Enhancing very long chain fatty acid production in Yarrowia lipolytica

doi:10.21203/rs.3.rs-1640913/v1

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Research Article

Enhancing very long chain fatty acid production in Yarrowia lipolytica

https://doi.org/10.21203/rs.3.rs-1640913/v1

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Background

Very long chain fatty acids and their derivatives are industrially attractive compounds. The most important are behenic and erucic fatty acids which are used as lubricants, moisturizers and has potential in biofuel production. These fatty acids are conventionally obtained from plant oils. Yarrowia lipolytica is an oleaginous yeast with long history of gene manipulations resulting in production of industrially interesting compounds, such as organic acids, proteins, and various lipophilic molecules. It has been shown previously that it has potential for production of very long chain fatty acids enriched single cell oils.

Results

Metabolism of Y. lipolytica has been redesigned to achieve increased production of very long chain fatty acids. Effect of native diacylglycerol acyltransferases of this yeast LRO1, DGA1, and DGA2 on accumulation of very long chain fatty acids was examined. It was found that DGA1 is the only enzyme with beneficial effect. Further improvement of accumulation was achieved by increasing of 3-ketoacyl-CoA synthase expression and blockage fatty acid degradation pathway. The best producing strain YL53 (Δmfe, pTEF-DGA1, 8UAS-pTEF-FAE1) produced 120 µg of very long chain fatty acids per g of produced biomass, which accounted for 34% of total fatty acids in biomass.

Conclusions

Recombinant strains of Y. lipolytica has proved to be good producers of very long chain fatty acids. Redesign of lipid metabolism pathways had positive effect on accumulation of erucic and behenic acids which are technologically attractive compounds.

Yarrowia lipolytica

erucic acid

behenic acid

diacylglycerol acyltransferase

Very long chain fatty acids (VLCFA) are fatty acids longer than 20 carbons synthesized by the complex fatty acid elongase. Fatty acid elongases are structurally and functionally very similar to fatty acid synthases. Both possess four enzymes performing a sequence of condensation, reduction, dehydratation, and reduction reactions. The major difference is that fatty acid synthases use acetyl-CoA as a starting substrate while elongases use exclusively fatty acyl-CoA instead. In plants substrate specificity of elongases is conferred by 3-ketoacyl-CoA synthase, which is the first enzyme in the four-step sequence, and it is performing condensation reaction. Plant genes responsible for 3-ketoacyl-CoA synthase activity are FATTY ACID ELONGATION (FAE1) [1]. Interestingly, FAE1-ketoacyl-CoA synthase are structurally different from yeast Elo proteins [2]. Compared to yeasts, which accumulate VLCFA only in minor quantities, plants are very good producers of VLCFA. Since VLCFA are components of cuticular waxes, they are essential for all plants. Especially plants from Brassicaceae family are exceptional producers of VLCFA which are accumulated in seed oils of these plants in the form of triacylglycerols. In seed oils of Simmondsia chinensis VLCFA are stored in the form of waxes (esters of fatty acids and fatty alcohols).

VLCFA and their derivatives have many industrial applications. For example, erucic acid is applicable as surfactant, lubricant, and constituent of nanocomposites [3, 4]. A study of Seames and colleagues has described beneficial impact of erucic acid on biodiesel yield produced by non-catalytic cracking of triacylglycerols [5]. Xu and colleagues described preparation of diesel like hydrocarbons from behenic acid [6]. Behenic acid and its derivative behenyl alcohol is common constituent of cosmetics such as moisturizers [7]. Also, derivatives of erucic and behenic acids erucamide and behenamide are used as slip agents [8].

Heterologous expression of plant FAE1-ketoacyl-CoA synthases in yeasts led to production of saturated and monounsaturated VLCFA. In the study of Fillet and colleagues Rhodosporidium toruloides was used as a host for heterologous expression of four FAE1 from Arabidopsis thaliana, Cardamine graeca, Crambe abyssinica, and Lunaria annua [9]. VLCFA profile and overall percentage of VLCFA was influenced by specific FAE1. Similarly, FAE1 from Thlaspi arvense was successfully expressed in Yarrowia lipolytica, which was cultivated on cheap feedstocks [10]. Yeasts have certain advantages over plants, especially much shorter generation periods and independence on climate and seasons. However, heterologous production of desired metabolites in yeasts often require several steps of genetic manipulations. It was shown previously, that besides genes directly responsible for fatty acid synthesis, genes regulating storage lipid metabolism such as acyltransferases are very important for specific fatty acid accumulation [11, 12].

Yarrowia lipolytica is ascomycetous, non-pathogenic, oleaginous yeast. Due to its natural ability to produce extracellular proteins, organic acids, and intracellular lipids [13, 14] it was predestined to become model yeast for studies concerned with production of such compounds. Moreover, there is plethora of genetic engineering tools developed for manipulating genome of Y. lipolytica [15]. Thanks to these features it has become the cellular factory for production of many biotechnologically interesting compounds [16]. As mentioned above VLCFA was already produced in Y. lipolytica in considerable quantities. In this study storage lipid metabolism of Y. lipolytica was redesigned to increase the production of VLCFA.

Co-expression of FAE1 with different diacylglycerol acyltransferase genes

Strains JMY1882 (pTEF-LRO1), JMY1884 (pTEF-DGA2) and JMY1892 (pTEF-DGA1) [17] were used as a host for FAE1 resulting in strains YL12 (pTEF-LRO1, pTEF-FAE1), YL13 (pTEF-DGA2, pTEF-FAE1) and YL15 (pTEF-DGA1, pTEF-FAE1). YL12, YL13 and YL15 has shown different growth patterns after 72 hours of cultivation in medium with 60 g/L glucose as a carbon substrate (Fig. 1). Higher biomass yields in YL13 and YL15 were accompanied by the significant difference in lipid accumulation. While the strain YL12 produced less than 10% of total fatty acids (TFA) in biomass, both YL13 and YL15 accumulated more than 30% of TFA (YL13 – 37%, YL15 – 43%). Expression of FAE1 resulted in effective production of VLCFA only in the strain YL15 (pTEF-DGA1, pTEF-FAE1) (Fig. 2). The other two strains exhibit neither increase in saturated (C22:0 and C24:0) nor in monounsaturated (C22:1 and C24:1) VLCFA.

Improvement of VLCFA accumulation by redesign of metabolism

Since accumulation of VLCFA by the strain YL15 was quite low (approximately 4.5% of TFA) strategy for increasing of VLCFA content was conducted. First, instead of pTEF promoter stronger 8UAS-pTEF promoter was used to control the expression of FAE1. This change resulted in more than 4-fold increase of VLCFA percentage in TFA of resulting strain YL62 (pTEF-DGA1, 8UAS-pTEF-FAE1) compared to strain YL15 (pTEF-DGA1, pTEF-FAE1). Another improvement was achieved by abolition of β-oxidation. Strain YL53 (Δmfe, pTEF-DGA1, 8UAS-pTEF-FAE1) had improved VLCFA percentage more than 7-fold compared to YL15. Strains YL15, YL62, YL53 were grown for 72 hours with 60 g/L glucose which was still present after the end of the cultivation (Table 1).

Table 1 Residual glucose (Glc), manitol (Man), and citric acid (CA) in medium

	Glc (g/L)	Man (g/L)	CA (g/L)
YL15	17.4±0.6	2.1±0.1	4.4±0.1
YL62	15.0±0.8	2.4±0.5	3.2±0.5
YL53	19.5±0.1	0.8±0.1	2.1±0.1

Yeast strains YL15 (pTEF-DGA1, pTEF-FAE1), YL62 (pTEF-DGA1, 8UAS-pTEF-FAE1), and YL53 (Δmfe, pTEF-DGA1, 8UAS-pTEF-FAE1) were grown in 60 g/L glucose medium with C/N ratio of 80 for 72 hours. Each value is an average of three experiments

It was observed, that increasing VLCFA accumulation was accompanied by drop in biomass yield (Fig. 3). Nonetheless, strain YL53 was the best VLCFA producer yielding up to 120 µg of VLCFA per g of produced biomass, which accounted for 34% of TFA in biomass.

Fatty acid profiles of VLCFA producing strains

Fatty acid profiles of constructed strain differed according to changes in the genome of cells (Fig. 4). Fatty acid profile of YL15 was very close to the standard fatty acid profile of wild type strain W29 [14] with oleic acid as the major fatty acid. The major difference was production of gondoic acid and erucic acid which are not common for Y. lipolytica. The situation was changed by overexpression of FAE1 under control of 8UAS-pTEF in YL62 when oleic acid dropped from over 50% to less than 30% of TFA in cells. This was accompanied by more than 2-fold increase in palmitoleic acid and significant increase in all VLCFA. The most profound change in fatty acid profile was seen in strain YL53, where the major fatty acid became palmitoleic acid instead of usual oleic acid. Behenic, erucic and lignoceric acids were the most accumulated VLCFA in YL53. Erucic acid was the most abundant VLCFA representing 13% of TFA.

In the previous study it was shown that expression of FAE1 from Thlaspi arvense in Y. lipolytica induced monounsaturated VLCFA production in this yeast [10]. Since the production of VLCFA in recombinant Y. lipolytica was quite low compared to plants, further manipulation was required to increase VLCFA content in this yeast. Generally, the accumulation of fatty acids is closely connected to the ability of the cell to sequester it to specific subcellular compartments such as lipid droplets. Fatty acids in lipid droplets are usually stored in the form of triacylglycerols, which are synthesized by diacylglycerol acyltransferases. Diacylglycerol acyltransferases are divided into phospholipid:diacylglycerol acyltransferases (PDAT) and acyl-CoA:diacylglycerol acyltransferases (DGAT) according to the acyl donor. DGATs are further categorized to DGAT1 and DGAT2 families according to their structure. In Y. lipolytica three genes are coding diacylglycerol acyltransferase: LRO1, DGA1, and DGA2. Previously, strains carrying single diacylglycerol acyltransferase enzyme were constructed [17]. In this study, these strains were used as a host for FAE1. It was observed that only strain co-expressing FAE1 and DGA1 (YL15) has shown considerable VLCFA accumulation. The other two strains (YL12 and YL13) exhibit neither increase in C22:0 and C24:0 which are naturally synthesized also in wild type strain W29 [18] nor in C22:1 and C24:1, which are synthesized exclusively by the action of FAE1. These results indicate that only DGA1 has the preference for such very long chain fatty acyl-CoA. LRO1 and DGA2 did not provide accumulation of VLCFA. LRO1 has the role of PDAT in Y. lipolytica and it has only minor contribution to triacylglycerol accumulation. Its main part is rather phospholipid remodelling through its phospholipase activity. On the other hand, DGA1 coding a DGAT2 acyltransferase and DGA2 coding a DGAT1 acyltransferase has the major impact on triacylglycerol accumulation in Y. lipolytica. While it is common for plants and animals to harbour both DGAT1 and DGAT2 enzymes, Y. lipolytica and Blastobotrys raffinosifermentans are the only two known microorganisms with both enzymes [17, 19]. DGA1 and DGA2 have different localisation in cells of Y. lipolytica [20] and obviously have different substrate specificities. Preference of DGAT1 and DGAT2 enzymes for certain types of acyl-CoA is not unusual and it was described for example in Vernicia fordii [21] and Claviceps purpurea [22]. More recently, results of the Sanya and colleagues indicates, that DGA1 and DGA2 from B. raffinosifermentans may have different specificities for substrates [19].

Further improvement of VLCFA production was achieved by tuning the FAE1 expression. The simple replacement of pTEF promoter for 8UAS-pTEF, which is stronger than pTEF [23], increased the VLCFA content from approximately 20 to more than 80 µg/mg of biomass in strains YL15 and YL62, respectively. This underlines the fact, that regulation of gene expression is particularly important since the gene expression must take place at right time, right place, and right level. When speaking about the regulation sequences, promoters are usually first in the row. This is due to their direct impact on expression of the certain gene. There are many promoters with different properties available for gene expression in Y. lipolytica. Few decades ago, the basic promoters for gene expression in Y. lipolytica were growth phase dependent hp4d [24], constitutive pTEF [25], and inducible pPOX2 [26]. As the research proceeded, novel promoters have been described which were either native or synthetic. Increasing the strength of promoter by adding upstream activating sequence 1 (UAS1) from XPR2 became extremely popular [23, 27–30]. However, the study of Dulermo and colleagues has shown, that the strongest promoter is not the best promoter in all circumstances [23]. Concerning inducible promoters, the study of Carly and colleagues has described the inducible promoter pEYK1, which is easier to use than pPOX2 [31]. The first needs hydrophilic inducer and the latter requires hydrophobic substrates, which is much more difficult to use in cultivation media. Taken together, nowadays, there is huge portfolio of promoters which could be used and none of them could be considered as universal for all applications. However, exchange of pTEF for 8UAS-pTEF has proven as the right option in this study.

Metabolic pathways other than those connected to fatty acid synthesis and formation of storage lipids present many times a serious obstacle in efforts leading to accumulation of fatty acids. Metabolic pathway which poses the major problem is certainly the β-oxidation of fatty acids. Therefore, prevention of fatty acid degradation by blocking of the β-oxidation is of high importance. There are two possibilities to achieve this goal. The first is to focus on acyl-CoA oxidase activity, which is in Y. lipolytica represented by six POX1-6 [32]. The second is to delete MFE1, the gene which is encoding multifunctional enzyme responsible for all β-oxidation reactions save acyl-CoA oxidation [33]. In this study deletion of MFE1 was employed, which resulted in further improvement of VLFA accumulation. It must be said that deletion of MFE1 had negative impact on biomass yield, which was observed previously as well [18]. Nonetheless, YL53 was the best producing strain yielding 120 µg of VLCFA per mg of biomass.

Overall percentage of VLCFA in our best strain YL53 was 34% which was comparable to strains constructed by Fillet and colleagues, where total VLCFA ranged from 30–40% of TFA [9]. Profile of VLCFA differed according to source of FAE1 since sequences were obtained from A. thaliana, C. graeca, C. abyssinica, and L. annua. They have found that 3-ketoacyl-CoA synthase from C. graeca was better for nervonic acid production, while 3-ketoacyl-CoA synthase from C. abyssinica increased erucic acid content. In our strains the most produced VLCFA was erucic acid. This is in line with VLCFA production in T. arvense, where erucic acid is represents almost 40% of TFA, while other VLCFA are produced only in minor quantities [34]. In our best producing strain Y. lipolytica YL53 also behenic and lignoceric acids are quite abundant in addition to erucic acid.

In this study Y. lipolytica was manipulated with the aim of VLCFA production. LRO1, DGA1, and DGA2 were co-expressed with FAE1 to determine the most suitable acyltransferase for VLCFA production. We have found, that DGA1 acyltransferase has positive impact on VLCFA accumulation. Further improvement in VLCFA accumulation was achieved by the expression of FAE1 using strong 8UAS-pTEF promoter. Deletion of MFE1 had negative impact on the growth, nonetheless further improved VLCFA accumulation. Thus, strain producing more than 30% of VLCFA was constructed with erucic acid as the most produced followed by behenic acid and lignoceric acid. Erucic and behenic acids are technologically interesting compounds and microbial production of oils enriched with these fatty acids is interesting alternative to their production by plants.

Strains, media composition, and culture conditions

Yarrowia lipolytica and Escherichia coli strains used in this study are summarized in Table 2.

Table 2

*E. coli* and *Y. lipolytica* strains and plasmids used in this study
Strain (host strain)	Plasmid, genotype	References
E. coli strains
EC11 (DH5a)	JMP62-pTEF-FAE1-LEU2ex	[10]
EC61 (DH5a)	JMP62-8UAS-pTEF-FAE1-LEU2ex	This work
JME1112 (DH5a)	JMP62-pTEF-DGA1-URA3ex	[17]
JME4305 (DH5a)	JMP62-URA3ex-8UASpTEF-FAR4	[35]
Y. lipolytica strains
JMY1882	MATA ura3-302 leu2-270 xpr2-322 Δdga1Δlro1Δare1Δdga2 pTEF-LRO1-URA3ex	[17]
JMY1884	MATA ura3-302 leu2-270 xpr2-322 Δdga1Δlro1Δare1Δdga2 pTEF-DGA2-URA3ex	[17]
JMY1892	MATA ura3-302 leu2-270 xpr2-322 Δdga1Δlro1Δare1Δdga2 pTEF-DGA1-URA3ex	[17]
JMY1915	MATA ura3-302 leu2-270 xpr2-322 Δdga1Δlro1Δare1Δdga2Δmfe1	[33]
YL12	JMY1882 pTEF-FAE1-LEU2ex	This work
YL13	JMY1884 pTEF-FAE1-LEU2ex	This work
YL15	JMY1892 pTEF-FAE1-LEU2ex	This work
YL51	JMY1915 pTEF-DGA1-URA3ex	This work
YL53	JMY1915 pTEF-DGA1-URA3ex 8UAS-pTEF-FAE1-LEU2ex	This work
YL62	JMY1892 8UAS-pTEF-FAE1-LEU2ex	This work

E. coli were cultured in lysogeny broth medium supplemented with 50 µg/mL kanamycin, according to a standard protocol [36]. All Y. lipolytica strains used in this study are derived from strain W29 (ATCC 20460). The plates of minimal YNB and YNBleu media agar were used for the selection of transformants. The minimal YNB medium consisted of 0.17% (w/v) yeast nitrogen base (without amino acids and ammonium sulfate; BD, Erembodegem, Belgium), 0.5% (w/v) NH₄Cl, 50 mM phosphate buffer (pH 6.8), and 2% (w/v) glucose. For YNBleu medium, leucine (0.1 g/L) was added to YNB medium. Agar plates were prepared by the addition of 20 g/L agar. Yeast inoculum was prepared in rich YPD medium containing 1% (w/v) yeast extract (BD, Erembodegem, Belgium), 1% (w/v) peptone (BD, Erembodegem, Belgium), and 2% (w/v) glucose (Mikrochem, Pezinok, Slovakia). The medium for lipid production (C/N ratio of 80) contained 60 g/L glucose, 1.5 g/L yeast extract, 0.5 g/L NH₄Cl, 7 g/L KH₂PO₄, 5 g/L Na₂HPO₄.12H₂O, 0.1 g/L CaCl₂, 1.5 g/L MgSO₄.7H₂O, 10 mg/L ZnSO₄.7H₂O, 0.6 mg/L FeCl₃.6H₂O, 0.07 mg/L MnSO4.H₂O, and 0.04 mg/L CuSO₄.5H₂O. The medium for lipid production was filter sterilized. Yeast inoculum was prepared in 20 mL of YPD medium in 100 mL flasks. Subsequently, 50 mL of production medium in 250 mL baffle flasks was inoculated with a 24-hour inoculum having an optical density (OD600) of 0.1. The cells were cultured at 28°C on an orbital shaker at 130 rpm.

Plasmid and strain construction

E. coli strain EC61 (JMP62-8UAS-pTEF-FAE1-LEU2ex) was constructed in this work. Plasmids (JMP62-pTEF-FAE1-LEU2ex) from the E. coli EC11 [10] and (JMP62-URA3ex-8UASpTEF-FAR4) from E. coli JME4305 [35] were double-digested by ClaI and BamHI to replace pTEF promoter in (JMP62-pTEF-FAE1-LEU2ex) by 8UAS-pTEF from (JMP62-URA3ex-8UAS-pTEF-FAR4). The resulting plasmid (JMP62-8UAS-pTEF-FAE1-LEU2ex) was stored in E. coli EC61. Insertion cassettes obtained by the digestion of plasmids from EC61 and JME1112 (JMP62-pTEF-DGA1-URA3ex) [17] using NotI (New England Biolabs, Ipswich, MA, USA) were used for transformation of yeast cells. Roti®-Prep Plasmid MINI and Roti®-Prep Gel Extraction kits (Carl Roth, Karlsruhe, Germany) were used for plasmid extraction and recovery of DNA fragments from agarose gel, respectively. The transformation of the yeast cells was done by the lithium acetate method [37]. The genomic DNA was prepared according to Lõoke and colleagues [38] and was then amplified by PCR in a Bio-Rad T100™ Thermal cycler using GoTaq® DNA polymerase (Promega, Madison, WI, USA). The successful insertion of DGA1 was verified using the primer pair URA3in and DGA1in with sequence 5'- TTGGTGGTGGTAACATCCAGAG-3' and 5'- AGCCAGATGATTCTCCACGG-3', respectively. The successful insertion of FAE1 was verified using the primer pair LEU2in and FAE1in with sequence 5'- TACGACGCATTGATGGAAGG-3' and 5'- TTCACCACCATAGCGGACAG-3', respectively.

Analytical methods

Isolation of biomass was performed as follows. Cell suspensions were centrifuged (2880 ×g, 5 min), washed twice with saline, once with deionized water, and freeze-dried. The freeze-dried cells were used for lipid analysis. The dry cell weight (DCW) was determined gravimetrically. The residual glucose (Agilent Technologies, Santa Clara, CA, USA) using an Aminex HPX87H column (Bio-Rad, Hercules, CA, USA) coupled to RI detector and UV detector, as described by [39]. FA from freeze-dried cells were transformed to FA methyl esters (FAME) and analyzed as described previously [18].

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

The work was supported by a Grant (APVV-17-0262) from the Slovak Research and Development Agency, Slovak Republic.

Authors' contributions

PG defined the concept of the study. MC acquired funding for this study. VU and MV designed and prepared the constructs, designed, and performed the cultivation experiments. PG prepared the manuscript. MC and VU critically revised the manuscript. All authors contributed to the article and approved the submitted version.

Acknowledgements

The authors would like to thank Dr. Jean-Marc Nicaud for providing yeast strains and vector used in this study.

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No competing interests reported.

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Editorial decision: Major revision
19 May, 2022
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18 May, 2022
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Enhancing very long chain fatty acid production in Yarrowia lipolytica

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