18]. TAGs, esters derived from glycerol and three chained fatty acids (FA) which are usually stored in cytosol-located lipid droplets [19], can be used to produce biodiesel following acid- or base-catalyzed transesterification reactions [20]. PUFAs, for their part, have well-proven beneficial health effects [21,22], especially Ω-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (Figure 2).

**Figure 2.** Examples of fatty acids of biotechnological interest. (**a**) Through various reactions of elongation and formation of double C-C bonds, poly-unsaturated fatty acids (PUFA) can be synthetized, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with nutraceutical or food applications; (**b**) Accumulation in triglycerides (TAGs) and biodiesel formation via chemical transesterification.

The most frequently studied enzyme involved in lipid synthesis is acyl-CoA diacylglycerol acyltransferase (DGAT), involved in the final reaction of the TAG biosynthetic pathway [23,24]. Three independent groups of enzymes, referred to as acyl-CoA diacylglycerol acyltransferases type 1, 2, and 3 (DGATs 1-2-3), take part in the acyl-CoA-dependent formation of TAGs from its precursor sn-1,2-diacylglycerol (DAG) [25]. The individual contribution of each DGAT isoenzyme to the fatty acid profile of TAG differs between species [24,26].

A gene encoding DGAT1 was initially discovered in the green alga *Chlorella ellipsoidea* by Guo et al. [27], and an experiment involving overexpression of DGAT1 was subsequently performed in the oleaginous microalgae *Nannochloropsis oceanica* [28]. The first DGAT2 sequence was obtained from the green alga *Ostreococcus tauri* [29], and different studies involving overexpression of DGAT2 were performed. In particular, DGAT2 overexpression led to an increase in TAG production in the diatoms *Phaeodactylum tricornutum* [30] and *Thalassiosira pseudonana* [31], and in the oleaginous microalgae *Neochloris oleoabundans* [32] and *N. oceanica* [33]. Different isoforms of DGAT2 (NoDGAT2A, 2C, 2D) have successively been identified in *N. oceanica* and different combinations of either overexpression or under-expression have been analyzed. These combinations gave different fatty acid-production profiles, with some optimized for nutritional applications and others for biofuel purposes [34]. Even if the green alga *Chlamydomonas reinhardtii* is considered a common biofuel feedstock, it showed no clear trends following overexpression of different DGAT2 isoforms, with increased levels of TAG in some reports [35], while levels were not increased in others [36]. Recently, Cui and coworkers [37] characterized a dual-function wax ester synthase (WS)/DGAT enzyme in *P. tricornutum,* whose overexpression led to an accumulation of both TAGs and wax esters. This was the first report of this particular enzyme in a microalga, and a patent involving the enzyme was subsequently filed (Patent Code: CN107299090A, 2017).

In addition to DGAT, other genes have been targeted in order to increase high-value added lipid production, including glucose-6-phosphate dehydrogenase (G6PD), Δ6-desaturase, 6-phosphogluconate dehydrogenase (6PGD), glycerol-3-phosphate acyltransferase (GPAT1-GPAT2), and acetyl-CoA synthetase 2 (ACS2). Overexpression of these enzymes resulted in increased lipid contents [38–42]. In particular, two patents for desaturases have been filed. One covers a Δ6-desaturase from *Nannochloropsis* spp., which converts linoleic acid to γ-linolenic acid (GLA) and α-linolenic acid (ALA) to stearidnoic acid (Patent Code: CN101289659A, 2010). The other covers a Ω6-desaturase from *Arctic chlamydomonas* sp. *ArF0006*, which converts oleic acid to linoleic acid (Patent Code: KR101829048B1, 2018).

Other approaches to increase lipid production and/or alter lipid profiles via gene disruption have been employed. Examples include the knock-out of a phospholipase A2 (PLA2) gene via CRISPR/Cas9 ribonucleoproteins in *C. reinhardtii* [43], microRNA silencing of the stearoyl-ACP desaturase (that forms oleic acid via addition of a double-bond in a lipid chain [44]) in *C. reinhardtii* [45], and meganuclease and TALE nuclease genome modification in *P. tricornutum* [46]. This last approach involved modifying the expression of seven genes, potentially affecting the lipid content (UDP-glucose pyrophosphorylase, glycerol-3-phosphate dehydrogenase, and enoyl-ACP reductase), the acyl chain length (long chain acyl-CoA elongase and a putative palmitoyl-protein thioesterase), and the degree of fatty acid saturation (Ω-3 fatty acid desaturase and Δ-12-fatty acid desaturase). In particular, a mutant for UDP-glucose pyrophosphorylase showed a 45-fold increase in TAG accumulation under nitrogen starvation conditions. Figure 3 provides an overview of the subcellular localization of metabolic pathways and engineered enzymes in the aforementioned examples.

Finally, Sorigué and coworkers [47] reported, for the first time, the presence of a photoenzyme named fatty acid photodecarboxylase (FAP) in *Chlorella variabilis* str microalgae. NC64A. FAP converts fatty acids to hydrocarbons and may be useful in light-driven production of hydrocarbons. It is worth mentioning that Misra et al. [48] have developed a database to catalogue the enzymes which have been identified as being responsible for lipid synthesis from available microalgal genomes (e.g., *C. reinhardtii*, *P. tricornutum*, *Volvox carteri*), called dEMBF (website: http://bbprof.immt.res.in/embf/). To date, the database has collected 316 entries from 16 organisms, while providing different browsing options (Search by: "Enzyme Classification", "Organism", and "Enzyme Class") and different web-based tools (NCBI's Blast software integrated, sequence comparison, Motif prediction via the MEME software). The enzymes discussed in this section are reported in Table 1.


**Table 1.** Enzymes from Microalgae for Lipid and Biodiesel Production. Marine and freshwater ecological strain sources are abbreviated as M or F, respectively. Algal classes of *Bacillariophyceae*, *Chlorophyceae*, *Trebouxiophyceae*, *Eustigmatophyceae*, *Mamiellophyceae*, *Coscinodiscophyceae*, and *Cyanidiophyceae* are abbreviated as BA, CH, TR, EU, MA, CO, and CY, respectively.


**Table 1.** *Cont*.

**Figure 3.** Main studied and engineered enzymes for TAGs and PUFAs in microalgae for the production of high value-added lipids. Enzymes are roughly divided in subcellular compartments. A single lipid droplet where TAGs are accumulated is added. Abbreviations: DGAT: Acyl-CoA diacylglycerol acyltransferase; G6PD: Glucose-6-phosphate dehydrogenase; 6PGD: 6-phosphogluconate dehydrogenase; GPAT: Glycerol-3-phosphate acyltransferase; ACS2: acetyl-CoA synthetase 2; PLA2: Phospholipase A2; Δ-6/Δ-12-Desaturase: delta-6/delta-12 fatty acid desaturase; Ω-3/Ω-6-desaturase: omega-2/omega-6 fatty acid desaturase; ENR: Enoyl-acyl carrier protein reductase; UGPase: UDP-glucose pyrophosphorylase; TAG: Triglyceride.
