Exploring industrial application of oleaginous yeast strains first requires versatile measuring tools to determine the physiological characteristics of the yeast and optimize them during fermentation. From the product perspective, the Nile red stain has been used with fluorescence microscopy to analyze intracellular lipid accumulation in Y. lipolytica (Poli et al., 2013, 2014). During fermentation, the online glucose biosensor has been successfully employed to monitor substrate concentration during long-term incubation of a repeated fed-batch process (Moeller et al., 2011). In this method, the ideal time duration of targeted bioproduct production can be estimated based on the consumption rate of the substrate. Finally, flow cytometry and flow particle image analysis were used together to analyze vitality, cell shape, and lipid development in other oleaginous yeast cultures (Raschke and Knorr, 2009).

A key strategy to improve the performance of Y. lipolytica in industrial applications is to optimize fermentation conditions. As a strict aerobic yeast, Y. lipolytica culture requires efficient aeration systems during its growth in bioreactors. In fact, the dissolved oxygen should be controlled based on the requirements of targeted bioproduct formation. For instance, high oxygen can cause overproduction of cell mass and lower production of some bioproducts (Huang and Zhang, 2011). However, production of oxidized bioproducts may require a sufficient amount of oxygen. For instance, the rate of oxygen consumption decreased during the transition from growth to lipogenic phases in engineered strains (Blazeck et al., 2014). The aerated cultures can boost oil biodegradation (Martins et al., 2012) or FFA oxidation to unsaturated ones (Sha, 2013). On the other hand, high agitation and aeration may intensify lipid degradation by increasing acyl-CoA oxidase activities (Papanikolaou et al., 2007). As a control, oxygen was adjusted to increase lipid content with a specific degree of saturation and regulate ω,β-oxidation enzymes (Choi et al., 1982; Hallenbeck, 2012). Some strategies have been developed to address oxygen as a typical limiting factor in the industrial aerobic process. For instance, perfluorodecalin can be used as an oxygen transporter or pressurized bioreactor to improve the growth rate and extracellular enzyme production in Y. lipolytica cultures (Coelho et al., 2010). Adjusting the culture media is also feasible to mitigate its oxygen requirement level. This strategy was practiced by using media with high iron (3.5 mg/L) (Kamzolova et al., 2003).

In addition to dissolved oxygen, other culture conditions should also be adjusted to increase biomass and bioproduct formation. These cultivation parameters include but are not limited to oxidoreduction potential, temperature, pH, the concentration of metal traces and minerals (e.g., Mg²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Ca²⁺), the type of carbon and nitrogen source, and the C/N ratio. For instance, the C/N ratio of 50–100 usually induces lipid accumulation (Ageitos et al., 2011; Hallenbeck, 2012). A recent study used the D-stat continuous system of cultivation to determine the appropriate ratio for lipid accumulation in glucose-grown cells. Lipid accumulation occurred at the C/N ratio of 11.7–47 when citric acid production was absent or limited (Ochoa-Estopier and Guillouet, 2014). Higher ratios may reduce lipid yield and increase citric acid and other undesired byproducts (Rossi et al., 2011; Thevenieau and Nicaud, 2013). However, another study found that total glucose amount plays a major role in lipid accumulation for engineered glucose-grown strains (Blazeck et al., 2014).

Proper fermentation modes should also be selected to improve production and cultivation conditions (Beopoulos and Nicaud, 2012). Typical fermentation modes include batch, single-stage and double-stage fed batches, and continuous culture. For continuous or fed-batch mode, the C/N ratio and dilution rate should be optimized to control lipid accumulation and acid production. The fed-batch system is a suitable mode to separate the growth and lipid accumulation phases because it offers control of the aforementioned factors (Beopoulos et al., 2009). The two-stage fed-batch system has been used to achieve high biomass for subsequent conversion of volatile FAs to the SCO by Y. lipolytica (Fontanille et al., 2012). However, this system is rather complex for industrial use. Lipid accumulation can also be achieved in a continuous culture under nitrogen-limiting conditions and with stable dilution rates (Ageitos et al., 2011). Traditional stirred-tank and air-lift bioreactors are often used to optimize and develop bioprocesses involving Y. lipolytica strains (Coelho et al., 2010). The biphasic system of fermentation can simplify and stabilize whole-cell bioconversion of the hydrophobic substrate to value-added products. For example, the aqueous and organic phases can be used to improve biotransformation of progesterone by recombinant Y. lipolytica in a stirred-tank bioreactor. In this strategy, ethyl oleate and dibutylphthalate were relatively more efficient at elevating the eventual production levels. This was facilitated by higher accessibility of the hydrophobic substrate, lower inhibitory effects of the hydrophobic compounds, and the facilitated co-uptake of the organic element and the substrate (Braun et al., 2012). Finally, the agitation rate can also affect the rate of biomass and bioproduct generation and should be adjusted accordingly (Rywinska et al., 2012). Generally, the Y. lipolytica culture should be properly maintained between 25 and 30°C and a pH range of 3–6 for lipid accumulation (Ageitos et al., 2011). Enshaeieh et al. (2013) reported that optimum temperature, pH, agitation rate, and incubation time for lipid accumulation in Y. lipolytica M7 were 25°C, 5, 200 rpm, and 72 h, respectively.

The fermentation process is typically followed by the extraction process, and various approaches to extracting oil from oleaginous microorganisms have been reviewed (Wang et al., 2012). Glass bead disruption followed by solvent extraction has been noted to extract oil from yeast. Ultrasound, microwave, and electroporation have been considered as novel methods to enhance extraction. However, these approaches are not practical at the industrial scale due to technological restrictions. Oil has also been extracted from oleaginous yeast through solvent extraction from dry biomass, vortex and glass beads, ultrasonic bath and glass beads, liquid nitrogen, and liquid nitrogen with sonication (Poli et al., 2013). The later approach was found to be the most efficient for lab use. To improve the lipid extraction from the Y. lipolytica biomass, applying subcritical water pretreatment at an optimal of 175°C and a water addition of 20 mL/g of biomass for 20 min has been suggested (Tsigie et al., 2012a). This enhances the crude lipid extraction from 51.54 to 84.79% in the subsequent solvent-assisted lipid extraction. This may be due to higher reactivity of water ions including H⁺ and OH⁻ as catalysts that facilitate the chemical reactions that break the cell wall. Finally, it is noteworthy that the most suitable lipid extraction method for the industrial scale has not yet been determined.

Using mixed cultures of organisms, including oleaginous and non-oleaginous ones, can also enhance the efficiency of fermentation and substrate utilization. For example, mixed cultures of microalgae and some oleaginous yeast species on sugarcane juice have been introduced as a desirable strategy for lipid accumulation. In this strategy, yeast as a CO₂ generator and microalgae as an O₂ generator supported one another’s growth under mixotrophic cultivation (Papone et al., 2012). The co-culture system of fermentation may demonstrate outstanding characteristic for more efficient utilization of feedstock, higher tolerance of inhibitory compounds, and higher production of biomass and target bioproducts.

Another approach to increase productivity during fermentation is biomass immobilization. An alginate bead carrier in a batch system and a k-carrageenan carrier in an air-lift bioreactor were used to immobilize Y. lipolytica for citric acid production (Kautola, 1991). Although this yeast shows promise for biofilm generation, this approach may be inefficient for lipid production under nutrient starvation. However, the Y. lipolytica biofilm on the selective substrate can be more efficiently used in a fixed bed biofilm reactor (Lehocký, 2007). The immobilization technique may also prevent reconsumption of secreted bioproducts. Another related application of immobilization technique is to enhance the effectiveness of yeast products. This yeast produces several types of lipases, and its enzymes can also be immobilized to enhance hydrolysis, esterification, interesterification, transesterification, and ring-opening polymerization processes. These processes have potential industrial applications for biodiesel production (Hallenbeck, 2012), digestive acid production (Marova et al., 2011), and flavor modification with the aid of free FAs and esters (Waché et al., 2006; Fickers et al., 2011). Immobilization strategies may also pave the way for continuous whole-cell or enzyme-based biotransformation processes.

To offset the cost of microbial lipid-based biofuel production, yeast biomass may be recycled for various purposes. For example, the direct use of residual yeast biomass by animals can improve the economy of SCO production, since the biomass of oleaginous yeast has considerable amounts of protein, carbohydrates, and other nutrient ingredients. The potential of a marine Y. lipolytica strain as a feed component for cultured marine animals has been examined (Wang et al., 2009). The anaerobic digestion of residual biomass is another means of reducing the production costs (Meng et al., 2009). In one study, a spent medium and yeast biomass from the first step of lipid accumulation and extraction were reused in the next steps of yeast and algae cultivation in an integrated system to maximize substrate utilization and improve the economy of the process (Chi et al., 2011). In a recent study, the hydrolyzate of Y. lipolytica biomass acted as a medium for bioethanol production using S. cerevisiae (Tsigie et al., 2013). Higher yields of glucose were obtained from hydrolyzate after lipid extraction, and 98.05% of this glucose was converted to bioethanol at a yield of 0.38 g ethanol/g glucose. These studies provide evidence for maximal exploitation of this yeast.

A key step for producing lipids and co-products by Y. lipolytica is to upgrade this platform to utilize pentoses from lignocellulose. There are contradictory reports on xylose utilization in this yeast. One study showed that this yeast can simultaneously utilize d-xylose and d-glucose by consuming xylose at a relatively higher rate. However, glucose could competitively inhibit the uptake of xylose (Tsigie et al., 2011). The co-fermentation of xylose and glucose may enhance biomass and lipid formation in this yeast (Sha, 2013). In another study, the xylose reductase encoding gene, XYL1, and xylitol dehydrogenase encoding gene, XYL2, were transferred from Scheffersomyces stipitis into Y. lipolytica (Tai, 2012). Actually, Y. lipolytica has the basic structure for this manipulation, since it possesses putative XYL12 genes within its genome. The combination of this strategy with several adaptation steps helped develop a strain that grows on xylose as the sole source of carbon and reach a lipid accumulation level of 42%. Further study may target this platform to enhance the uptake of pentose and oligo-sugars, which are present in pretreated lignocellulosic materials, while maintaining the appropriate growth rate.

Yu et al. (2011) used wheat straw hydrolyzate from a dilute sulfuric acid pretreatment for lipid accumulation in several oleaginous yeast strains including Y. lipolytica. This strain could use both detoxified and non-detoxified forms of the substrate to grow and reach a lipid content of about 4.6% w/w (Yu et al., 2011). Sugarcane bagasse hydrolyzate was also used to grow Y. lipolytica and achieve a maximum lipid content of 58.5% (Tsigie et al., 2011). Defatted rice bran hydrolyzate and sugarcane bagasse hydrolyzates as carbon and nitrogen source have been used to cultivate this yeast to reach a lipid content of 48.02 and 58.5%, respectively (Tsigie et al., 2011, 2012b). These studies indicate that the lipid content of this yeast varies, depending on strain and feedstock.

One promising strategy is to express heterologous polysaccharide cleaving enzymes and target them to the cell surface in order to enable Y. lipolytica to grow on lignocellulosic feedstocks. The advantage of this strategy lies in the fact that Y. lipolytica is a non-cellulose degrading yeast. On the other hand, the cost of hydrolyzing enzymes is still high, which can hamper the development of economically viable cellulosic biofuel technology. In an attempt to upgrade this platform to utilize plant polysaccharides, the gene for exo-inulinase was cloned from the yeast Kluyveromyces marxianus and expressed into Y. lipolytica. This resulted in direct bioconversion of inulin into 48.3% (w/w) oil (Zhao et al., 2010a). In fact, a remarkable lipid accumulation and small citric acid production were observed during fermentation of inulin containing a substrate by Y. lipolytica. In another study, the gene encoding exo-inulinase from the yeast K. marxianus was expressed and immobilized on Y. lipolytica cells using a surface-display plasmid (Liu et al., 2010). This strategy was used for simultaneous utilization of inulin and production of citric acid. By targeting heterologous proteins at the surface of the cell, yeast cells become more suitable for other applications such as the development of live vaccines, antibody libraries, whole-cell biocatalysts, and absorbent. The combination of fungal α-amylase gene, together with its signal peptide, successfully led to the extracellular secretion of this enzyme in Y. lipolytica (Park et al., 1997). The bacterial chitosanase is another example of a sugar-hydrolyzing enzyme whose gene, chi, was transformed into Y. lipolytica for the overexpression of bacterial chitosanase. A remarkable level of expression (i.e., 9.0 U/ml) and activity were achieved in the conversion of chitin into constituent oligosaccharides (Liu et al., 2012a). Heterologous mannanase has also been produced on the cell surface of this yeast, reaching the high activity level of 62.3 IU/g DCW during the 96 h of cultivation period (Yang et al., 2009). Molasses, a byproduct of sugar refinery, have also been used at an optimum concentration of 8% to achieve lipid accumulation of 59.9% by this yeast (Karatay and Dönmez, 2010). In order to achieve efficient utilization of molasses, the invertase-positive strain of Y. lipolytica is required. Alternatively, the heterologous invertase gene can be introduced into this yeast (Lazar et al., 2013). Recently, several heterologous cellulases including endoglucanase, cellobiohydrolase, and β-glucosidase were expressed in this yeast. This enhanced the enzymatic hydrolysis of cellulosic substance, which can be further converted to biofuels. This strategy may enable the platform to simultaneously utilize lignocellulosic sugars and produce targeted bioproducts in consolidated bioprocessing (CBP). This strategy may also improve enzyme stability and the economics of downstream processing.

To improve the economy of the microbial lipid technology, simultaneous hydrolysis of cellulosic biomass and lipid accumulation in the oleaginous yeast Cryptococcus curvatus has been suggested (Gong et al., 2013). In this integrated system, a high dosage of inoculum combined with hydrolytic enzymes could improve the level of sugar utilization and mitigate the susceptibility of oleaginous yeast culture to contamination. Additionally, the absence of supplementary nutrients aids in lipid accumulation and higher cell resistance. One study used CBP to produce cellulosic bioethanol with a non-oleaginous yeast consortium was also reported (Kim et al., 2013). Such studies can spark further research on the Y. lipolytica platform in advanced CBP to produce cellulosic lipid-based bioproducts. Therefore, overexpressing heterologous sugar-hydrolyzing enzymes in this GRAS yeast can help facilitate the use of CBP for the production of cellulosic lipid and functional oligosaccharides. These oligosaccharides can be used as prebiotics in various food products to boost proliferation of probiotic bacteria in the colon and improve human health (Abghari et al., 2011).

Yarrowia lipolytica can also be potentially used to enhance the biorefinery of lignocellulose-based feedstock and utilization of byproducts. Y. lipolytica has been used to develop a biological detoxification process, due to its natural capability to produce extracellular laccase (Lee et al., 2012). This capability was harnessed to oxidize the lignin-derived phenolic compounds for pretreated rice straw to mitigate their inhibitory impacts on cellulase. Other studies have explored the suitability of Y. lipolytica as an expression system for recombinant laccase (Madzak et al., 2005b). However, the high concentration of Y. lipolytica biomass can potentially maintain volatile FAs, the byproducts of pretreatment process, below the inhibitory level for recycling (Fontanille et al., 2012).

Yarrowia lipolytica has shown promise for converting glycerol to SCO and citric acid (André et al., 2009; Makri et al., 2010). Recently, lipid accumulation has been found in the Y. lipolytica strains grown on glycerol in the range of 37.1–54.4% of cell dry weight (Sriwongchai et al., 2013). The introduction of heterologous glycerol dehydratase genes and several alcohol oxidoreductases under the control of glycerol inducible promoter, G3P dh, gave rise to a higher growth rate and a lipid yield at13 g/L of culture volume (40% of DCW) (Celinska and Grajek, 2013). This could be enhanced by the codon optimization of the heterologous genes and the prevention of further degradation of accumulated lipid. The mixed substrate utilization of glycerol and other substrates also show promise for enhancing lipid accumulation (Papanikolaou et al., 2003). However, the inhibitory impact of glycerol on the metabolism of hydrophobic biomass and the regulatory mechanisms should be addressed in mixed carbon sources (Mori et al., 2013).

Selecting optimum cultivation conditions should be accompanied with the appropriate metabolic and genetic engineering of Y. lipolytica. These engineering strategies have mainly been used to intensify carbon flux to the targeted bioproduct, produce new FA-based co-products, and extend the range of utilizable feedstock. Most of the strategies can be found tabulated elsewhere (Liang and Jiang, 2013). Successful application of these engineering techniques requires understanding of the roles of contributory metabolic enzymes, regulatory mechanisms, and the mechanisms of secretion and/or re-uptake of bioproducts. Moreover, the application of next generation DNA sequencing, microarray analysis, and mass spectrometry can also help to comprehend how the Y. lipolytica platform works and how it should be manipulated.

The major engineering strategies to improve the Y. lipolytica platform include but are not limited to the expansion of the pools of G3P and acyl-CoA, the promotion of key steps in TAG biosynthesis pathway, and the prevention of lipid degradation through the competitive pathway (Courchesne et al., 2009). Some critical contributory enzymes to lipid biosynthesis are ACC, Δ 9 stearoyl-ACP desaturase (D9), and DGA, and ACL. The foregoing enzymes were ordered based on the highest to the lowest degree of contribution to lipid overproduction as DGA, ACC, D9, and ACL (Tai, 2012). The transporting enzyme can also be targeted to enhance production of lipid-derived bioproducts. This strategy was used in other oleaginous yeasts, and appears to be simpler than manipulating the central metabolic pathway (Cao et al., 2006).

Researchers have transformed the genes encoding acetyl-CoA synthase and ATP:citrate lyase from S. cerevisiae and Mus musculus into Y. lipolytica to increase the acetyl-CoA pool (Zhou et al., 2012). Upregulation of the latter gene is important, since its enzyme activity seems to increase as intracellular carbon flux increases (André et al., 2009). TAG accumulation was also enhanced in this yeast by elevating the G3P pool. This was achieved by modifying the G3P shuttle by disrupting GUT2 and the overexpression of GPD1 genes. These genes govern the reverse reaction between G3P and DHAP. Overexpression of the GPD1gene, in combination with deletion of POX1–6 genes, may cause lipid accumulation of up to 65% dry weight in Y. lipolytica (Dulermo and Nicaud, 2011). Others have disrupted the putative acyl-CoA synthetase gene YAL1 in order to identify the function of one of the fatty acyl-CoA synthetases in Y. lipolytica. This led to a 1.47-fold increase in lipid production and an elevation of saturated to unsaturated FAs. This gene may play a role in elongating and desaturating FA (Wang et al., 2011b). However, more research is needed to determine the function of various acyl-CoA synthetase genes in Y. lipolytica organelles, as well as their selectivity toward activation of specific FAs and how activated FAs are directed to lipid accumulation or degradation pathways. The disruption of TAG lipases coding genes TGL3 and TGL4 may also improve lipid content by repressing TAG hydrolysis (Dulermo and Nicaud, 2011). The POX genes and corresponding Aox proteins can be manipulated in different ways to produce various lipid bioproducts, control of FA chain length, and supplement expression of heterologous FA modifying enzymes in Y. lipolytica. In fact, POX deficiency may heighten modification to lipid-associated genes. For example, the impact of G3P shuttle modification on improvement of TAG content is intensified when modification is followed by POX deficiency. This combinational strategy promotes the pool of G3P and FA, altering gene expression in TAG homeostasis and enhanced lipid accumulation (Dulermo and Nicaud, 2011). The disruption of POX genes can also limit TAG biosynthesis thorough the feedback inhibitory effect (Mlickova et al., 2004).

Genetic modification of oleaginous microorganism is considered to be a sustainable and economical strategy for producing essential FAs, such as linoleic acid (LA), α-linolenic acid, and other functional poly unsaturated FAs that are not synthesized by the human body. Y. lipolytica and some other oleaginous yeasts are naturally capable of producing LA acid or linolenic acid. Introducing specific desaturase, elongase, hydroxylase coding genes, and their facilitator genes can convert FAs to PUFA and other functional FAs. Tuned expression of the heterologous Δ 9 pathway containing Δ-9 elongase, Δ-8 desaturase, Δ-5 desaturase, and Δ-17 desaturase, along disrupting PEX10, involved in the biogenesis of peroxisome, produces a high yield of eicosapentaenoic acid (EPA) in Y. lipolytica (Xue et al., 2013).

Some research has targeted the intrinsic regulatory system of this yeast. The inhibitory effect of glucose on transcript levels of lipid biosynthesis-associated genes should be mitigated to improve lipid content in glucose-grown Y. lipolytica. To this end, researchers have disrupted Mig1 protein as a global transcriptional repressor, which is the major constituent of the glucose repression pathway in this yeast. This disruption could enhance lipid content from 36 to 48.7% (w/w) at the cost of a lower growth rate (Wang et al., 2013). This could be due to the activation of more metabolic pathways when they are no longer repressed. Other researchers have intensified and hastened the lipid accumulation process by manipulating the SNF1 complex and some of its subunits. Ylsnf1 is one of the major negative regulators of lipid accumulation in Y. lipolytica. Deleting the Ylsnf1 gene has been found to enhance FA content 2.6 times in the absence of nutrient starvation. This is because this gene plays a role in the transcriptional regulation of key lipid metabolism-associated genes (Seip et al., 2013). The combined manipulation of the SNF1 and lipid degradation pathway may help continuously produce FAs of specific functionality during exponential growth and stationary phases. Identifying and manipulating these regulatory pathways will improve the ability of this yeast to produce lipid-based biofuel.

In addition to directing flux to the engineered pathway, a balance should be maintained between substrates and cofactors and also between the upstream and downstream flux. In a recent study on Y. lipolytica, balance was established between these two fluxes by amplifying ACC1 and overexpressing DGA (DGA1). This balance minimized the accumulation of intermediate metabolites and created a synergistic effect of gene co-expression. This co-expression, in tandem gene construction, resulted in a fivefold increase of lipid content to 41.4%, and subsequently to 61.7% of DCW during 120 h of fermentation in a 2-L bioreactor (Tai and Stephanopoulos, 2013). This diverted the flux toward the desirable product and created a driving force. A de novo synthesized lipid has recently been enhanced to 90% DCW in glucose-grown cells, through upregulation of key lipid-associated genes along with the suppression of peroxisomal FA degrading pathway and application of leucine, an intracellular inducer signal. Leucine may facilitate lipogenesis and alleviate dependency on nitrogen starvation (Blazeck et al., 2014). This rises beyond the maximum ex novo lipid accumulation of 70% DCW, which was obtained from an engineered strain fed on pure oleic acid (Beopoulos et al., 2008). This shows great promise for application of this platform for the production of cellulosic biofuel.

Another promising strategy is to compartmentalize specific metabolic pathways in one organelle. Targeting the enzymes of the Ehrlich pathway to mitochondria was recently found to be more efficient than overexpressing contributory enzymes to achieve higher yield of isobutanol (Avalos et al., 2013). This advantage is due to the concentrated availability of the precursors and enzymes, obviating the need to transport metabolites. These strategies inspire more exploration of the heterologous lipid-associated metabolic pathway in the lipid body, peroxisome, or mitochondria of this oleaginous yeast, and pave the way for purposeful and concentrated degradation of lipid and lipid precursors to lipid-derived bioproducts of higher value.

The FA biosynthesis pathway can be dynamically controlled by identifying key contributory transcriptional factors and developing tunable hybrid promoters. For instance, the synthetic regulator system is well-constructed for enhanced production of biodiesel in E. coli (Zhang et al., 2012b). IPTG has been used to activate the hybrid promoter, and the induction level was finely tuned by FA/acyl-CoA level based on naturally existing FA-sensing protein. The invention of such synthetic regulators can be inspiring for the dynamic control of FA-associated pathways in the Y. lipolytica platform.

Finally, after successful optimization of the metabolic and culture conditions for the maximal production of bioproducts, the scale-up process should be properly carried out for consistent production of targeted byproducts at a larger scale.

The intrinsic FA and lipid production of oleaginous yeasts make them ideal for production of lipid-derived bio fuel. Several potential routes for FA-based biofuel production are shown in Figure 3 (Brown and Shanks, 2012; Hallenbeck, 2012; Wang and Lu, 2013). Some of these routes have already been investigated for Y. lipolytica, while others need further exploration.

Yarrowia lipolytica shows promise in the conversion of low-cost materials to microbial oil and biodiesel, which is regarded as the first feasible resolution for the shortage of oily resources (Katre et al., 2012; Huang et al., 2013a; Blazeck et al., 2014). However, direct production of biodiesel has not yet been achieved with this yeast. However, this was achieved in recombinant E. coli and S. cerevisiae. Table 5 presents major achievements in microbial biodiesel production.

Genetic strategies for producing FAEE and FA methy ester (FAME) have typically targeted de novo or ex novo FA biosynthesis, ethanol production, and phosphoketolase pathways by introducing heterologous genes for higher production of acetyl-CoA, NADPH, ethanol, S-adenosylmethionine, acyltransferase, including FA methyltransferase and wax ester synthase, and disruption of FA degrading genes.

Another strategy was introduced by Blazeck et al. (2013a) who produced short-chain n-alkane in Y. lipolytica by introducing soybean lipoxygenase enzyme to cleave LA to pentane and tridecadienoic acid. In fact, many oxygenases can be used in specific biooxidation of FAs and lipids to functional metabolites, including alkanes and aldehydes. These membrane-associated enzymes require cofactors such as NADPH and NADH to introduce oxygen at single or double bond locations in the presence of electron transfer partner such as flavin reductase and iron sulfur protein (Urlacher and Schmid, 2006). Introducing such heterologous lipid-related enzymes into Y. lipolytica can pave the way for production of new lipid-derived biofuels. Nonetheless, complexity in the membrane-associated metabolic steps can complicate the introduction of such pathways into the yeast.

Some genes known as ole genes were found in Gram-negative and Gram-positive bacteria and encoded head-to-head FA condensation pathways. In this pathway, two FAs become connected through their carboxyl group releasing the carboxyl group and generating double bonds at the linkage point. Heterologous expression of these genes allows for long-chain olefinic hydrocarbon or monoketone production to the host cell (Sukovich et al., 2010). FA-overproducing E. coli has been used as platform for the biosynthesis of large chain alkenes. Heterologous three-gene cluster encoding for the homologous of the condensing enzymes, involved in FA biosynthesis (i.e., β-ketoacyl-ACP synthases), has been expressed in the host (Beller et al., 2010). In the suggested biosynthesis pathway, β-ketoacyl-CoA, a product of the β-oxidation pathway, and acyl-CoA, a product of FA biosynthesis, undergo condensation reaction. The product of the reaction goes through a pathway that is homologous to the FA elongation pathway in the presence of different reductases and dehydratases, resulting in long-chain alkene production (Ruffing, 2013). The suggested pathway can potentially be installed on the oleaginous yeast platform, since peroxisomal β-oxidation and FA biosynthesis are inherently and efficiently present in this platform. However, successful installation and compartmentalization requires comprehensive understanding of the peroxisome, its metabolic components, and corresponding regulatory and transformational mechanisms. These strategies may be employed in the future to produce advanced biofuels by manipulating oleaginous yeast platform.

Reducing fatty acyl-CoA to the corresponding aldehyde through eukaryotic acyl-CoA reductase followed by enzymatic decarbonylation can open the door to the production of alkane and alkenes from FAs with this oleaginous platform. However, it should be noted that fatty aldehyde decarbonylation is not well-understood. Some genes with aldehyde decarbonylase activity have been discovered in microbial species such as cyanobacteria. Codon optimization may be necessary to achieve an acceptable expression level. On the other hand, the above-mentioned multistep process may be compartmented into specific organelles to boost efficiency and conversion yield. A recent study shows that introducing specific heterologous FA reductase complex and aldehyde decarbonylase enables E. coli to directly use FFA for the production of linear and branched-chain alkane and alkene (Howard et al., 2013). The intracellular pool of FFA, and consequently the composition of the produced alkanes and alkenes were modified by supplementing media exogenously, and introducing heterologous thioesterase, branched-chain α-keto acid dehydrogenase, and β-keto acyl carrier protein synthase. FA intermediates may also undergo decarboxylation to hydrocarbon by FA decarboxylases of the P450 enzyme family (Rude et al., 2011). This is another inspiring way to make this platform a hydrocarbon producer.

Yarrowia lipolytica has been engineered to produce various functional FAs. For instance, arachidonic acid, as a ω-6 polyunsaturated FA, was produced with recombinant strains. This was achieved by transferring several heterologous desaturases, elongases, and acyltransferases into the yeast (Damude et al., 2006). Y. lipolytica was also recently engineered for the production of EPA at 56.6 and 15% of lipid content and DCW, respectively, the highest titer reported so far (Xue et al., 2013). Y. lipolytica has also been used to produce up to 5.9% of trans-10, cis-12 conjugated linoleic acid (CLA) of the total FA yield from glucose. The heterologous gene encoding LA isomerase was codon optimized and introduced into the yeast platform that is capable of free LA production, to covert the free FAs to the CLA (Zhang et al., 2012a). That study showed that codon optimization, integration sites, and the gene’s copy number are major factors for modifying the expression level. Also, the manipulation of heterologous and homologous desaturase, elongase, and acyltransferase system enabled this yeast to produce docosahexaenoic acid at 5.6% of total accumulated lipid (DHA) (Damude et al., 2014).

This yeast has also been used to produce other lipid-derived neutraceuticals. Some examples of these products are sterols (Bailey et al., 2010), carotenoids, and antioxidants (Sharpe et al., 2008). In the lipoyeasts project, various heterologous carotenoid genes (crt genes) were placed under the control of pPOX2 promoter in the integration expression vector and transformed into Y. lipolytica. In spite of the disruption of β-oxidation pathway and the restriction of lipid accumulation, the modification did not yield an outstanding production level of carotenoids (Sabirova, 2011). Y. lipolytica has also been engineered to direct the products of its isoprenoid biosynthesis pathway to β-carotene as lipophilic bioproducts (Grenfell-Lee, 2009). In a recent study, Y. lipolytica β-carotene did not display a safety profile that differed from other commercial carotenoid products, including microbial and synthetic ones (Grenfell-Lee et al., 2014). However, its application as a safe food additive may require further long-term oral toxicity analysis. This yeast has also been engineered for lycopene production (Ye et al., 2012; Matthäus et al., 2014). In a recent study, disruption of POX1–6 and GUT2 genes were followed by the introduction of heterologous genes and upregulation of native genes to achieve a maximum lycopene yield of 16 mg/g DCW in a fed-batch system under oil accumulation conditions. In fact, stimulation of lipid accumulation may play a key role in accommodating such hydrophobic carotenoids.

Recombinant Y. lipolytica also shows potential for expression of various heterologous P450 systems (Obiero, 2006; Theron, 2007). This expression may aid in production of active steroids (Mauersberger et al., 2013) or other oxygenated FAs with antimicrobial properties.

Several subcellular metabolic pathways of Y. lipolytica have been used to produce various lipid-derived biochemicals. For instance, the pathway of microsomal ω-oxidation, peroxisomal β-oxidation, FA synthesis, and TCA cycle have been engineered and harnessed in favor of specific bioproducts. In fact, these pathways accommodate a remarkable amount of carbon flux in this yeast.

The addition of functional groups such as hydroxy, carboxyl, or epoxy groups to FAs, can be achieved through ω-oxidation. The resultant products have a tendency to polycondense and cyclisize (Thevenieau et al., 2010). The ω-oxidation pathway can be manipulated to produce a variety of α,ω-dicarboxylic acids (DCAs), ω-hydroxy acids, and lactones. Production of these compounds using engineered Y. lipolytica has been recently discussed (Wache, 2013). Y. lipolytica has also been selected as a host for the expression of various CYP540s (Obiero, 2006). DCA production requires that a pool of FA is converted to DCA through ω-oxidation. The typical strategy for producing DCA from alkanes or FAs is to improve the flux toward ω-oxidation pathway by amplifying the first limiting step in this pathway (e.g., ALK1 and/or ALK2, and CPR). The next step is to reduce the flux toward the competitive β-oxidation pathway by partially disrupting POX genes (e.g., POX2, POX 3, POX 4, and POX 5). DCA9 and DCA11 have been produced from the β-oxidation of DCA13 when Aox1p and Aox6p are present (Thevenieau et al., 2010). Finally, it is challenging to produce DCAs with chain lengths of more than 16 carbons, since contributory oxidizing enzymes have not been fully characterized. On the other hand, it is typically challenging to purify P450 enzymes and study their functions in vitro, due to their co-factor dependency, sensitivity to oxygen species, and product inhibition (Van Beilen and Funhoff, 2007; Meng et al., 2009).

The β-oxidation pathway can potentially be engineered to produce β-hydroxyl FAs, lactones, methyl ketones, and vanillin with the applications in flavor, fragrance, and biofuel industries. To achieve this aim, directing the substrate to the correct pathway and favoring its exit at a specific level of degradation is necessary (Waché et al., 2006).

Some researchers have produced several types of lactone with aromatic characteristic from ricinoleic acid (C18:1-OH) or methyl ricinoleate using Y. lipolytica (Waché et al., 2003). In fact, γ-decalactone can reach the production level of 10 g/L. Ricinoleic acid is converted to 4-hydroxydecanoic acid through four cycles of β-oxidation, and then undergoes lactonization under acidic conditions (Swizdor et al., 2012). However, it is not clear whether lactonization occurs on acyl-CoA or on the FFAs. Intact β-oxidation genes can degrade lactone, highlighting the need to modify this pathway and avoid further degradation of the products (Thevenieau et al., 2009). The disruption of POX3, along with overexpression of POX2, can be used to continuously produce γ-decalactone from methyl ricinoleate (Guo et al., 2012). More specifically, disrupting acyl-CoA oxidase with short-chain specificity (i.e., <10 carbons) can promote the production of γ-decalactone (Waché et al., 2006; Thevenieau et al., 2009). In fact, many types of lactone can be produced from different hydroxyl FAs released at different stages of β-oxidation cycle. Y. lipolytica is one of the most promising candidates for production of γ-decalactone. This yeast was also recently engineered to produce ricinoleic acid. This was achieved through the disruption of β-oxidation, native Δ12 desaturase, and the majority of native TAG acyltransferases, except for Lro1p, followed by the heterologous expression of fungal hydroxylase. This FA reached 43% of total lipid at 15% CDW (Beopoulos et al., 2014).

β-hydroxy FAs are important chiral building blocks in the asymmetric synthesis of drugs, vitamins, fragrances, pheromones, and other bioproducts. They can be the products of partial β-oxidation cycle through the reduction of β-ketoacids, oxidation of 1,3-diol, and subsequently, hydration of α,β-unsaturated acids (Swizdor et al., 2012). When using β-oxidation, further metabolization of β-hydroxy acids to the corresponding acids should be avoided by genetic engineering to increase molar conversion yield.

Another type of lipid-derived flavor compound is green note, including a variety of aldehyde and alcohols of six to nine carbons. These flavor compounds can be produced through successive action of lipoxygenase, hydroperoxide lyase, and if necessary, alcohol dehydrogenase on LA and linolenic acids, which are naturally found in yeast lipids. Different tactics have been reviewed for the heterologous expression of the above-mentioned enzymes from plants, fruits, and conventional yeast into oleaginous yeasts such as Y. lipolytica (Waché et al., 2006).

Methyl ketones are reduced compounds with applications in the fragrance, flavor, and pharmacological industries. They can be produced from various sources, such as fungal biotransformation of medium chain FAs or oxidation of secondary alcohols by yeast cell suspension (Patel et al., 1979; Hagedorn and Kaphammer, 1994). Recently, E. coli was genetically manipulated to convert glucose to methyl ketones using 3-ketoacyl-ACP as precursor (Park et al., 2012). In another strategy, a β-oxidation pathway of E. coli was engineered for methyl ketone production. In these studies, overexpression and interruption of some native genes along with the expression of heterologous genes were employed (Goh et al., 2012). The presence of strong infrastructure for the production and degradation of FA flux makes the Y. lipolytica a good candidate to produce methyl ketone. However, the successful application of such strategies requires comprehensive understanding of contributory genes, proteins, regulatory mechanisms, and points of action in this oleaginous yeast.

Polyhydroxyalkanoate synthase can be used to polymerize the released hydroxylated acyl-CoA from β-oxidation to produce PHAs (Beopoulos and Nicaud, 2012). Although bacteria can produce PHA, the oleaginous yeast platform is also highly competitive, due to its ability to utilize a wider range of cheap feedstock, and intrinsically accumulate lipid and convert it to PHA. For example, the expression of PHA synthetase from Pseudomonas aeruginosa in this yeast under different POX genetic profiles has been reported (Haddouche et al., 2010). Based on the results, only Aox3p and Aox5p have been found to be involved in PHA biosynthesis from specific FAs. The hydroxylated FAs can also be obtained directly from hydroxy acyl-CoA and used as chiral building blocks to synthesize some chemicals (Sabirova et al., 2011).

Among the oleaginous yeasts, some Y. lipolytica strains show a good tolerance to different metals and possess inherent reductive compounds (e.g., reductases, proteases, melanin, and specific protein metallothionein). These compounds improve the potential for these strains to produce metal nanoparticles as prominent building blocks in nanotechnology (Pimprikar et al., 2009). Some potential strains of Y. lipolytica may be used to generate elemental metal nanoparticles (Agnihotri et al., 2009; Apte et al., 2013). This field of research is still in its infancy. Further studies will shed light on the molecular mechanism of nanoparticle biosynthesis (Chi et al., 2010a) and engineer Y. lipolytica strains. The use of Y. lipolytica to produce nanoproducts and lipid-based bioproducts will transform this platform into an effective tool for bridging between biotechnology and nanotechnology through development of novel biocatalysts and nano-oils.