Fatty alcohols (FA-OH) are aliphatic unbranched primary alcohols with varying chain lengths ranging from 4 to 28 carbon atoms and containing either saturated or unsaturated carbon bonds (McNaught and Wilkinson, 1997). The properties and the potential applications of fatty alcohol depend on their molecular structure. Generally, fatty alcohols are used as fuels, solvents, detergents, cosmetics, lubricants, and pharmaceuticals, or may serve as precursors for other compounds such as waxes or polymers (Rutter and Rao, 2016; Wang G. et al., 2016; Wang W. et al., 2016; Borodina et al., 2018a; Cordova et al., 2019). In 2019, the global demand for fatty alcohols was estimated to be over two million tons, with an annual growth rate of 4.3%. Traditionally, these molecules are produced through the catalytic hydrogenation of petrochemicals or plant oils, which currently relies on fossil fuels or unsustainable palm farming and has significant environmental consequences such as deforestation or contribution to global warming (Rutter and Rao, 2016; Shah et al., 2016; Cordova et al., 2019). Therefore, there is an urgent requirement for a further efficient and ecologically-friendly process.

Fatty alcohols and their derivatives also have important biological functions. Insects have evolved an efficient mate-finding system that is based on volatile sex pheromones. In most species, sex pheromones are released either as a single component (Jurenka, 2004; Groot et al., 2016; Tupec et al., 2017) or as a specific blend of molecules in specific ratios, most of which are fatty acid (FA) derivatives, usually alcohols, aldehydes, or acetates. The sex pheromone blend of the bumblebees mainly comprises saturated, mono-unsaturated, and poly-unsaturated fatty alcohols with a chain length of 16–18 carbon atoms along with terpenoid compounds (Ayasse and Jarau, 2014). Sex pheromones are synthesized de novo in specialized cells known as pheromone glands, which have evolved from epidermal cells (Žáček et al., 2013; Tittiger and Blomquist, 2017). The pheromone biosynthesis process involves several key enzymes. In addition, the saturated FAs such as stearic and palmitic must undergo processing mediated by chain-shortening enzymes, desaturases, reductases, acetyltransferases, and oxidases, among others (Tillman et al., 1999; Jurenka, 2004; Matoušková et al., 2008; Matsumoto, 2010; Buček et al., 2013; Koutroumpa and Jacquin-Joly, 2014; Tupec et al., 2017).

The first pheromone to be identified and purified was a fatty alcohol named bombykol (10E,12Z- hexadeca-10,12-dien-1-ol), which was isolated from the silkmoth Bombyx mori (Butenandt et al., 1961). Enzymes involved in bombykol biosynthesis were described later (Moto et al., 2003, 2004). Since then, pheromones have been identified in several thousands of insect species and are known for their potential as attractants or repellents in agriculture (Koutroumpa and Jacquin-Joly, 2014; El-Sayed, 2019). Pheromones represent the most suitable substitute for insecticides. Insecticides have been in use in agriculture for over 50 years. However, the environmental damage caused by the insecticides and the development of insecticide resistance among insects and pests are emerging as serious threats. The most significant problems associated with the use of insecticides include: (a) harmful effects on other organisms, including humans and the plants that rely on insects for pollination, (b) persistence of the insecticides in the biosphere, (c) worldwide spread, and (d) significant levels of pollution associated with the current methods of insecticide production (Hagström et al., 2013; Borodina et al., 2018b). In order to resolve some of these issues, synthetic pheromones were developed to control the pest insects in a species-specific manner and to maintain healthy agricultural practices (Hagström et al., 2013). For instance, tetradec-9-enyl acetate (C14:1Δ⁹-OAc) was reported to disrupt the mating efficiency of the fall armyworm when applied alone (Mitchell and McLaughlin, 1982). However, most agricultural applications of pheromones are limited by their high cost. Pheromones are expensive because their purity is paramount for eliciting a response in an insect, and the production of pheromones with such high levels of purity through chemical processes requires expensive and complicated methods while generating waste in huge quantities, which again requires disposal and increases costs (Borodina et al., 2018b).

The development of various genetic tools has allowed the characterization of heterologously produced fatty acyl-CoA reductases (FARs), which catalyze the reduction of fatty acyl-CoA precursors into the corresponding alcohols (Moto et al., 2003; Liénard et al., 2010; Antony et al., 2016; Ding et al., 2016; Tupec et al., 2019). The FAR genes are present in several species, including vertebrates, non-insect invertebrates, and fungi, with a particularly high number of FAR gene families reported in plant and insect genomes (Eirín-López et al., 2012; Buček et al., 2016; Tupec et al., 2019). Within the class Insecta, large quantities of long-chain alcohols have been identified in the pheromone mixtures of different bumblebees, including Bombus lucorum, Bombus lapidarius, and Bombus terrestris. Tupec et al. (2019) used heterologous expression in S. cerevisiae to demonstrate that bumblebees have evolved a specific FAR gene group that encodes reductases with unusual specificities and contributes to the biosynthesis of different fatty alcohols that form a part of bumble-specific pheromones.

The recent advancements in metabolic engineering and synthetic biology have enabled an environment-friendly production of FA-derived compounds, including FA-OHs and the biofuels from renewable feedstock using microbial biomass (Guo et al., 2016; Rutter and Rao, 2016). Yarrowia lipolytica is an oleaginous non-pathogenic yeast belonging to the Ascomycota phylum of kingdom Fungi (Abdel-Mawgoud et al., 2018), which could serve as a perfect cell factory for industrial applications (Groenewald et al., 2014). This yeast species is of great importance to researchers due to its high tolerance to a variety of organic substrates, higher salt levels in the environment, and a broad range of pH (Miller and Alper, 2019). Since the genome of Y. lipolytica was unraveled long ago and the tools for manipulating genomes and the knowledge of genetic engineering has also progressed dramatically, Y. lipolytica has become a suitable representative model organism for the production of natural biosynthetic products in the laboratory (Ledesma-Amaro and Nicaud, 2016). In this context, the present study was aimed to evaluate the ability of Y. lipolytica in the production of FA-OHs of different lengths, for which two bumblebee FARs (Tupec et al., 2019) were expressed in this yeast. The BlapFAR4 from B. lapidarius is capable of preferentially catalyzing the production of shorter FA-OHs (14–16 carbons), while BlucFAR1 from B. lucorum prefers acyl chains containing 18–26 carbon atoms.

In the present study, we engineered multiple strains of Y. lipolytica for a redesigned lipid metabolism, with genes encoding BlucFAR1 and BlapFAR4, to produce the FARs and obtain the corresponding fatty alcohols. The expression of BlucFAR1 (JMY7086) stimulated the production of 18–24 carbon atom-long fatty alcohols, presenting the highest fatty alcohol production among all the strains (166.6 mg/L). Unlike JMY7086, the BlapFAR4-expressing strain (JMY7090) produced only 14.6 mg/L of fatty alcohols with a chain length of 16 carbons.

Fatty alcohol production using microorganisms could reduce the production costs for insect pheromones and render the production process environmentally safer (Borodina et al., 2018b; Petkevicius et al., 2020). Although the literature has reported several approaches for producing fatty alcohols using E. coli and S. cerevisiae, one promising tool is the use of oleaginous microorganisms. So far, among all the oleaginous yeasts, Y. lipolytica is the most researched one (Borodina et al., 2018a). Host systems, either bacterial (E. coli or cyanobacteria) or yeast (S. cerevisiae, R. toruloides, or Y. lipolytica) that express non-insect enzyme sequences are reported to have reached several grams of fatty alcohols per liter of cultivation medium after optimization (Tupec et al., 2017). A fatty alcohol titer of 6.33 g/L was achieved in fed-batch fermentation in E. coli via the deletion of all fatty acyl-CoA thioestarases and ldhA, pta, and ackA genes, to starve cells of fatty acids and remove competing pathways (Liu et al., 2016). The expression of Tyto alba FAR (TaFAR1) in Y. lipolytica resulted in the production of 690 mg/L of hexadecan-1-ol (Wang G. et al., 2016), while the Y. lipolytica transformed with the MhFAR gene encoding the reductase from Marinobacter hydrocarbonoclasticus produced 6 g/L of alcohols representing an accumulation of 36 mg alcohols/g of cells (Cordova et al., 2019). The same reductase stimulated the production of 770 mg/L of fatty alcohols in L. starkeyi (Wang W. et al., 2016) and 8 g/L of fatty alcohols in Rhodosporidium toruloides (Fillet et al., 2015). Since the pheromone blends often contain unusual components, it is an advantage to use insect fatty acyl biosynthetic enzymes for their production.

In the present work, Y. lipolytica strains were engineered to have different genetic backgrounds with genes encoding reductases from Bombus lucorum (BlucFAR1) and Bombus lapidarius (BlapFAR4). When these genes were introduced into the Po1d strain under the control of the pTEF promoter, alcohols were detected only in trace amounts (in strains JMY6697 and JMY6698). Therefore, the second strategy was adopted, which involved redesigning the strain JMY3820, which had all the six POX genes and TGL4 lipase deleted and the DGA2 and GPD1 overexpressed using the pTEF promoter, with the incorporation of FAR genes driven by a stronger promoter 8UAS-pTEF. This genotype is particularly effective in accumulating high amounts of lipids (Dulermo et al., 2015). Deletions of POX genes and TGL4 gene prevents degradation of storage lipids and oxidation of fatty acids. On the other hand, overexpression of the DGA2 and GPD1 drives the formation of the TAGs and incorporation of fatty acids into the TAGs. The premise was to implement the push and pull strategy (Tai and Stephanopoulos, 2013), with the higher lipid production as the driving force for the higher fatty acid synthesis. With the higher fatty acyl-CoA pool, more fatty acyl-CoAs can be redirected to the reduction by FARs. As the cells become more saturated with TAGs, more fatty acyl-CoA becomes available as a substrate for reductases. This strategy produced three new strains: JMY7086 (8UAS-pTEF-BlucFAR1), JMY7090 (8UAS-pTEF-BlapFAR4), and JMY7094 (8UAS-pTEF-BlucFAR1, 8UAS-pTEF-BlapFAR4). A co-expression of both the reductases (JMY7094) did not meet the expectations. It might have affected the FA profiles or reduced the activity of the elongases (Figure 3). This could have led to limited availability of the substrate and ultimately a lower yield of alcohols. For instance, co-expression of BlucFAR1 and BlapFAR4 resulted in a higher level of C16:0-OH and C16:1Δ⁹-OH and a decreased level of longer FA-OHs (Table 2). Another explanation could be as a consequence of an increased degree of FA-OH degradation. Table 3 presents the degradation of long-chain fatty alcohol between Day 4 and Day 5, which might have been induced by the presence of C16 fatty alcohols, which would also explain the lower level of total FA-OHs in JMY7090. It may also be interpreted by the level of BlucFAR1 expression, possibly due to the integration site of BlucFAR1 in the double mutant or titration of the pTEF promoter. All of the results did not indicate alcohol toxicity on the cells and rather indicated the inhibition of FA formation and lipid accumulation by the alcohols or the by-products of their metabolism. It is also possible that in addition to being affected by the total FA-OH amount, the FA profile is affected by the FA-OH composition as well. For example, Figure 3 clearly shows that the JMY7086 strain produces more long chain fatty acids (>C18). Indirectly, this tells us that the production of long-chain fatty alcohols may well be driving the elongation of fatty acids - the so-called metabolic driving force. This also corresponds to the push and pull strategy described by Tai and Stephanopoulos (2013). If FAR is taking substrates (long-chain fatty acyl-CoAs) from lipid metabolism, the cells are overcompensating by producing more long-chain fatty acyl-CoAs. Although the metabolic engineering of the selected Y. lipolytica strains enabled the production of the desired FA-OHs, there is scope for further improvement. A combination of lipid metabolism manipulation in Y. lipolytica and engineering of the FA-OH metabolism could produce higher amounts of specific FA-OHs synthesized by a particular FAR in low concentrations. It is well recognized that in certain cases, Y. lipolytica secretes fatty acids and fatty alcohols into the medium, which increases the production while simplifying the purification. Most often, this occurs spontaneously when the cells accumulate high amounts of toxic products that they are unable to degrade. The excretion of fatty alcohols may also be achieved by optimizing the fermentation process. The deletion of FAO1 (fatty alcohol oxidase) and HFD4 (fatty alcohol dehydrogenase) could prevent product degradation by the cells (Dahlin et al., 2019; Holkenbrink et al., 2020). Another approach is to manipulate the FAA1 gene (fatty acyl-CoA synthetase) or the lipases to increase the substrate availability for the functioning of FARs, or to use dodecane-mediated extraction fermentation for the secretion of alcohols into the medium (Cordova et al., 2019).

In a previous study, the FARs used in the present work were expressed in Saccharomyces cerevisiae (Tupec et al., 2019), and several discrepancies were observed between the composition of FA-OH synthesized by the FARs in labial gland of bumblebee and in the S. cerevisiae expression system. The reductase BlapFAR4 produced poly-unsaturated fatty alcohols in the yeast cells, which are not present in the labial gland of male B. lapidarius. It is possible that a pool of fatty acyl CoA precursors in the yeasts differs from those present in the labial gland. The concentrations of C18:1Δ⁹-CoA, C18:2Δ^9,12-CoA, and C18:3Δ^9,12,15-CoA in the labial gland of B. lapidarius might be extremely low, to the extent that detectable accumulation of the corresponding alcohols is not possible. This implies that the ratio of fatty alcohols in the pheromones is dependent not only on the FAR substrate specificity, rather also on substrate availability, which is affected greatly by the other mechanisms such as the conversion of fatty acyls into fatty acyl-CoAs, fatty acyl-CoAs hydrolysis, etc. The differences in the quantity and ratio of fatty alcohols between Y. lipolytica and S. cerevisiae (Table 4) most likely reflects the differences in metabolism of these species. While Y. lipolytica is considered as oleaginous yeast, S. cerevisiae do not accumulate significant quantities of lipids. FA composition of lipids of these two yeast species is also different. While Y. lipolytica naturally prefers to accumulate the FAs with a chain length of 18 carbon atoms, S. cerevisiae prefers 16 carbon atom-long FAs. It could, therefore, be assumed that this difference in the accumulation of FAs was reflected in the fatty acyl-CoA pool as well. Since the fatty acyl-CoA pool is the source of substrates for FAR, a higher quantity of either 16 or 18 carbon long FA could influence the quantity and quality of FA-OHs. While the enzyme BlucFAR1 produced the highest FA-OH content in Y. lipolytica (166.6 mg/L), the same enzyme produced only a small amount of FA-OH in S. cerevisiae (6.9 mg/L). On the contrary, BlapFAR4 worked much better in S. cerevisiae (79 mg/L) than in Y. lipolytica (14.6 mg/L). Therefore, variations in yields could be attributed to the difference in the substrate availability between Y. lipolytica and S. cerevisiae. However, the expression level of the enzymes and in particular their activities also might have an effect on the synthesis of FA-OH.

Taken together, the results of the present work demonstrated that bioengineered Y. lipolytica strain with deleted POX and TGL4 lipase genes and overexpressed DGA2 and GPD1 genes is suitable for the efficient production of FA-OHs with different chain lengths. To our knowledge, this is the first study showing the ability of Y. lipolytica to produce FA-OHs of different lengths and the first study achieving production of FA-OHs longer than 20 carbons by Y. lipolytica.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

IP, RL-A, J-MN, and MC defined the concept of the study. MT isolated the genes. IP provided the gene sequences. RL-A designed and prepared the constructs. PG and JH designed and performed the cultivation experiments and performed the GC MS analyses. JH prepared the manuscript. IP, MT, RL-A, J-MN, MC, and PG revised the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.