De novo synthesis of nervonic acid and optimization of metabolic regulation by Yarrowia lipolytica

De novo synthesis of nervonic acid and optimization of metabolic regulation by Yarrowia lipolytica | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article De novo synthesis of nervonic acid and optimization of metabolic regulation by Yarrowia lipolytica LiuJing Wei, Xin-Ru Zhao, Xin-Liang Chen, Jing-Lin Yang, Qi Gao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3107416/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Oct, 2023 Read the published version in Bioresources and Bioprocessing → Version 1 posted 4 You are reading this latest preprint version Abstract Nervonic acid, a natural fatty acid compound and also a core component of nerve fibers and nerve cells, has been widely used to prevent and treat related diseases of the brain nervous system. At present, fatty acids and their derivatives are mainly obtained by natural extraction or chemical synthesis which are limited by natural resources and production costs. In this study, the de novo synthetic pathway of nervonic acid was constructed in Yarrowia lipolytica by means of synthetic biology, and the yield of nervonic acid was further improved by metabolic engineering and fermentation optimization. Specially, heterologous elongases and desaturases derived from different organism were successfully expressed and evaluated for their potential for the production of nervonic acid in Y. lipolytica . Meanwhile, we overexpressed the genes involving in the lipid metabolism to increase the nervonic acid titer to 111.6 mg/L. In addition, the potential of adding oil as auxiliary carbon sources for nervonic acid production by the engineered Y. lipolytica were analyzed. The results indicated that supplementation with colleseed oil as an auxiliary carbon source can be beneficial for the nervonic acid productivity, which led to a highest concentration of 185.0 mg/L in this work. To summary, this study describes that the Y. lipolytica can potentially be used for a promising platform to produce nervonic acid and other very long chain fatty acid. nervonic acid Yarrowia lipolytica de novo metabolic engineering colleseed oil Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nervonic acid is a natural fatty acid compound and also a core component of nerve fibers and nerve cells. As a necessary fatty acid nervonic acid is essential for brain development and maintenance of neuronal biosynthesis and improvement. It can be used to prevent and treat related diseases of the brain nervous system such as mental disorders, cognitive disorders and so on (Tanaka et al. 2007 ). Studies have shown that nervonic acid can inhibit HIV-1 RT activity in a dose-dependent manner as a non-competitive inhibitor (Kasai et al. 2002 ). Individual neurotic acid levels are strongly associated with a higher risk of psychiatric disorders, and therefore several neurological disorders, such as demyelinating diseases, can be treated by neurotic acid supplementation (Amminger et al. 2012 ; Raoul et al. 2001; Vozella et al. 2017 ). Nervonic acid is also a natural component of breast milk, it can assist in the development of the infant’s nervous system and promoting their growth. It has proved that increasing the content of nervonic acid in the daily diet of mice can improve the energy metabolism in mice, which may be an effective strategy for the treatment of obesity and obesity complications (Kepple Y et al. 2020). Nervonic acid has a valuable biological function, which makes it play an important role in pharmacological and nutritional applications (Li et al. 2019 ). Currently, nervonic acid has been extracted from plant tissues or obtained by chemical synthesis. These extraction methods have different limitations. By means of chemical synthesis, the yield of nervonic acid is very low and there are many byproducts (Rongkai et al. 2018 ). The most commonly used method extracting neurotic acid from plants is the at present, but the process is limited by the growth cycle and climatic condition. Therefore, it is time to explore a green and feasible way to biosynthesis nervonic acid. In the past, great progress has been made in the biosynthesis of fatty acids by means of microorganisms (Rongkai et al. 2018 ). The development of synthetic biology and metabolic engineering has greatly facilitated the manipulation of microbial metabolic pathways and has significantly contributed to the production of various chemicals (Li et al. 2019 ). For example, the filamentous fungi Mortierella capitata RD000969 isolated from soil can accumulated nervonic acid for 6.94% of the total fatty acid (Umemoto et al. 2014 ). In Saccharomyces cerevisiae , β-estradiol inducible expression system (EIES) was used to enhance the intracellular production of neuronic acid. Then the level of neuronic acid was further increased by overexpression of KCS and ELOVL1 genes and knockout of ELO2 (Liu et al. 2020 ). It has been reported that through the screening and expressing of elongation genes (3-ketoacyl-CoA synthases, KCS) from different plant sources, the production of neuronic acid was realized in Rhodosporidium toruloides (Fillet et al. 2017 b). The study has proved that the copy number of KCS gene and the push/pull strategy for KCS gene preference increased the contents of C24:1 and C22:1 fatty acid. By optimizing the fermentation conditions, the yield of erucic acid and neuronic acid in the 7 L bioreactor reached 20–30% of the yield of very-long chain fatty acids. Yarrowia lipolytica , as a GRAS grade yeast strain, is one of the most studied "unconventional" yeast species (Bourdichon et al. 2012 ). Yarrowia lipolytica , due to its capacity for synthesizing and secreting hydrolytic enzymes like proteases and lipases (Fickers et al. 2005), is predominantly found in oily sewage and soil (Hassanshahian et al. 2012 ). It is also commonly exists in fatty and protein-rich foods such as cheese (Groenewald et al. 2014 ), dairy products, meat and sausages (Fickers et al. 2005). Y. lipolytica has complex intimal structure which enables it to have a high storage capacity of neutral lipids (mainly triacylglycerol), and to grow rapidly and produce lipids at a rapid rate (Beopoulos et al. 2009 ). Y. lipolytica has a wide carbon source spectrum, and since it often exists in an environment rich in hydrophobic substrates (such as alkanes or lipids), it has developed a complex mechanism to efficiently use hydrophobic substrates as the only carbon source (Fickers et al. 2005). Moreover, there are several gene families involved in the metabolic pathway of hydrophobic substrates, which are conducive to the uptake of more diverse hydrophobic substrates and lipid accumulations. Based on the above mechanism, strain can accumulate lipids that exceed 50% of cell dry weight (Beopoulos et al. 2009 ). The strong tolerance of Y. lipolytica to fluctuating pH values, salt concentrations and various organic compounds simplifies and optimizes biological processes and promotes the use of non-glucose-based feedstock (Miller et al. 2019). The genome of Y. lipolytica has been sequenced and gene-editing tools developed and used are becoming more sophisticated (Liu et al. 2014). There is a natural fatty acid synthesis pathway in Y. lipolytica . Once the glucose enters the cytoplasm, it goes through glycolytic pathway (EMP) and eventually becomes pyruvate, which has three carbon atoms, and then it goes to the mitochondria, where it ends up with acetyl-CoA (Vorapreeda et al. 2012 ). Acetyl-CoA which is an important precursor involved in fatty acid biosynthesis, can be produced through a variety of metabolic pathways, such as ATP citrate lyase (ACL) catalyzing the degradation of citric acid, fatty acids through β-oxidative degradation and acetyl-CoA synthase conversion to acetic acid. Under nitrogen restriction, mitochondria secrete citric acid and then forms malonyl-CoA catalyzed by acetyl-CoA carboxylase (ACC1). Acyl-CoA with 16 and 18 chain lengths which generated with acetyl-CoA as the starting point and malonyl-CoA as the elongation unit was further extended and desaturated with 16:0 and 18:0 activated molecules as the precursor to obtain fatty acids with various chain lengths and saturation (Beopoulos et al. 2009 ). Each elongation consumes two molecules of NADPH, of which NADPH is derived in two ways. One is through the malate dehydrogenase catalyzed decarboxylation reaction in the cytoplasm another is pentose phosphate pathway (Wasylenko et al. 2015 ). During the preparation of this manuscript, Wang et al. engineered Y. lipolytica to produce up to 57.48 g/L of microbial oil with 23.44% nervonic acid in fed-batch fermentation; the highest production titer so far described in Y. lipolytica . The authors combined orthogonal plant and non-plant fatty acid biosynthesis pathways in Y. lipolytica , used a “block-pull-restrain” strategy to increase precursor production, and strengthened TAGs synthesis to improve lipid pool (Wang et al. 2023 ). In this work, we constructed the de novo synthesis of nervonic acid in oleaginous yeast Y. lipolytica (Fig. 1 ). In order to further improved the production of nervonic acid, the elongation genes and desaturation genes in the process of nervonic acid synthesis were screened and overexpressed in Y. lipolytica . Meanwhile, the expression patterns of different combinations of key genes were explored to further enhance the production of nervonic acid. Moreover, we analyzed the potential of different auxiliary carbon sources for the production of nervonic acid by Y. lipolytica , and first found that colleseed oil as auxiliary carbon source was helpful to increase nervonic acid production. Materials and methods Plasmids, strains and medium Y. lipolytica strain ATCC MYA2613 (Po1f), which was the initial strain of the engineered strains. Construction and amplification of plasmids were dependent on E. coli strain JM109, which was cultured in Luria-Bertani (LB) medium and grew at 37°C. LB medium (10 g/L yeast extract, 20 g/L peptone, 10 g/L NaCl, and 15 g/L Bacto agar) was added with different resistance to construct plasmids such as 50 mg/L of kanamycin and 100 mg/L of ampicillin. The Y. lipolytica strains were cultivated at 30℃ in YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 15 g/L Bacto agar). The YNB medium, which contains 6.7 g/L yeast nitrogen base without amino acids, 20 g/L glucose and 15 g/L Bacto agar, was used to screen transformants by adding 100 mg/L leucine or uracil. In this study, 5-fluoroorotic acid (1 g/L of 5-FOA) was added to YPD medium for the recovery of URA3 screening markers. All strains constructed and used in this study are listed in Table 1 . Table 1 Strains used in this study Strains Descriptions Source E. coli JM109 recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, Δ(lac-proAB)/F[traD36, proab + , lacI q , lacZΔM15] Invitrogen Y. lipolytica Po1f MatA, leu2-270, ura3-302, xpr2-322, axp1-2 (Nicaud 2012 ) GQY-∆PEX10 Po1f-∆PEX10 (Qi Gao et al. 2018 ) GQ06 Po1f-∆PEX10 integrated MaELO3 by CRISPR/Cas9 at F1 site (Gao et al. 2020 b) NA01 GQ06 integrated optimized AtKCS by CRISPR/Cas9 at A3 site This study NA02 NA01 integrated optimized CraKCS by CRISPR/Cas9 at F1-3 site This study NA03 NA01 integrated optimized CgKCS by CRISPR/Cas9 at AXP site This study NA04 NA03 integrated optimized CraKCS by CRISPR/Cas9 at F1-3 site This study NA05 NA04 cells harboring pINA1312-P UT - MaD15D This study NA06 NA04 cells harboring pINA1312-P UT - CsD15D This study NA07 NA04 integrated optimized CgKCS-L-MaD15D by CRISPR/Cas9 at A1-2 site This study NA08 NA07 integrated optimized CgKCS-L-MaD15D by CRISPR/Cas9 at E1-3 site This study NA09 NA04 cells harboring pINA1312-P UT - DGA1 This study NA10 NA08 cells harboring pINA1312-P UT - OLE1 This study NA11 NA08 cells harboring pINA1312-P UT - DGA1-L-OLE1 This study NA12 NA08 cells harboring pINA1312-P UT - OLE1-L-DGA1 This study NA13 NA12 cells harboring pINA1269- OLE1-L-DGA1 This study NA14 NA10 cells harboring pINA1269-DGA1 This study NA15 NA12 cells harboring pINA1269-DGA1 This study NA16 NA12 cells harboring pINA1269-MaELO3 This study NA17 NA12 cells harboring pINA1269-MaELO3-AtKCS This study NA18 NA12 cells harboring pINA1269-MaELO3-CraKCS This study NA19 NA12 cells harboring pINA1269-CgKCS This study NA20 NA04 cells harboring pINA1269-ACL This study NA21 NA04 cells harboring pINA1269-ACS2 This study NA22 NA04 cells harboring pINA1269-ACC1 This study NA23 NA20 cells harboring pINA1312-ACS2 This study NA24 NA04 integrated FAA1 by CRISPR/Cas9 at MFE site This study Construction of plasmids and yeast transformation In this study, two integrative plasmids, pINA1312 and pINA1269, and CRISRPR/Cas9 system were used for metabolic engineering modification of the strains. All constructed strains are shown in Table 1 . The elongation enzyme gene ( CgKCS ) from Cardamine graeca and the ∆15 desaturase genes (MaD15D/CsD15D) from Mortierella alpine and Cannabis sativa were synthesized and coded optimally. Primers were designed to amplify target genes by PCR, and the amplified genes were linked to plasmids pINA1312 or pINA1269 that had been digested by the ClonExpress® II One Step Cloning Kit (Vazyme Biotech, Nanjing, China). Then the recombinant plasmid with the target gene expression cassette was obtained. The primers involved in this study are all shown in Table S1 . In order to achieve efficient gene expression, expression cassttes with different promoter strength and different terminator were constructed. After construction of the recombinant plasmid, it was linearized by the corresponding enzyme and then transferred into yeast cells by Frozen-EZ yeast transformation II Kit (Zymo Research, Irvine, CA). The CRISPR/Cas9 system is able to knock out the gene and knock-in the target gene at the same time. Taking the CgKCS gene which was inserted into the AXP site as an example, primers with 20 bp homologous sequences at both ends of insertion site were used to obtain the amplified CgKCS expression cassette by PCR. Then plasmid pHR_AXP_hrGFP digested with SpeI and AvrII connected with CgKCS expression cassette to obtain recombinant plasmid pHR_AXP_CgKCS. Finally, the single gRNA and recombinant plasmid pHR_AXP_CgKCS were transformed into corresponding yeast cell together. All the primers used and plasmids constructed were shown in Table S2 -S3. Growth condition and auxiliary carbon source Y. lipolytica strains were cultured in 2 mL YPD at 30°C (220 rpm) and then inoculated in 250 mL triangular flask containing 50 mL YPD with an initial OD 600 of 0.01. The strains were cultured for 72 hours under the same conditions. Adding 0.25 mL of different carbon sources (ω-9 octadecanoic acid, soybean oil, colleseed oil, sunflower seed oil, waste cooking oil) to 50 mL YPD. On this basis, gradient experiments of colleseed oil supplemental levels were designed, such as 0, 0.25, 0.5, 0.75, 1.0, and 1.25 mL added to 50 mL YPD. Extraction of VLCFAs 20 mL of the fermentation medium was taken into a 50 mL centrifuge tube and centrifuged at 6,000 rpm for 5 min. The supernatant was discarded, and then 15 mL of ddH 2 O was added to the centrifuge tube. The mixture was thoroughly mixed and subjected to centrifugation under the same conditions. After repeating the above procedure, added 5 mL 4 M HCl to the collected cells. The mixture was oscillated and then held for 30 minutes at 37℃ at 220 rpm. Next, the test tube was kept in boiling water bath and ice for 5 minutes, and the operation was repeated again. Then 20 mL of methanol and chloroform mixed solution was added into the test tube, in which the volume ratio of methanol to chloroform was 1:2. After 30 minutes at 37°C, the underlying liquid was centrifuged (4800 rpm, 5 min) and then sucked into a glass tube and dried in an oven at 105°C. After about 12 h, taking the test tube out and then adding 3 mL of 0.5 mol/L methanol potassium hydroxide solution into the test tube when the test tube is restored to room temperature. Ultrasound was used to dissolve the oil in the tube, and the tube was kept in a water bath at 75℃ for 20 min. Adding 3 ml of 14% boron trichloride solution to the test tube and keep the same condition for 20 min. Then taking out the tube, and adding 1 mL saturated NaCl and 0.5 mL n-hexane in it. The mixture was thoroughly mixed and the upper solution was centrifuged at 12000 rpm for 2 min. Then dilute the upper liquid and mix it with the internal standard at a volume ratio of 1:4 to get the sample to be tested. Gas chromatography coupled with mass spectrometry (GC − MS) analysis of VLCFAs The sample was analyzed by GC-MS which was carried out using an Agilent System 6890 gas chromatograph (GC) with an Agilent 5975 quadrupole mass selective detector (MSD) equipped with a HP-5 column (30 m × 0.25 mm × 0.25 µm, Agilent, Santa Clara, CA, USA). The initial temperature of GC was held at 150°C for 2 min, and then at a rate of 20°C/min to 180°C. And then it went up to 200°C at a rate of 8°C/min. Then in 18 minutes the temperature reached 218°C, raised to 250°C at 8°C/min. The temperature subsequently raised to 300°C in 3.4 min. The split ratio was 20:1. The quantitative analysis was carried out by the corresponding fatty acid methyl ester standards. Results and discussion 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. 2017 a). 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. 2017 a). 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. 3 A, 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. 3 B). 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. 4 A). 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. 4 B). 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. 4 C). We also tried different combinations of DGA1 , OLE1 and OLE1-L-DGA1 obtained three different strains. In comparison, strain NA15, which simultaneously overexpressed DGA1 and 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. 5 A, 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. 2017 b), 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. 6 A). 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. 6 A, 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. 6 B). 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. 4 S). 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. 6 C). 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. Conclusion In summary, we engineered the oleaginous yeast Y. lipolytica following multi-level strategies for efficient accumulation of neurotic acid production. Specifically, we reconstructed the elongation pathway as well as desaturation pathway, optimized the key gene expression in fatty acid metabolism through adding gene copy and protein fusion. Furthermore, we first demonstrated that supplementing the colleseed oil as auxiliary carbon benefited the neurotic acid production. The yeast engineering strategy of pathway assembling presented in this study may be employed to optimize microbial production of other valuable VLCFA chemistry. Declarations Ethics approval and consent to participate Not applicable. Consent for publication All of the authors have read and approved to submit it to Bioresources and Bioprocessing . Availability of data and materials All data generated or analyzed during this study are included in this article. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the National Key R&D Program of China (2021YFC2102805). Author contributions XRZ designed experiments; XRZ and XLC conducted experiments, XRZ, XLC and JLY collected data; XRZ, XLC, JLY, GQ and JTS analyzed data; QH and LJW conceived the idea and supervised the research; XRZ, XLC and LJW drafted the manuscript and contributed to data interpretation. All authors read and approved the final manuscript. Acknowledgments The authors would like to thank Prof. Er-Zheng Su for guidance at the beginning of this study. References Amminger GP, Schafer MR, Klier CM, Slavik JM, Holzer I, Holub M, Goldstone S, Whitford TJ, McGorry PD, Berk M (2012) Decreased nervonic acid levels in erythrocyte membranes predict psychosis in help-seeking ultra-high-risk individuals. Mol Psychiatry 17(12):1150–1152. 10.1038/mp.2011.167 Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM (2009) Yarrowia lipolytica as a model for bio-oil production. 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J biotechnol 281:106–114. 10.1016/j.jbiotec.2018.07.001 Supplementary Files GraphicalAbstract.docx SupplemetaryFilesWLJ.docx Cite Share Download PDF Status: Published Journal Publication published 06 Oct, 2023 Read the published version in Bioresources and Bioprocessing → Version 1 posted Reviewers agreed at journal 01 Jul, 2023 Reviewers invited by journal 01 Jul, 2023 Editor assigned by journal 01 Jul, 2023 First submitted to journal 25 Jun, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3107416","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":214878091,"identity":"523096aa-c6cf-4a11-ae7c-03f1f85603e2","order_by":0,"name":"LiuJing Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYFACxsYDCQxsPAwMzAdgQgaEtDRAtbAlgLgSRGhhYICazmNAnBaD480NBx628cmY86/5+OBnW10dA3vzNgmGmju4tZw52HAgsY2Nx3LG282GvW2HJRh4jpVJMBx7hlOL2Y1EiBaDG2e3STO2HZBgkMgxk2BsOIxby/2HMC1nnv9mbKuTYJB/Q0DLDUaolvM9bMyMbcxAW3jwa7E/A3RYwjmQLWzGkj3nDku28aQVWyQcw61Fsv34w4c/yo7ZG5w//PDDj7I6fn72wxtvfKjBrQUKjgEjJAHCZAMRCYQ0MDDUMDDwHyCsbBSMglEwCkYmAADU/1hg+4MHuQAAAABJRU5ErkJggg==","orcid":"","institution":"ECUST: East China University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"LiuJing","middleName":"","lastName":"Wei","suffix":""},{"id":214878092,"identity":"0f55eb84-abec-40ac-a6b2-f11ca1e49f48","order_by":1,"name":"Xin-Ru Zhao","email":"","orcid":"","institution":"East-China Institute of Technology: East China University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin-Ru","middleName":"","lastName":"Zhao","suffix":""},{"id":214878093,"identity":"117d9a0d-0d33-4272-8afa-1b11e156aac8","order_by":2,"name":"Xin-Liang Chen","email":"","orcid":"","institution":"East-China Institute of Technology: East China University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin-Liang","middleName":"","lastName":"Chen","suffix":""},{"id":214878094,"identity":"0893843f-90e0-4619-9008-be5486781d68","order_by":3,"name":"Jing-Lin Yang","email":"","orcid":"","institution":"East-China Institute of Technology: East China University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing-Lin","middleName":"","lastName":"Yang","suffix":""},{"id":214878095,"identity":"2942fdf4-13fe-4248-b6f9-6969347686eb","order_by":4,"name":"Qi Gao","email":"","orcid":"","institution":"East-China Institute of Technology: East China University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Gao","suffix":""},{"id":214878096,"identity":"02acf36f-f9c8-452f-a69e-3fd7f4b9bf2f","order_by":5,"name":"Jiang-Ting Shi","email":"","orcid":"","institution":"East-China Institute of Technology: East China University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiang-Ting","middleName":"","lastName":"Shi","suffix":""},{"id":214878097,"identity":"b11edc79-3f42-4b1f-82bf-210bf7396aad","order_by":6,"name":"Qiang Hua","email":"","orcid":"","institution":"East-China Institute of Technology: East China University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Hua","suffix":""}],"badges":[],"createdAt":"2023-06-25 15:32:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3107416/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3107416/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40643-023-00689-6","type":"published","date":"2023-10-06T15:01:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":39652102,"identity":"7b4af32a-5d33-42ad-a4ba-d5d65d21a404","added_by":"auto","created_at":"2023-07-06 17:39:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":264523,"visible":true,"origin":"","legend":"\u003cp\u003eBiosynthesis pathway for neurotic acid production in the yeast \u003cem\u003eY. lipolytica\u003c/em\u003e. Neurotic acid is biosynthesized from the initiation unit of acetyl-CoA and the extension unit of malonyl-CoA with fatty acid synthetase. Green words represent the heterogeneous expression pathways; black words represent the native pathways; and red words represent selected for disruption in this study. Gene abbreviations: Pyr, pyruvic acid; Ac-CoA, acetyl coenzyme A; CIT, citric acid; Mal-CoA, Malonyl coenzyme A; FAS, fatty acid synthase; TEs, thioesterase; ACL, ATP-citrate lyase; FAA1, acyl-CoA synthetase; FFA, free fatty acid; KS, ketoacyl-CoA synthase; KR, 3-ketoacyl-CoAreductase; DH, 3-hydroxyacyl-CoA dehydratase; ER, enoyl-CoA reductase; D15D, ∆15 desaturase; TAG, triacylglycerol.\u003c/p\u003e","description":"","filename":"FigWLJ1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/1317c2c9c27f24a62b619b1b.jpg"},{"id":39652965,"identity":"5dc01b5b-5b8d-45a8-a4bd-4d5be2eada45","added_by":"auto","created_at":"2023-07-06 17:47:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164799,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of overexpression of elongation genes from different sources on nervonic acid production on solid medium (YPD). The data shows the average of two independent experiments, with the error bars representing standard deviations. \u003cem\u003eMaELO3\u003c/em\u003e: \u003cem\u003eMortierella alpina\u003c/em\u003e \u003cem\u003eELO3\u003c/em\u003e gene; \u003cem\u003eAtKCS\u003c/em\u003e: \u003cem\u003eArabidopsis thaliana\u003c/em\u003e \u003cem\u003eKCS\u003c/em\u003e gene; \u003cem\u003eCgKCS\u003c/em\u003e: \u003cem\u003eCardamine graeca KCS\u003c/em\u003e gene; \u003cem\u003eCraKCS\u003c/em\u003e: \u003cem\u003eCrambe abyssinica\u003c/em\u003e \u003cem\u003eKCS\u003c/em\u003e gene.\u003c/p\u003e","description":"","filename":"FigWLJ2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/5726516983b13a232f4a0770.jpg"},{"id":39652964,"identity":"fb2e687e-deb2-4697-8600-0f67286f4091","added_by":"auto","created_at":"2023-07-06 17:47:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":389073,"visible":true,"origin":"","legend":"\u003cp\u003eHeterologous desaturase expression in \u003cem\u003eY. lipolytica\u003c/em\u003e. (A) Screening the vary version of ∆15 desaturase and elongase for nervonic acid production. \u003cem\u003eMaD15D\u003c/em\u003e: \u003cem\u003eMortierella alpine\u003c/em\u003e ∆15 desaturase gene; \u003cem\u003eCsD15D\u003c/em\u003e: \u003cem\u003eCannabis sativa\u003c/em\u003e ∆15 desaturase gene. (B) Percentage of FA distribution in the engineered \u003cem\u003eY. lipolytica\u003c/em\u003e strains NA04 and NA08. The data are the averages of two biological replicates with error bars representing standard deviations.\u003c/p\u003e","description":"","filename":"FigWLJ3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/47099784352d00695ef169cc.jpg"},{"id":39652963,"identity":"81f25aad-6bc4-4000-86b9-eb0c3f4a4e7a","added_by":"auto","created_at":"2023-07-06 17:47:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":382361,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of overexpression of genes \u003cem\u003eOLE1\u003c/em\u003eand \u003cem\u003eDGA1\u003c/em\u003e for the biosynthesis of nervonic acid in \u003cem\u003eY. lipolytica\u003c/em\u003e. (A) Overexpression of \u003cem\u003eDGA1\u003c/em\u003e gene to improve nervonic acid production. (B) Percentage of FA distribution in the engineered \u003cem\u003eY. lipolytica\u003c/em\u003e strains NA04 and NA09. (C) Different combinations of \u003cem\u003eDGA1\u003c/em\u003eand \u003cem\u003eOLE1\u003c/em\u003e to increase nervonic acid production. The data are the averages of two biological replicates with error bars representing standard deviations.\u003c/p\u003e","description":"","filename":"FigWLJ4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/93de425845ee52b8fc1d6023.jpg"},{"id":39651339,"identity":"21f0fb8a-5de1-4be0-a150-2ed9fcd86e7c","added_by":"auto","created_at":"2023-07-06 17:31:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":336963,"visible":true,"origin":"","legend":"\u003cp\u003eImprovement of nervonic acid production in \u003cem\u003eY. lipolytica\u003c/em\u003e through elongation \u003cem\u003ekcs\u003c/em\u003e gene copy number adjustment. (A) An extra copy of \u003cem\u003eKCS\u003c/em\u003e gene was overexpressed in NA12. \u003cem\u003eMaELO3\u003c/em\u003e: \u003cem\u003eMortierella alpina\u003c/em\u003e \u003cem\u003eELO3\u003c/em\u003e gene; \u003cem\u003eAtKCS\u003c/em\u003e: \u003cem\u003eArabidopsis thaliana\u003c/em\u003e \u003cem\u003eKCS\u003c/em\u003egene; \u003cem\u003eCgKCS\u003c/em\u003e: \u003cem\u003eCardamine graeca KCS\u003c/em\u003e gene; \u003cem\u003eCraKCS\u003c/em\u003e: \u003cem\u003eCrambe abyssinica\u003c/em\u003e \u003cem\u003eKCS\u003c/em\u003e gene. (B) Percentage of FA distribution in the engineered \u003cem\u003eY. lipolytica\u003c/em\u003e strains NA04, NA16-18. The data are the averages of two biological replicates with error bars representing standard deviations.\u003c/p\u003e","description":"","filename":"FigWLJ5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/7708c8afe586cab621ba0e6c.jpg"},{"id":39651341,"identity":"a0eaf8ba-8ca0-4611-b58e-f06a7bb0a671","added_by":"auto","created_at":"2023-07-06 17:31:09","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":517611,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of oil/oleic acid addition on nervonic acid production in engineered \u003cem\u003eY. lipolytica\u003c/em\u003e. (A) Adding different auxiliary carbon sources on nervonic acid production. (B) \u0026nbsp;colleseed oil supplemental level on the yield of nervonic acid. (E) Fermentation of strains NA15 in the YPD medium with 0.5 mL colleseed oil. The data are the averages of two biological replicates with error bars representing standard deviations.\u003c/p\u003e","description":"","filename":"FigWLJ6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/1c9883e2dda0787469b02444.jpg"},{"id":44301642,"identity":"6482bf17-f11d-4961-9881-e9c127b4dc09","added_by":"auto","created_at":"2023-10-09 15:05:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":863844,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/a7ffabb1-52d0-422b-b3e5-00742f07a4e1.pdf"},{"id":39652105,"identity":"771f9996-bcd1-4f4b-9a14-fbbf4eb9ac9d","added_by":"auto","created_at":"2023-07-06 17:39:09","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":36325,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/6dbb28aaa3a226e5ccbf91bd.docx"},{"id":39651346,"identity":"2013bf1f-5c61-4729-918e-5576819aad39","added_by":"auto","created_at":"2023-07-06 17:31:09","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":221124,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemetaryFilesWLJ.docx","url":"https://assets-eu.researchsquare.com/files/rs-3107416/v1/772df559e3d7fba489fe0a79.docx"}],"financialInterests":"","formattedTitle":"De novo synthesis of nervonic acid and optimization of metabolic regulation by Yarrowia lipolytica","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNervonic acid is a natural fatty acid compound and also a core component of nerve fibers and nerve cells. As a necessary fatty acid nervonic acid is essential for brain development and maintenance of neuronal biosynthesis and improvement. It can be used to prevent and treat related diseases of the brain nervous system such as mental disorders, cognitive disorders and so on (Tanaka et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Studies have shown that nervonic acid can inhibit HIV-1 RT activity in a dose-dependent manner as a non-competitive inhibitor (Kasai et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Individual neurotic acid levels are strongly associated with a higher risk of psychiatric disorders, and therefore several neurological disorders, such as demyelinating diseases, can be treated by neurotic acid supplementation (Amminger et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Raoul et al. 2001; Vozella et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Nervonic acid is also a natural component of breast milk, it can assist in the development of the infant\u0026rsquo;s nervous system and promoting their growth. It has proved that increasing the content of nervonic acid in the daily diet of mice can improve the energy metabolism in mice, which may be an effective strategy for the treatment of obesity and obesity complications (Kepple Y et al. 2020). Nervonic acid has a valuable biological function, which makes it play an important role in pharmacological and nutritional applications (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Currently, nervonic acid has been extracted from plant tissues or obtained by chemical synthesis. These extraction methods have different limitations. By means of chemical synthesis, the yield of nervonic acid is very low and there are many byproducts (Rongkai et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The most commonly used method extracting neurotic acid from plants is the at present, but the process is limited by the growth cycle and climatic condition. Therefore, it is time to explore a green and feasible way to biosynthesis nervonic acid.\u003c/p\u003e \u003cp\u003eIn the past, great progress has been made in the biosynthesis of fatty acids by means of microorganisms (Rongkai et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The development of synthetic biology and metabolic engineering has greatly facilitated the manipulation of microbial metabolic pathways and has significantly contributed to the production of various chemicals (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, the filamentous fungi \u003cem\u003eMortierella capitata RD000969\u003c/em\u003e isolated from soil can accumulated nervonic acid for 6.94% of the total fatty acid (Umemoto et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, β-estradiol inducible expression system (EIES) was used to enhance the intracellular production of neuronic acid. Then the level of neuronic acid was further increased by overexpression of \u003cem\u003eKCS\u003c/em\u003e and \u003cem\u003eELOVL1\u003c/em\u003e genes and knockout of ELO2 (Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It has been reported that through the screening and expressing of elongation genes (3-ketoacyl-CoA synthases, KCS) from different plant sources, the production of neuronic acid was realized in \u003cem\u003eRhodosporidium toruloides\u003c/em\u003e (Fillet et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003eb). The study has proved that the copy number of KCS gene and the push/pull strategy for KCS gene preference increased the contents of C24:1 and C22:1 fatty acid. By optimizing the fermentation conditions, the yield of erucic acid and neuronic acid in the 7 L bioreactor reached 20\u0026ndash;30% of the yield of very-long chain fatty acids.\u003c/p\u003e \u003cp\u003e \u003cem\u003eYarrowia lipolytica\u003c/em\u003e, as a GRAS grade yeast strain, is one of the most studied \"unconventional\" yeast species (Bourdichon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eYarrowia lipolytica\u003c/em\u003e, due to its capacity for synthesizing and secreting hydrolytic enzymes like proteases and lipases (Fickers et al. 2005), is predominantly found in oily sewage and soil (Hassanshahian et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). It is also commonly exists in fatty and protein-rich foods such as cheese (Groenewald et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), dairy products, meat and sausages (Fickers et al. 2005). \u003cem\u003eY. lipolytica\u003c/em\u003e has complex intimal structure which enables it to have a high storage capacity of neutral lipids (mainly triacylglycerol), and to grow rapidly and produce lipids at a rapid rate (Beopoulos et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). \u003cem\u003eY. lipolytica\u003c/em\u003e has a wide carbon source spectrum, and since it often exists in an environment rich in hydrophobic substrates (such as alkanes or lipids), it has developed a complex mechanism to efficiently use hydrophobic substrates as the only carbon source (Fickers et al. 2005). Moreover, there are several gene families involved in the metabolic pathway of hydrophobic substrates, which are conducive to the uptake of more diverse hydrophobic substrates and lipid accumulations. Based on the above mechanism, strain can accumulate lipids that exceed 50% of cell dry weight (Beopoulos et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The strong tolerance of \u003cem\u003eY. lipolytica\u003c/em\u003e to fluctuating pH values, salt concentrations and various organic compounds simplifies and optimizes biological processes and promotes the use of non-glucose-based feedstock (Miller et al. 2019). The genome of \u003cem\u003eY. lipolytica\u003c/em\u003e has been sequenced and gene-editing tools developed and used are becoming more sophisticated (Liu et al. 2014). There is a natural fatty acid synthesis pathway in \u003cem\u003eY. lipolytica\u003c/em\u003e. Once the glucose enters the cytoplasm, it goes through glycolytic pathway (EMP) and eventually becomes pyruvate, which has three carbon atoms, and then it goes to the mitochondria, where it ends up with acetyl-CoA (Vorapreeda et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Acetyl-CoA which is an important precursor involved in fatty acid biosynthesis, can be produced through a variety of metabolic pathways, such as ATP citrate lyase (ACL) catalyzing the degradation of citric acid, fatty acids through β-oxidative degradation and acetyl-CoA synthase conversion to acetic acid. Under nitrogen restriction, mitochondria secrete citric acid and then forms malonyl-CoA catalyzed by acetyl-CoA carboxylase (ACC1). Acyl-CoA with 16 and 18 chain lengths which generated with acetyl-CoA as the starting point and malonyl-CoA as the elongation unit was further extended and desaturated with 16:0 and 18:0 activated molecules as the precursor to obtain fatty acids with various chain lengths and saturation (Beopoulos et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Each elongation consumes two molecules of NADPH, of which NADPH is derived in two ways. One is through the malate dehydrogenase catalyzed decarboxylation reaction in the cytoplasm another is pentose phosphate pathway (Wasylenko et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the preparation of this manuscript, Wang et al. engineered \u003cem\u003eY. lipolytica\u003c/em\u003e to produce up to 57.48 g/L of microbial oil with 23.44% nervonic acid in fed-batch fermentation; the highest production titer so far described in \u003cem\u003eY. lipolytica\u003c/em\u003e. The authors combined orthogonal plant and non-plant fatty acid biosynthesis pathways in \u003cem\u003eY. lipolytica\u003c/em\u003e, used a \u0026ldquo;block-pull-restrain\u0026rdquo; strategy to increase precursor production, and strengthened TAGs synthesis to improve lipid pool (Wang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this work, we constructed the \u003cem\u003ede novo\u003c/em\u003e synthesis of nervonic acid in oleaginous yeast \u003cem\u003eY. lipolytica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In order to further improved the production of nervonic acid, the elongation genes and desaturation genes in the process of nervonic acid synthesis were screened and overexpressed in \u003cem\u003eY. lipolytica\u003c/em\u003e. Meanwhile, the expression patterns of different combinations of key genes were explored to further enhance the production of nervonic acid. Moreover, we analyzed the potential of different auxiliary carbon sources for the production of nervonic acid by \u003cem\u003eY. lipolytica\u003c/em\u003e, and first found that colleseed oil as auxiliary carbon source was helpful to increase nervonic acid production.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids, strains and medium\u003c/h2\u003e \u003cp\u003e \u003cem\u003eY. lipolytica\u003c/em\u003e strain ATCC MYA2613 (Po1f), which was the initial strain of the engineered strains. Construction and amplification of plasmids were dependent on \u003cem\u003eE. coli\u003c/em\u003e strain JM109, which was cultured in Luria-Bertani (LB) medium and grew at 37\u0026deg;C. LB medium (10 g/L yeast extract, 20 g/L peptone, 10 g/L NaCl, and 15 g/L Bacto agar) was added with different resistance to construct plasmids such as 50 mg/L of kanamycin and 100 mg/L of ampicillin. The \u003cem\u003eY. lipolytica\u003c/em\u003e strains were cultivated at 30℃ in YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 15 g/L Bacto agar). The YNB medium, which contains 6.7 g/L yeast nitrogen base without amino acids, 20 g/L glucose and 15 g/L Bacto agar, was used to screen transformants by adding 100 mg/L leucine or uracil. In this study, 5-fluoroorotic acid (1 g/L of 5-FOA) was added to YPD medium for the recovery of URA3 screening markers. All strains constructed and used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStrains used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescriptions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e JM109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erecA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, Δ(lac-proAB)/F[traD36, proab\u003csup\u003e+\u003c/sup\u003e, lacI\u003csup\u003eq\u003c/sup\u003e, lacZΔM15]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY. \u003cem\u003elipolytica\u003c/em\u003e Po1f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMatA, leu2-270, ura3-302, xpr2-322, axp1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(Nicaud \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGQY-∆PEX10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePo1f-∆PEX10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(Qi Gao et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGQ06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePo1f-∆PEX10 integrated MaELO3 by CRISPR/Cas9 at F1 site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(Gao et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003eb)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGQ06 integrated optimized AtKCS by CRISPR/Cas9 at A3 site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA01 integrated optimized CraKCS by CRISPR/Cas9 at F1-3 site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA01 integrated optimized CgKCS by CRISPR/Cas9 at AXP site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA03 integrated optimized CraKCS by CRISPR/Cas9 at F1-3 site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA04 cells harboring pINA1312-P\u003csub\u003eUT\u003c/sub\u003e- MaD15D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA04 cells harboring pINA1312-P\u003csub\u003eUT\u003c/sub\u003e- CsD15D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA04 integrated optimized CgKCS-L-MaD15D by CRISPR/Cas9 at A1-2 site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA07 integrated optimized CgKCS-L-MaD15D by CRISPR/Cas9 at E1-3 site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA04 cells harboring pINA1312-P\u003csub\u003eUT\u003c/sub\u003e- DGA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA08 cells harboring pINA1312-P\u003csub\u003eUT\u003c/sub\u003e- OLE1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA08 cells harboring pINA1312-P\u003csub\u003eUT\u003c/sub\u003e- DGA1-L-OLE1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA08 cells harboring pINA1312-P\u003csub\u003eUT\u003c/sub\u003e- OLE1-L-DGA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA12 cells harboring pINA1269- OLE1-L-DGA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA10 cells harboring pINA1269-DGA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA12 cells harboring pINA1269-DGA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA12 cells harboring pINA1269-MaELO3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA12 cells harboring pINA1269-MaELO3-AtKCS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA12 cells harboring pINA1269-MaELO3-CraKCS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA12 cells harboring pINA1269-CgKCS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA04 cells harboring pINA1269-ACL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA04 cells harboring pINA1269-ACS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA04 cells harboring pINA1269-ACC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA20 cells harboring pINA1312-ACS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA04 integrated FAA1 by CRISPR/Cas9 at MFE site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of plasmids and yeast transformation\u003c/h2\u003e \u003cp\u003eIn this study, two integrative plasmids, pINA1312 and pINA1269, and CRISRPR/Cas9 system were used for metabolic engineering modification of the strains. All constructed strains are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The elongation enzyme gene (\u003cem\u003eCgKCS\u003c/em\u003e) from \u003cem\u003eCardamine graeca\u003c/em\u003e and the ∆15 desaturase genes (MaD15D/CsD15D) from \u003cem\u003eMortierella alpine\u003c/em\u003e and \u003cem\u003eCannabis sativa\u003c/em\u003e were synthesized and coded optimally. Primers were designed to amplify target genes by PCR, and the amplified genes were linked to plasmids pINA1312 or pINA1269 that had been digested by the ClonExpress\u0026reg; II One Step Cloning Kit (Vazyme Biotech, Nanjing, China). Then the recombinant plasmid with the target gene expression cassette was obtained. The primers involved in this study are all shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. In order to achieve efficient gene expression, expression cassttes with different promoter strength and different terminator were constructed. After construction of the recombinant plasmid, it was linearized by the corresponding enzyme and then transferred into yeast cells by Frozen-EZ yeast transformation II Kit (Zymo Research, Irvine, CA).\u003c/p\u003e \u003cp\u003eThe CRISPR/Cas9 system is able to knock out the gene and knock-in the target gene at the same time. Taking the \u003cem\u003eCgKCS\u003c/em\u003e gene which was inserted into the AXP site as an example, primers with 20 bp homologous sequences at both ends of insertion site were used to obtain the amplified \u003cem\u003eCgKCS\u003c/em\u003e expression cassette by PCR. Then plasmid pHR_AXP_hrGFP digested with \u003cem\u003eSpeI\u003c/em\u003e and \u003cem\u003eAvrII\u003c/em\u003e connected with \u003cem\u003eCgKCS\u003c/em\u003e expression cassette to obtain recombinant plasmid pHR_AXP_CgKCS. Finally, the single gRNA and recombinant plasmid pHR_AXP_CgKCS were transformed into corresponding yeast cell together. All the primers used and plasmids constructed were shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e-S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGrowth condition and auxiliary carbon source\u003c/h2\u003e \u003cp\u003e \u003cem\u003eY. lipolytica\u003c/em\u003e strains were cultured in 2 mL YPD at 30\u0026deg;C (220 rpm) and then inoculated in 250 mL triangular flask containing 50 mL YPD with an initial OD\u003csub\u003e600\u003c/sub\u003e of 0.01. The strains were cultured for 72 hours under the same conditions. Adding 0.25 mL of different carbon sources (ω-9 octadecanoic acid, soybean oil, colleseed oil, sunflower seed oil, waste cooking oil) to 50 mL YPD. On this basis, gradient experiments of colleseed oil supplemental levels were designed, such as 0, 0.25, 0.5, 0.75, 1.0, and 1.25 mL added to 50 mL YPD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of VLCFAs\u003c/h2\u003e \u003cp\u003e20 mL of the fermentation medium was taken into a 50 mL centrifuge tube and centrifuged at 6,000 rpm for 5 min. The supernatant was discarded, and then 15 mL of ddH\u003csub\u003e2\u003c/sub\u003eO was added to the centrifuge tube. The mixture was thoroughly mixed and subjected to centrifugation under the same conditions. After repeating the above procedure, added 5 mL 4 M HCl to the collected cells. The mixture was oscillated and then held for 30 minutes at 37℃ at 220 rpm. Next, the test tube was kept in boiling water bath and ice for 5 minutes, and the operation was repeated again. Then 20 mL of methanol and chloroform mixed solution was added into the test tube, in which the volume ratio of methanol to chloroform was 1:2. After 30 minutes at 37\u0026deg;C, the underlying liquid was centrifuged (4800 rpm, 5 min) and then sucked into a glass tube and dried in an oven at 105\u0026deg;C. After about 12 h, taking the test tube out and then adding 3 mL of 0.5 mol/L methanol potassium hydroxide solution into the test tube when the test tube is restored to room temperature. Ultrasound was used to dissolve the oil in the tube, and the tube was kept in a water bath at 75℃ for 20 min. Adding 3 ml of 14% boron trichloride solution to the test tube and keep the same condition for 20 min. Then taking out the tube, and adding 1 mL saturated NaCl and 0.5 mL n-hexane in it. The mixture was thoroughly mixed and the upper solution was centrifuged at 12000 rpm for 2 min. Then dilute the upper liquid and mix it with the internal standard at a volume ratio of 1:4 to get the sample to be tested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGas chromatography coupled with mass spectrometry (GC\u0026thinsp;\u0026minus;\u0026thinsp;MS) analysis of VLCFAs\u003c/h2\u003e \u003cp\u003eThe sample was analyzed by GC-MS which was carried out using an Agilent System 6890 gas chromatograph (GC) with an Agilent 5975 quadrupole mass selective detector (MSD) equipped with a HP-5 column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m, Agilent, Santa Clara, CA, USA). The initial temperature of GC was held at 150\u0026deg;C for 2 min, and then at a rate of 20\u0026deg;C/min to 180\u0026deg;C. And then it went up to 200\u0026deg;C at a rate of 8\u0026deg;C/min. Then in 18 minutes the temperature reached 218\u0026deg;C, raised to 250\u0026deg;C at 8\u0026deg;C/min. The temperature subsequently raised to 300\u0026deg;C in 3.4 min. The split ratio was 20:1. The quantitative analysis was carried out by the corresponding fatty acid methyl ester standards.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eDe novo\u003c/b\u003e \u003cb\u003esynthesis of nervonic acid in\u003c/b\u003e \u003cb\u003eY. lipolytica\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe previously engineered \u003cem\u003eY. lipolytica\u003c/em\u003e to produce VLCFAs with carbon chain lengths up to 24 by co-expression heterologous C16/18-elongase from \u003cem\u003eMortierella alpina\u003c/em\u003e (MaELO3), β-ketoacyl-CoA synthases (KCSs) from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (AtKCS) and \u003cem\u003eCrambe abyssinica\u003c/em\u003e (CraKCS) combining with the deletion of \u003cem\u003ePEX10\u003c/em\u003e (Gao et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). 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. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The use of hybrid promoter UAS4B-TEF (UT) provided an excellent platform for high gene expression in \u003cem\u003eY. lipolytica\u003c/em\u003e (Gao et al. 2018). We therefor used this for the over-expression of the codon-optimized \u003cem\u003eMaELO3\u003c/em\u003e, \u003cem\u003eAtKCS\u003c/em\u003e, and \u003cem\u003eCraKCS\u003c/em\u003e genes into the integration sites F1, A3, F1-3 of \u003cem\u003eY. lipolytica\u003c/em\u003e GQY-∆PEX10 strain, respectively. Previous studies confirmed that \u003cem\u003eCardamine graeca\u003c/em\u003e KCS enzyme has the ability to elongate erucoyl-CoA (C22:1-CoA) to nervonic acid by in vitro activity assays (Taylor et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In another work, heterologous expression of \u003cem\u003eC. graeca\u003c/em\u003e KCS in \u003cem\u003eRhodosporidium toruloides\u003c/em\u003e efficiently catalyzed all elongation steps to produce nervonic acid (Fillet et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003ea). To elucidate the effects of \u003cem\u003eCgKCS\u003c/em\u003e overexpression on nervonic acid production in \u003cem\u003eY. lipolytica\u003c/em\u003e, the codon-optimized \u003cem\u003eCgKCS\u003c/em\u003e was integrated into the AXP stie by CRISPR/Cas9 technology in the \u003cem\u003eMaELO3\u003c/em\u003e, \u003cem\u003eAtKCS\u003c/em\u003e-expressing background strain (NA01), yielding strain (NA03). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e, 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, \u003cem\u003eCgKCS\u003c/em\u003e gene could efficiently push elongation of the erucoyl-CoA pool to nervonic acid (Fillet et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003ea). Simultaneously overexpression \u003cem\u003eMaELO3\u003c/em\u003e, \u003cem\u003eAtKCS\u003c/em\u003e, \u003cem\u003eCraKCS\u003c/em\u003e and \u003cem\u003eCgKCS\u003c/em\u003e 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.\u003c/p\u003e\n\u003ch3\u003eExplore desaturase of neurotic acid synthesis\u003c/h3\u003e\n\u003cp\u003eThe fatty acid profile of the engineered \u003cem\u003eY. lipolytica\u003c/em\u003e 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 \u003cem\u003eMortierella alpina\u003c/em\u003e (MaD15D) and \u003cem\u003eCannabis sativa\u003c/em\u003e (CsD15D) for expression and characterization in \u003cem\u003eY. lipolytica\u003c/em\u003e NA04 strain under the control of hybrid promoter UAS4B-TEF (UT) using plasmid pINA1312(Wang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bielecka et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To ensure efficient expression of the D15D, the gene sequences were codon optimized for expression in \u003cem\u003eY. lipolytica\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, 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 \u003cem\u003eY. lipolytica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo 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 (\u003cem\u003eCgKCS-L-MaD15D\u003c/em\u003e) in the chromosome of \u003cem\u003eY. lipolytica\u003c/em\u003e NA07 strain using established CRISPR/Cas9 technology. However, while one copy of \u003cem\u003eCgKCS-L-MaD15D\u003c/em\u003e was introduced into the A1-2 site of \u003cem\u003eY. lipolytica\u003c/em\u003e 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 \u003cem\u003eCgKCS-L-MaD15D\u003c/em\u003e was introduced into the E1-3 site of \u003cem\u003eY. lipolytica\u003c/em\u003e NA07 strain resulting strain NA08. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, increasing the fusion copy of \u003cem\u003eCgKCS-L-MaD15D\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of genes\u003c/b\u003e \u003cb\u003eOLE1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eDGA1\u003c/b\u003e \u003cb\u003eleads to significant increases in nervonic acid accumulation\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eDiacylglycerol-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. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gajdos et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tai et al. 2013). The integrative vector pINA1312 carrying the \u003cem\u003eDGA1\u003c/em\u003e 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 \u003cem\u003eDGA1\u003c/em\u003e significantly enhanced neurotic acid-producing level, which increase 1.8-fold compared to the NA04 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Meanwhile, percentage of FA distribution showed a very different between the two engineered \u003cem\u003eY. lipolytica\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Therefore, the target gene \u003cem\u003eDGA1\u003c/em\u003e was selected for subsequent genetic modification.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOLE1\u003c/em\u003e of \u003cem\u003eY. lipolytica\u003c/em\u003e 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. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, \u003cem\u003eOLE1\u003c/em\u003e serve as an attractive engineering target to overproduce nervonic acid. To implement the identified target, we overexpressed the \u003cem\u003eOLE1\u003c/em\u003e in the \u003cem\u003eY. lipolytica\u003c/em\u003e NA08 strain by introducing a native copy of the \u003cem\u003eOLE1\u003c/em\u003e gene through integrated plasmid pINA1312 under the control of strong promoter UT resulting stain NA10. As shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, overexpression of \u003cem\u003eOLE1\u003c/em\u003e 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. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Here, we investigated the effects of overexpressing the key genes in acety-CoA metabolic pathway on the nervonic acid productivity in \u003cem\u003eY. lipolytica\u003c/em\u003e. The \u003cem\u003eACL\u003c/em\u003e, encoding the ATP-dependent citrate lyase, the \u003cem\u003eACC1\u003c/em\u003e, encoding the acetyl-CoA carboxylase from \u003cem\u003eY. lipolytica\u003c/em\u003e, and \u003cem\u003eACS2\u003c/em\u003e, encoding the acetyl-CoA synthetase gene, from \u003cem\u003eS. cerevisiae\u003c/em\u003e 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 \u003cem\u003eACC1\u003c/em\u003e led to a C24:0 titer 4-fold higher than the control strain NA04 (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). 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 \u003cem\u003eFAA1\u003c/em\u003e was thus overexpressed on the \u003cem\u003eMFE\u003c/em\u003e 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 \u003cem\u003eY. lipolytica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eSince the single overexpression of \u003cem\u003eDGA1\u003c/em\u003e or \u003cem\u003eOLE1\u003c/em\u003e boosted the titer of nervonic acid in flask culture, we then reasoned that simultaneous co-overexpression of \u003cem\u003eDGA1\u003c/em\u003e and \u003cem\u003eOLE1\u003c/em\u003e 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 \u003cem\u003eY. lipolytica\u003c/em\u003e NA08 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We also tried different combinations of \u003cem\u003eDGA1\u003c/em\u003e, \u003cem\u003eOLE1\u003c/em\u003e and \u003cem\u003eOLE1-L-DGA1\u003c/em\u003e obtained three different strains. In comparison, strain NA15, which simultaneously overexpressed \u003cem\u003eDGA1\u003c/em\u003eand \u003cem\u003eOLE1-L-DGA1\u003c/em\u003e, had the highest yield of nervonic acid (111.6 mg/L) among all combinations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eElongation\u003c/b\u003e \u003cb\u003ekcs\u003c/b\u003e \u003cb\u003egene copy number adjustment increased nervonic acid production in\u003c/b\u003e \u003cb\u003eY. lipolytica\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo 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 \u003cem\u003eMaELO3\u003c/em\u003e, \u003cem\u003eCraKCS\u003c/em\u003e, \u003cem\u003eAtKCS\u003c/em\u003e and \u003cem\u003eCgKCS\u003c/em\u003e thought integrated plasmid pINA1269 to the strain NA12. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, only the extra copy of \u003cem\u003eMaELO3\u003c/em\u003e 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 \u003cem\u003eCgKCS\u003c/em\u003e. Since previous reports showed that increasing the copy number of \u003cem\u003eCgKCS\u003c/em\u003e could boost the concentration of nervonic acid in \u003cem\u003eR. toruloides\u003c/em\u003e (Fillet et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003eb), the inconsistent results might be caused by different genetic background of the stains.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of oily substrates as auxiliary carbon sources for nervonic acid production by the engineered\u003c/b\u003e \u003cb\u003eY. lipolytica.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs an oleaginous yeast, \u003cem\u003eY. lipolytica\u003c/em\u003e 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. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Poli et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In order to screen the most suitable carbon source for the production of neuronic acid by \u003cem\u003eY. lipolytica\u003c/em\u003e, 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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, 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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). 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.\u003c/p\u003e \u003cp\u003eTo 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. \u003cem\u003eY. lipolytica\u003c/em\u003e GQY-∆PEX10 was the strain that only deleted \u003cem\u003ePEX10\u003c/em\u003e, a gene encoding a major peroxisomal matrix protein, from \u003cem\u003eY. lipolytica\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eS).\u003c/p\u003e \u003cp\u003eFinally, 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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Despite complex multistep engineering efforts, production titers in this study still lower than that of previous report.\u003csup\u003e24\u003c/sup\u003e However, the systematic engineering strategies of \u003cem\u003eY. lipolytica\u003c/em\u003e 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 \u003cem\u003eY. lipolytica\u003c/em\u003e will be expected using higher biomass concentrations and controlled bioreactor.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we engineered the oleaginous yeast \u003cem\u003eY. lipolytica\u003c/em\u003e following multi-level strategies for efficient accumulation of neurotic acid production. Specifically, we reconstructed the elongation pathway as well as desaturation pathway, optimized the key gene expression in fatty acid metabolism through adding gene copy and protein fusion. Furthermore, we first demonstrated that supplementing the colleseed oil as auxiliary carbon benefited the neurotic acid production. The yeast engineering strategy of pathway assembling presented in this study may be employed to optimize microbial production of other valuable VLCFA chemistry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the authors have read and approved to submit it to \u003cem\u003eBioresources and Bioprocessing\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key R\u0026amp;D Program of China (2021YFC2102805).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXRZ designed experiments; XRZ and XLC conducted experiments, XRZ, XLC and JLY collected data; XRZ, XLC, JLY, GQ and JTS analyzed data; QH and LJW conceived the idea and supervised the research; XRZ, XLC and LJW drafted the manuscript and contributed to data interpretation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Prof. Er-Zheng Su for guidance at the beginning of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmminger GP, Schafer MR, Klier CM, Slavik JM, Holzer I, Holub M, Goldstone S, Whitford TJ, McGorry PD, Berk M (2012) Decreased nervonic acid levels in erythrocyte membranes predict psychosis in help-seeking ultra-high-risk individuals. 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J biotechnol 281:106\u0026ndash;114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jbiotec.2018.07.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jbiotec.2018.07.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"nervonic acid, Yarrowia lipolytica, de novo, metabolic engineering, colleseed oil","lastPublishedDoi":"10.21203/rs.3.rs-3107416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3107416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNervonic acid, a natural fatty acid compound and also a core component of nerve fibers and nerve cells, has been widely used to prevent and treat related diseases of the brain nervous system. At present, fatty acids and their derivatives are mainly obtained by natural extraction or chemical synthesis which are limited by natural resources and production costs. In this study, the \u003cem\u003ede novo\u003c/em\u003e synthetic pathway of nervonic acid was constructed in \u003cem\u003eYarrowia lipolytica\u003c/em\u003e by means of synthetic biology, and the yield of nervonic acid was further improved by metabolic engineering and fermentation optimization. Specially, heterologous elongases and desaturases derived from different organism were successfully expressed and evaluated for their potential for the production of nervonic acid in \u003cem\u003eY. lipolytica\u003c/em\u003e. Meanwhile, we overexpressed the genes involving in the lipid metabolism to increase the nervonic acid titer to 111.6 mg/L. In addition, the potential of adding oil as auxiliary carbon sources for nervonic acid production by the engineered \u003cem\u003eY. lipolytica\u003c/em\u003e were analyzed. The results indicated that supplementation with colleseed oil as an auxiliary carbon source can be beneficial for the nervonic acid productivity, which led to a highest concentration of 185.0 mg/L in this work. To summary, this study describes that the \u003cem\u003eY. lipolytica\u003c/em\u003e can potentially be used for a promising platform to produce nervonic acid and other very long chain fatty acid.\u003c/p\u003e","manuscriptTitle":"De novo synthesis of nervonic acid and optimization of metabolic regulation by Yarrowia lipolytica","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-06 17:31:04","doi":"10.21203/rs.3.rs-3107416/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-07-01T14:02:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-01T13:43:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-07-01T06:29:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2023-06-25T11:32:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90fb3ce0-f8f3-4883-ab80-eba51cf4c83a","owner":[],"postedDate":"July 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-09T15:03:18+00:00","versionOfRecord":{"articleIdentity":"rs-3107416","link":"https://doi.org/10.1186/s40643-023-00689-6","journal":{"identity":"bioresources-and-bioprocessing","isVorOnly":false,"title":"Bioresources and Bioprocessing"},"publishedOn":"2023-10-06 15:01:02","publishedOnDateReadable":"October 6th, 2023"},"versionCreatedAt":"2023-07-06 17:31:04","video":"","vorDoi":"10.1186/s40643-023-00689-6","vorDoiUrl":"https://doi.org/10.1186/s40643-023-00689-6","workflowStages":[]},"version":"v1","identity":"rs-3107416","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3107416","identity":"rs-3107416","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}