De novo synthesis of nervonic acid in Y. lipolytica
We previously engineered Y. lipolytica to produce VLCFAs with carbon chain lengths up to 24 by co-expression heterologous C16/18-elongase from Mortierella alpina (MaELO3), β-ketoacyl-CoA synthases (KCSs) from Arabidopsis thaliana (AtKCS) and Crambe abyssinica (CraKCS) combining with the deletion of PEX10 (Gao et al. 2020). Although VLCFAs metabolism was successfully engineered, the resulting strain GQ07 only accumulates marginal nervonic acid (C24:1), and the titer needs to be further improved. Owing to the limitation of auxotrophic markers of plasmids, here, we re-engineered VLCFAs metabolism pathway into chromosome using the recently established CRISPR/Cas9 technology without the selection marker (Schwartz et al. 2016). The use of hybrid promoter UAS4B-TEF (UT) provided an excellent platform for high gene expression in Y. lipolytica (Gao et al. 2018). We therefor used this for the over-expression of the codon-optimized MaELO3, AtKCS, and CraKCS genes into the integration sites F1, A3, F1-3 of Y. lipolytica GQY-∆PEX10 strain, respectively. Previous studies confirmed that Cardamine graeca KCS enzyme has the ability to elongate erucoyl-CoA (C22:1-CoA) to nervonic acid by in vitro activity assays (Taylor et al. 2009). In another work, heterologous expression of C. graeca KCS in Rhodosporidium toruloides efficiently catalyzed all elongation steps to produce nervonic acid (Fillet et al. 2017a). To elucidate the effects of CgKCS overexpression on nervonic acid production in Y. lipolytica, the codon-optimized CgKCS was integrated into the AXP stie by CRISPR/Cas9 technology in the MaELO3, AtKCS-expressing background strain (NA01), yielding strain (NA03). As shown in Fig. 2, strain NA03 can produce about 18.2 mg/L of nervonic acid, which is approximately 4-fold than that of strain NA01. These results clearly showed that the chain length of VLCFs could be selectively modulated by engineering different source of KCS. Consisting with previous reports, CgKCS gene could efficiently push elongation of the erucoyl-CoA pool to nervonic acid (Fillet et al. 2017a). Simultaneously overexpression MaELO3, AtKCS, CraKCS and CgKCS genes obtaining strain NA04 led to the production of 20.8 mg/L neurotic acid, this strain was used as a host strain for the following genetic manuscript.
Explore desaturase of neurotic acid synthesis
The fatty acid profile of the engineered Y. lipolytica NA04 strains revealed that the rewritten the elongation pathway can improve the accumulation of nervonic acid. However, cells engineered also resulted in high amounts of C24:0 saturated fatty acid (lignoceric acid), which indicated that the desaturation step from lignoceric acid to nervonic acid was rate limiting. We thus speculated that introduction of heterologous desaturation pathway would further enhance nervonic acid production. Nervonic acid is produced from lignoceric acid catalyzed by the enzyme ∆-15 desaturase (D15D). Several D15D have been identified until now, out of which we selected two D15D from Mortierella alpina (MaD15D) and Cannabis sativa (CsD15D) for expression and characterization in Y. lipolytica NA04 strain under the control of hybrid promoter UAS4B-TEF (UT) using plasmid pINA1312(Wang et al. 2011; Bielecka et al. 2014). To ensure efficient expression of the D15D, the gene sequences were codon optimized for expression in Y. lipolytica. As shown in Fig. 3, CsD15D gave the less effects on titer of nervonic acid, while MaD15D gave the better performance on production of nervonic acid with a titer of 49.4 mg/L, 2.4-fold increase. These results illustrated that both of the elongation pathway and desaturation pathway are important for nervonic acid biosynthesis in Y. lipolytica.
To optimize the KCS and D15D expression, and release the auxotrophic markers as well, we tried to fuse CgKCS with MaD15D with a (GSG) linker between CgKCS and MaD15D (CgKCS-L-MaD15D) in the chromosome of Y. lipolytica NA07 strain using established CRISPR/Cas9 technology. However, while one copy of CgKCS-L-MaD15D was introduced into the A1-2 site of Y. lipolytica NA07 strain, not necessarily improve nervonic acid production was found, instead a slight decrease in nervonic acid titer was observed. The reason could be due to the low expression of the fusion. As such, an extra copy of CgKCS-L-MaD15D was introduced into the E1-3 site of Y. lipolytica NA07 strain resulting strain NA08. As shown in Fig. 3A, increasing the fusion copy of CgKCS-L-MaD15D in strain NA04 significantly enhanced the production of nervonic acid to 32.1 mg/L in shake flask culture. At the meantime, the amount of lignoceric acid produced by NA08 were 255.1 mg/L, which were 7.3-fold than that for control strain NA04. The FA profiles of the new engineering strain and the control strain were compared. The strain NA08 was found to synthesize more VLCFA (C20-C24) than the control strain NA04, while the C18:2/1 fatty acid content was reduced (Fig. 3B).
Overexpression of genes OLE1 and DGA1 leads to significant increases in nervonic acid accumulation.
Diacylglycerol-acyltransferase (DGAT) catalyzes the acylation of diacylglycerol using acyl-CoA as the acyl donor. This enzyme has been postulated to be a main enzyme in boosting lipogenesis because it catalyzes the last step in TAG synthesis (Blazeck et al. 2014; Gajdos et al. 2016; Tai et al. 2013). The integrative vector pINA1312 carrying the DGA1 gene under the control of hybrid promoter UAS4B-TEF (UT) was successfully integrated into the chromosome of NA04 strain. After 96 h cultivation, homologous recombinant of DGA1 significantly enhanced neurotic acid-producing level, which increase 1.8-fold compared to the NA04 strain (Fig. 4A). Meanwhile, percentage of FA distribution showed a very different between the two engineered Y. lipolytica strains NA04 and NA09. A large reduction in C16:0 and C18:1/2 content was observed in strain NA09 resulting in an increase in the VLCFA fraction (Fig. 4B). Therefore, the target gene DGA1 was selected for subsequent genetic modification.
OLE1 of Y. lipolytica encodes the sole and essential ∆-9 stearoyl-CoA desaturase catalyzing the conversion of saturated to unsaturated fatty acids. Previous studies have shown that OLE1 is important for lipogenesis (Flowers et al. 2008; Qiao et al. 2015). Therefore, OLE1 serve as an attractive engineering target to overproduce nervonic acid. To implement the identified target, we overexpressed the OLE1 in the Y. lipolytica NA08 strain by introducing a native copy of the OLE1 gene through integrated plasmid pINA1312 under the control of strong promoter UT resulting stain NA10. As shown in Fig. S1, overexpression of OLE1 led to 24.4% increase in nervonic acid level over the control strain NA08. Acetyl-CoA is a critical metabolite carbon and energy metabolism involving in multiple key metabolic function (Gao et al. 2018; Huang et al. 2018). Here, we investigated the effects of overexpressing the key genes in acety-CoA metabolic pathway on the nervonic acid productivity in Y. lipolytica. The ACL, encoding the ATP-dependent citrate lyase, the ACC1, encoding the acetyl-CoA carboxylase from Y. lipolytica, and ACS2, encoding the acetyl-CoA synthetase gene, from S. cerevisiae were overexpressed in the background strain through integrated plasmid pINA1269. Though no obviously different of nervonic acid production was observed among the engineered strain, the overexpression of ACC1 led to a C24:0 titer 4-fold higher than the control strain NA04 (Fig. S2). We wanted to evaluate whether increased supply of the precursor acetyl-CoA level could increase nervonic acid production. The main fatty acyl-CoA synthetase encoding gene FAA1 was thus overexpressed on the MFE loci, which involving in ꞵ-oxidation, in the background strain NA04 by CRISPR/Cas9 system. The resulting strain NA22 produce 28.3 mg/L nervonic acid in shake flasks, which was 1.36-fold higher than that of the control strain NA04 (Fig. S3). This strategy might be a potential way to improve nervonic acid production in Y. lipolytica.
Since the single overexpression of DGA1 or OLE1 boosted the titer of nervonic acid in flask culture, we then reasoned that simultaneous co-overexpression of DGA1 and OLE1 would further increase nervonic acid accumulation. And we also performed the fusion strategy to evaluate if the covalent joining of these two enzymes could improve the productivity level of nervonic acid. DGA1 and OLE1 were fused with an artificial flexible linker (GSG) as either DGA1-L-OLE1 or OLE1-L-DGA1, but only the OLE1-L -DGA1 fusion protein resulted in a 1.7-fold increased acid in engineered Y. lipolytica NA08 (Fig. 4C). We also tried different combinations of DGA1, OLE1 and OLE1-L-DGA1 obtained three different strains. In comparison, strain NA15, which simultaneously overexpressed DGA1and OLE1-L-DGA1, had the highest yield of nervonic acid (111.6 mg/L) among all combinations.
Elongation kcs gene copy number adjustment increased nervonic acid production in Y. lipolytica
To further develop a high-level nervonic acid production strain, we evaluated the impact of adjusting the gene dosage on nervonic acid yield. For this purpose, we adding an extra copy of four elongation genes MaELO3, CraKCS, AtKCS and CgKCS thought integrated plasmid pINA1269 to the strain NA12. As shown in Fig. 5A, only the extra copy of MaELO3 enhanced the production of nervonic acid, the yield of nervonic acid increased by 63.9% and reached 90.6 mg/L. Meanwhile, the production of fatty acids C20:1 and C22:1 was significantly improved in the strain with extra copy of CgKCS. Since previous reports showed that increasing the copy number of CgKCS could boost the concentration of nervonic acid in R. toruloides (Fillet et al. 2017b), the inconsistent results might be caused by different genetic background of the stains.
Effect of oily substrates as auxiliary carbon sources for nervonic acid production by the engineered Y. lipolytica.
As an oleaginous yeast, Y. lipolytica can quickly grow to high densities with a high lipid content and utilize a large number of renewable substrates and inexpensive materials such as hydrophobic substrates, crude glycerol and lignocellulosic biomass as carbon sources (Ledesma-Amaro et al. 2016; Nambou et al. 2014; Poli et al. 2014). In order to screen the most suitable carbon source for the production of neuronic acid by Y. lipolytica, an auxiliary carbon sources such as colleseed oil, soybean oil, sunflower seed oil, waste cooking oil or oleic acid was supplemented to YPD medium (Fig. 6A). In this screening experiment of the auxiliary carbon sources, the strain NA02 was first used as the fermentation strain, and 0.25 mL of the auxiliary carbon source was added into the 50 mL YPD medium. As shown in Fig. 6A, the culture with colleseed oil as auxiliary carbon exhibited the highest nervonic acid productivity among all the auxiliary substrates used. In the medium with colleseed oil added, the yield of nervonic acid in strain NA09 reached 132.6 mg/L, which was 2.5-fold higher than that of control YPD medium. The effect of the concentration of colleseed oil as an auxiliary carbon source on neurotic acid production was evaluated by supplementation with colleseed oil at 0.25 to 1.25 mL in shake flask (Fig. 6B). The fermentation results showed that when the colleseed oil supplemental level was 0.5 mL or 0.75 mL, the yield of neuronic acid was the highest, which was 132.6 mg/L and 138.4 mg/L, respectively, about 3.6-fold improvement over the level observed in the medium without colleseed oil. Considering the cost efficiency, for the following experiments we selected 0.5 mL colleseed oil adding into 50 mL fermentation medium.
To explore the reason why colleseed oil was the most suitable auxiliary carbon source for neuronic acid production in this study, the VLCFA profile of colleseed oil was analysis. For colleseed oil used here, C20:1 and C22:1 were the most abundant portion of the VLCFA, little amount of C24:1 and C24:0 were observed. Y. lipolytica GQY-∆PEX10 was the strain that only deleted PEX10, a gene encoding a major peroxisomal matrix protein, from Y. lipolytica Po1f. When this strain was cultured in YPD supplemented with colleseed oil, for without any modification on elongation and desaturation, its profile of VLCFA was similar to that of colleseed oil. After metabolic engineering of the strain, the carbon flux was significantly drain to VLCFA, which demonstrated that the successful of our strategies to generate high neuronic acid production (Fig. 4S).
Finally, the performance of the best neuronic acid-producing strain NA15 in this study was assessed in YPD medium with or without colleseed oil. After 3 days fermentation, a neuronic acid production of 185.0 mg/L was achieved in the medium adding colleseed oil, approximately 1.6-fold higher than that without colleseed oil, which was the highest yield of neurotic acid in this study (Fig. 6C). Despite complex multistep engineering efforts, production titers in this study still lower than that of previous report.24 However, the systematic engineering strategies of Y. lipolytica introduced in this study may provide a deep understanding of the biosynthesis of neurotic acids and other VLCFAs. It should nevertheless be pointed out that further improvements of neurotic acids production in Y. lipolytica will be expected using higher biomass concentrations and controlled bioreactor.