Enhanced synthesis of 1, 3-medium chain-2-long chain triacylglycerols by engineered Saccharomyces cerevisiae
Zhuangju Peng, Zikun Zhang, Rihan Gao, Li Deng, Fang Wang, Junfeng Liu
TL;DR
This paper describes a new method to produce a specific type of fat using engineered yeast, which could be more cost-effective than current methods.
Contribution
The study introduces an engineered yeast strain that efficiently produces MLM-TAGs through iterative genetic and environmental optimizations.
Findings
Overexpression of fas1R1834K increased medium-chain fatty acid production to 0.41 mol% of total TAGs.
Introduction of RnACSM4 and deletion of GAT2 and LRO1 raised MLM-TAGs to 6.7 mol%.
Optimized conditions achieved 34.4 mol% MLM-TAGs with a 135-fold yield improvement over the original strain.
Abstract
Enzymatic synthesis is currently the primary method for preparing 1,3-medium chain- 2-long-chain triacylglycerols (MLM-TAGs), which serve as both a dietary component and clinical nutrient for specific populations. The application of MLM-TAGs is obviously constrained by the high cost of catalysts. Hence, a novel approach was proposed for MLM-TAGs production by engineered yeast. Overexpressing the mutated fas1R1834K increased the production of medium-chain fatty acids (C8–C12) and resulted in an MLM content of 0.41 mol% of the total TAGs. The introduction of RnACSM4 enabled the recombinant to produce MLM-TAGs at a level of 4.2 mol% when supplemented with 0.2 mM sodium laurate. Further deletion of GAT2 and LRO1 increased the content of MLM-TAGs to 6.7 mol%. Iterative optimization involving sodium laurate dosage, culture temperature, and amino acid addition elevated the MLM-TAGs content to…
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Figure 7- —http://dx.doi.org/10.13039/501100001809National Natural Science Foundation of China
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Taxonomy
TopicsLipid metabolism and biosynthesis · Microbial Metabolic Engineering and Bioproduction · Enzyme Catalysis and Immobilization
Introduction
Oils and fats derived from plants and animals are extensively used in the food, cosmetics, energy, and other industries. These lipids are predominately composed of triacylglycerols (TAGs), which are esters formed by the esterification of glycerol with three fatty acids (He et al. 2017). The degree of saturation, chain length, and positional distribution of fatty acids significantly influence the physical properties and health benefits of TAGs (Guo et al. 2020). Structured TAGs, such as 1, 3-dioleoyl-2-palmitoyl (OPO) triacylglycerol and TAGs rich in medium chain fatty acids (MCFAs) or polyunsaturated fatty acids, possess distinct nutritional advantages (Wu et al. 2025; Yuan et al. 2020; Zhang et al. 2010). Notably, 1, 3-medium chain-2-long chain triglycerols (MLM-TAGs) are regarded as ideal dietary components for individuals undergoing weight management and as clinical nutrients for postoperative patients (Matsuo et al. 2022; Yuan et al. 2020). Although some natural oils and fats contain high levels of MCFAs, the proportion of MLM-TAGs in these products remains low (More et al. 2019). Currently, MLM-TAGs are primarily prepared through enzymatic catalysis (Utama et al. 2019). However, their application is severely constrained by high catalyst costs and limited availability of raw materials (Li et al. 2023a, b; Peng et al. 2020; Heil et al. 2019). Therefore, it is imperative to explore alternative approaches to overcome these limitations.
Given that neutral TAGs serve as storage lipids in yeasts, enabling yeast to directly synthesize MLM-TAGs presents an attractive alternative strategy. In yeasts, long-chain fatty acids such as C16 and C18 dominate the fatty acid profile, whereas MCFAs ranging from C6 to C14 are present in much lower proportions (Liu et al. 2013; Xu et al. 2016). Thus, enhancing MCFAs synthesis is a prerequisite for MLM-TAGs production. Type I fatty acid synthase (FAS), which is responsible for de novo fatty acid synthesis in yeast, is an integrated multi-domain enzyme complex that typically terminates fatty acid elongation at C16–C18, although shorter-chain fatty acids (C12 and C14) are produced in small amounts (Garces Daza et al. 2023). To augment the synthesis of MCFAs in fungi, multiple engineering strategies have been developed, including the construction of orthogonal FAS system, site-directed mutations in FAS domains, introduction of short-chain-specific thioesterase, engineering of the membrane transporter, establishment of reverse β-oxidation pathway, disruption of β-oxidation, and modifications of fatty acid metabolic pathway (Garces Daza et al. 2023; Zhu et al. 2020; Xu et al. 2016).
The assembly of acyl-CoA onto the glycerol backbone is relatively conserved across organisms (Parsons and Rock 2013). Glycerol-3-phosphate undergoes three acylation reactions and one dephosphorylation step to form TAG, a process sequentially catalyzed by glycerol-3-phosphate acyltransferase (GAT), lysophosphatidic acid acyltransferase (LPAAT), phosphatidyl phosphatase, and diacylglycerol acyltransferase (DGA). OPO-TAG has been successfully synthesized in multiple eukaryotic cells by exploiting the substrate preference of acyltransferase involved in TAG biosynthesis (Peng et al. 2025; Zhou et al. 2024; Bhutada et al. 2022; van Erp et al. 2021). Above information underscores the feasibility of biosynthesizing MLM-TAGs and suggest a promising alternative to conventional production methods.
By expressing specific thioesterase, phosphatidyl phosphatase, and acyltransferases, a bacterial cell factory capable of producing MLM-TAGs has been developed (Chen et al. 2022). However, due to the limited lipid synthesis capacity and the generation of endotoxins, E. coli is not considered an ideal host for producing edible oils. In contrast, Saccharomyces cerevisiae, a eukaryotic organism that is generally regarded as safe, is widely employed for the production of fatty acids and their derivatives (Li et al. 2023a, b; Kathryn et al. 2021; Bergenholm et al. 2018). Although the feasibility of MLM-TAGs synthesis in yeast has been preliminarily demonstrated (Peng et al. 2025), further investigation is required to optimize synthesis strategies and improve the yield of MLM-TAGs. In this study, two approaches for MLM-TAGs production, de novo synthesis and substrate feeding, were compared. Through metabolic engineering and optimization of culture conditions, a maximum MLM-TAGs yield of 18.5 mg/g DCW was achieved by feeding sodium laurate.
Materials and methods
Strains, media and plasmids
S. cerevisiae YS58 (MATα, ura3-52, his4-519, trp1-789, leu2-3,112) was used as the expression host. The plasmid pRS425 (Amp^R^, leu2) served as expression vector. YS58 was cultured in YPD medium containing 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract. Recombinants were screened on synthetic dextrose solid medium lacking leucine and uracil (SC-Leu/Ura), which consisted of 6.7 g/L yeast nitrogen base without amino acids (YNB), 5 g/L (NH_4_)2_SO_4, 20 g/L glucose, 1.29 g/L drop-out supplement (Beijing Coolaber Company), and 1.7 g/L agar.
Plasmid construction
The gene encoding acyl-CoA synthetase medium-chain family member 4 (ACSM4) from Rattus norvegicus was chemically synthesized and ligated into plasmid pRS425 (GENEWIZ, Suzhou, China). A point mutation (Arg1834Lys) was introduced into FAS1 via PCR using two pairs of primers containing the desired mutation site (Table S2). The resulting PCR fragments and digested pRS425 vector were assembled by homologous recombination to generate the plasmid pRS425-fas1^*^. After confirmation by DNA sequencing (GENEWIZ, Suzhou, China), the recombinant plasmids were transformed into YS58 using the lithium acetate method. Positive recombinants were selected on SC-Leu plates and further confirmed by sequencing analysis. All recombinants used in this study are listed in Table S1. YS58 harboring the empty plasmid was employed as the control. The primers used in this study are detailed in Table S2.
Gene knockout
Gene knockout was carried out via homologous recombination. The recombination cassette comprised the upstream homologous arm of the target gene, PURA3 promoter, URA3, TADH1 terminator, and the downstream homologous arm of the target gene. The entire cassette was chemically synthesized by GENEWIZ (Suzhou, China). Transformants were selected on SC-Ura plates and verified by DNA sequencing. The primers used for validation are listed in Table S2.
Batch fermentation
The positive transformants were cultured in a 250 mL flask containing 50 mL of SC-Leu medium at 30 °C and 200 rpm for 72 h. The medium consisted of 20 g/L glucose, 1.29 g/L dropout-Leu supplement, 1.7 g/L YNB, and 5 g/L (NH_4_)2_SO_4. The resulting broth was used to optimize the culture conditions and served as the served as the inoculum for subsequent bioreactor experiments. The seed culture was transferred into a sterilized 5-L bioreactor (Shanghai Bailun Bio-Technology Co., China) at a 1:10 (v/v) inoculation ratio. Fermentation was carried out at 30 °C for 96 h with an agitation speed of 250 rpm and an aeration rate of 2 vvm. After 24 h of cultivation, sodium laurate was added to the medium, and the temperature was reduced to 23 °C.
Quantitation of total fatty acids
Following centrifugation at 5000 rpm and 25 °C for 5 min, the harvested cells were resuspended in sterile water and subjected to a second centrifugation. The cell pellet was then mixed with a KOH (10 wt%)—methanol solution at a ratio of 1:9 (v/v) and refluxed for 2 h. Subsequently, 6 mol/L HCl and n-hexane were added sequentially. After extraction at 20 °C and 180 rpm for 2 h on a shaker, the upper organic layer containing fatty acids was collected and esterified with methanol to generate fatty acid methyl esters (Khosa et al. 2024). Fatty acid content was analyzed using a GC-2012 gas chromatograph (Shimadzu, Japan) equipped with a DB-Wax column (30 m × 0.25 mm × 0.25 μm, Agilent) according to the method described by Peng et al. (2025). A mixture of fatty acid standards was used as external reference for quantification.
Analysis of acyl distribution in TAGs
Four freeze–thaw cycles were performed on cell pellets obtained from 800 mL of broth, followed by ultrasonication using an ultrasonic homogenizer (Ningbo Scientz, China). Main parameters of ultrasonic treatment were as follows: power 70%, total time 15 min, ultrasound time 15 s, ultrasound interval time 10 s. Yeast lipids were cextracted with a chloroform/methanol solution (2:1, v/v), and TAGs were subsequently separated by thin layer chromatography. The sample was applied onto a silica gel plate, which was then developed using a n-hexane/ether/glacial acetic acid solvent system (v/v/v = 50:50:1). After development, the TAG band was scraped from the plate, and TAGs were extracted using a chloroform/methanol (2:1, v/v) solution. Following hydrolysis with pancreatic lipase and esterification with methanol (Zhang et al. 2016), the acyl composition of TAGs was analyzed using a GC-2012 (Shimadzu, Japan) equipped with a DB-WAX column (30 m × 0.25 mm × 0.25 μm, Agilent). A mixture of fatty acid standards was used as external reference. The MLM-TAGs content was calculated based on the amounts of sn-2 monoglyceride and fatty acids released from sn-1,3 positions of TAGs according to the following formula (Chandler et al. 1998) (1):
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{aligned} {\mathrm{MLM}} - {\text{TAGs }}\left( \% \right) & = \left[ {{\mathrm{mol}}\% \;{\mathrm{medium}} - {\mathrm{chain}}\;{\text{ acyl }}\;{\mathrm{at}}sn - {1}} \right] \, \\ & \quad \times \left[ {{\mathrm{mol}}\% \;{\mathrm{long}} - {\mathrm{chain}}\;{\text{ acyl }}\;{\mathrm{at}}sn - {2}} \right] \\ & \quad \times {\mathrm{mol}}\% \;{\mathrm{medium}} - {\text{chain }}\;{\mathrm{acyl}}\;{\text{ at}}sn - {3} \times { 1}0^{{ - {4}}} \\ \end{aligned} $$\end{document}Quantitative analysis of MLM-TAGs
The quantitative analysis of MLM-TAGs was completed by the Institute of Oil Crops, Chinese Academy of Agricultural Sciences. In accordance with established protocols (Yang et al. 2023), approximately 20 mg of lipids were extracted from freeze-dried yeast cells and analyzed using a UPLC 30A system (Shimadzu, Japan) equipped with a Kinetex C18 column (100 mm × 2.1 mm, 2.6 µm, Phenomenex, America) and coupled to a Triple TOF® 6600 system (AB Sciex, Canada). Raw mass spectrometry data were processed using MS-DIAL and commercial software packages from AB Sciex (Canada), including MarkView, MasterView, PeakView, and MultiQuant. A standard mixture of TAG 48:1 (C15:0/C18:1/C15:0), DAG 33:1 (C15:0/C18:1), and monoglyceride 18:1 (C18:1), purchased from Avanti Polar Lipids (Alabaster, USA), was employed as internal standard. The internal standard concentration was 10 μg/mL and its recovery efficiency was 80%.
Glucose detection
After centrifugation at 5000 rpm for 5 min, the supernatant was collected and analyzed for glucose concentration by HPLC (Shimadzu, Japan) equipped with a differential detector and an organic acid column (Zhang et al. 2022). The mobile phase was 5 mM sulfuric acid, delivered at a flow rate of 0.6 mL/min. The column temperature was maintained at 50 °C, and the detection time was set to 25 min. Cell growth was monitored by measuring the OD_600_ of the fermentation broth using a spectrophotometer (Shanghai Metash Instruments, China).
Statistical analysis
Three independent biological samples were used in each experiment, and the final data was expressed as mean ± standard deviation (SD). The difference is considered statistically significant at p < 0.05 with a two-side t-test.
Results and discussion
Effect of mutated FAS1 on MLM production
The synthesis of MCFAs is essential for the intracellular production of MLM. Budding yeast naturally synthesizes only trace amounts of MCFAs (Liu et al. 2013). Overexpression of a mutant Fas1 (R1834K) markedly enhances MCFA production, whereas overexpression of the wild-type Fas1 fails to increase MCFA synthesis in brewing yeast (Zhu et al. 2020; Gajewski et al. 2017). Such, the mutant allele fas1^^ was directly introduced into the host strain to augment MCFA biosynthesis. Compared to host strain, overexpression of fas1^^ imposed a noticeable growth burden on the recombinant (Fig. 1A). Meanwhile, the introduction of fas1^^ led to elevated intracellular levels of MCFAs, with particularly pronounced increases in C8:0, C12:0, and C14:1 fatty acids (Fig. 1B). Given the high membrane permeability of MCFAs, we further examined their extracellular concentrations. As expected, recombinants expressing fas1^^ exhibited elevated extracellular levels of MCFAs (C8:0, C10:0, and C12:0) relative to the control. Notably, C8:0 reached a titer of 2.1 mg/L, representing a 13-fold increase over the host strain (Fig. 1C). Additionally, a small amount of short-chain fatty acid C4:0 was detected. The total extracellular MCFAs (C8:0, C10:0, and C12:0) titer in recombinant reached 2.83 mg/L, which is fourfold higher than the intracellular MCFA content and 5.6-fold greater than that of the parent strain. Despite this, the MLM content increased from 0.33% to 0.41% of total TAGs in recombinant compared to the control, indicating that Fas1^*^ augmented the cellular synthesis of MLM (Fig. 1D). It should be noted that intracellular MLM level remained very low. Several plausible explanations may account for this observation: (i) limited production of MCFAs by the Fas1 mutant; (ii) insufficient capacity of the cell to incorporate MCFAs into TAGs; (iii) active efflux or passive diffusion of free MCFAs into the extracellular space due to their cytotoxicity (Yang et al. 2022). Taken together, these findings suggest that achieving high-yield MLM via de novo biosynthetic pathways remains a significant challenge.
Fig. 1. Enhanced MLM synthsis by expressing FAS1^*^. A Growth curves, B intracellular and C extracellular fatty acid levels, and D MLM contents of recombinant and host strain. Samples were taken at 72 h of fermentation. *, **, and *** represent statistically significant with p < 0.05, p < 0.01, and p < 0.001, respectively
The role of acyltransferase in MLM synthesis
To boost the cellular synthesis of MLM, we employed yeast cells as catalysts for MLM production through the supplementation of MCFAs. However, it’s worth noting that exogenous fatty acids must first be activated by acyl-CoA synthetase to generate acyl-CoA thioesters, which serve as substrates for the biosynthesis of structured TAGs (van der Sluis 2018). To this end, we heterologously expressed the medium-chain acyl‑CoA synthetase ACSM4 from Rattus norvegicus in the yeast host. Upon addition of 0.2 mM sodium laurate (SL), the C12:0 content in total TAGs of the engineered strain reached 14.1% (v/v), representing a threefold increase compared to the control (Fig. S1). The expression of RnACSM4 not only compensated for the deficiency of endogenous medium-chain acyl‑CoA synthetase but also enhanced the intracellular utilization efficiency of lauric acid. Thus, the exogenous feeding lauric acid presents a viable alternative to de novo synthesis for MLM production.
To further improve the incorporation of lauric acid into glycerolipids, we systematically evaluated how the substrate specificity of acyltransferases in the TAG biosynthetic pathway influences MLM accumulation. Analysis of acyl distribution in TAGs revealed that the abundance of C12:0 at the sn‑1,3 positions reached 26.3% (v/v) and 23.0% (v/v) in recombinants ACSM4‑Δgat2 and ACSM4‑Δlro1, respectively (Fig. 2A). It can be speculated that Gat1 and Dga1 exhibit relatively high selectivity toward lauric acid at these positions, though long-chain monounsaturated fatty acids (C16:1 and C18:1) remained predominant. In contrast, saturated fatty acids C16:0 and C18:0 were primarily esterified at the sn‑2 position, where C12:0 accounted for only a minor proportion (Fig. 2B). The deletion of specific acyltransferase had negligible effects on cell growth (Fig. 2C), but to varying degrees impaired MLM biosynthesis. Among all tested strains, the highest MLM content of approximately 6.7 mol% was observed in recombinants ACSM4‑Δgat2 and ACSM4‑Δlro1, which increased by 60.4% compared to the control (Fig. 2D). These observations indicated that acyltransferases responsible for acylation at the sn-1 and sn-3 positions play a more dominant role in regulating MLM synthesis compared to those acting at the sn-2 position. Though expression of LPAAT with palmitoyl-CoA specificity has been shown to enhance OPO-TAGs synthesis in eukaryotic cells (Zhou et al. 2024; Bhutada et al. 2022; van Erp et al. 2021), deletion of LPAAT (SLC1 or ALE1) resulted in decreased levels of C12:0 at *sn-*1,3 positions and a concomitant reduction in MLM content (Fig. 2A, D). It is speculated that the interactions between LPAAT and other acyltransferases may disrupt the distribution of acyl groups on glycerol backbone (Chen et al. 2022; Rosalind and Douglas 2004). These findings revealed that exogenous C12:0 could be effectively mobilized for incorporation into TAGs, and C12:0 was preferentially utilized by Gat1 and Dga1 at the sn‑1 and sn‑3 positions during TAG formation.
Fig. 2. Effect of acyltransferase on MLM synthesis. A, B Acyl composition at sn-1,3 positions and sn-2 position of TAGs. C Growth curves and D MLM contents of recombinants deleting acyltransferase. All recombinants were cultivated in a shake flask and supplemented with 0.2 mM sodium laurate after 24 h of cultivation. Samples were taken at 72 h of fermentation. ACSM4, medium-chain acyl‑CoA synthetase from Rattus norvegicus; Gat1/Gat2, glycerol-3-phosphate O-acyltransferase; Slc1/Ale1, lysophosphatidic acid acyltransferase; Dga1, diacylglycerol acyltransferase; Lro1, phospholipid: diacylglycerol acyltransferase. “Others” refers to all fatty acids except fatty acid C12:0, C14:0, C16:0, C16:1, C18:0 and C18:1
Effect of sodium laurate dosage
Given the predominance of long-chain acyls at the sn-2 position (Fig. 2B), the dosage of exogenous SL emerged as a critical determinant influencing intracellular MLM synthesis. Therefore, we investigated the effect of different amounts of SL on MLM production. As the SL dosage increased, the content of C12:0 at the sn-1,3 positions of TAGs initially rose and then declined, reaching a maximum of 43.8% (v/v) when supplemented with 0.8 mM SL (Fig. 3A). Concurrently, the levels of saturated fatty acids (C16:0 and C18:0) at these positions exhibited an overall increase, whereas those of unsaturated fatty acids (C16:1 and C18:1) generally decreased, except upon supplementation with 1.6 mM SL. Notably, the C12:0 content at the sn-2 position exhibited a gradual decline with increasing SL concentration, coinciding with the rise in saturated fatty acids (C16:0 and C18:0), with the exception observed at 1.2 mM (Fig. 3B). Moreover, the increasing SL addition exerted a progressively negative effect on the growth of recombinants (Fig. 3C). At 0.8 mM SL, lauric acid and MLM reached peak levels, accounting for 32.2% (v/v) and 18.8 mol% of the total TAGs, respectively (Fig. S2, Fig. 3D). Excessive SL supplementation led to a reduction in MLM content, suggesting that cells may adjust their lipid composition in response to exogenous fatty acid availability, thereby regulating the accumulation of MCFAs and maintaining optimal growth (McDonough et al. 2002). An additional cellular response to the passive influx of exogenous fatty acids could involve remodeling membrane lipid composition to either enhance or restrict their diffusion (Hibbs and Marzuki 1986). Based on these observations, 0.8 mM SL was selected as the optimal concentration for subsequent experiments.
Fig. 3. Increased MLM production by feeding appropriate dosage of sodium laurate. A, B Acyl composition at sn-1,3 positions and sn-2 position of TAGs. C Growth curves and D MLM content of recombinant ACSM4-ΔLRO1. “Others” refers to all fatty acids except fatty acid C12:0, C16:0, C16:1, C18:0 and C18:1. Recombinant ACSM4-ΔLRO1 was cultivated in a shake flask and supplemented with different dosage of sodium laurate after 24 h of cultivation. Samples were taken at 96 h of fermentation
Effect of culture temperature
As the primary intracellular energy storage molecules, TAGs are influenced by a variety of extracellular and intracellular factors (Ferreira et al. 2018; Gossing et al. 2018). Temperature not only directly affects yeast growth but also modulates the composition of cellular fatty acids (Yang et al. 2023; Redón et al. 2011). Compared to the standard cultivation temperature (30 °C), both elevated (37 °C) and reduced (23 °C) temperatures exerted measurable effects on the biomass of the recombinant (Fig. 4A). With decreasing culture temperature, the C12:0 content in total TAGs increased from 21.8 (v/v) to 40.9% (v/v) (Fig. S3), accompanied by a corresponding rise in MLM content from 7 to 30.6 mol% (Fig. 4B). These findings suggested that culturing the engineered yeast at lower temperature enhances its capacity to utilize SL and incorporate it into the sn-1,3 positions of TAGs, thereby promoting MLM synthesis. Further analysis uncovered that lower culture temperature also reduced the C16:0 content at the sn-2 position while increasing the C16:1 content (Fig. 4C). Notably, the C12:0 content in sn-1,3 positions reached a maximum of 55.6% (v/v) at 23 °C, which was 2.2-fold higher than that observed at 37 °C (Fig. 4D). On one hand, the increased C12:0 content may improve membrane phase behavior and fluidity, helping maintain membrane functionality under low temperature conditions (Stephanie and Robert 2017; Ernst et al. 2016). On the other hand, low temperature appears to promote the synthesis of MCFAs and TAGs (Froissard et al. 2015; Redón et al. 2011), facilitating the incorporation of C12:0 into the glycerol backbone. Additionally, temperature variations can induce changes in the average fatty acid chain length in budding yeast (Péter et al. 2021). Collectively, these results demonstrated that reduced culture temperature effectively enhanced the enrichment of C12:0 at the sn-1,3 positions of TAGs, thereby increasing MLM production.
Fig. 4. Promoted MLM synthesis by low temperature. A Growth curves of recombinant cultured at different temperature. B MLM content in total TAGs. C, D Acyl composition at sn-2 position and sn-1,3 positions of TAGs. “Others” refers to all fatty acids except fatty acid C12:0, C16:0, C16:1, C18:0 and C18:1. Recombinant ACSM4-ΔLRO1 was cultivated in a shake flask and supplemented with 0.8 mM sodium laurate after 24 h of cultivation. Samples were taken at 96 h of fermentation
Effect of amino acid addition
Amino acids serve as nitrogen sources and growth factors, exerting a direct and profound impact on microbial growth and metabolism. Therefore, we investigated the effect of exogenous amino acid addition on MLM synthesis. Among the tested amino acids (histidine, cysteine, glutamate, arginine, lysine, serine, and tyrosine), supplementation with 1 g/L glutamate yielded the highest MLM content, reaching 30.8 mol% of total TAGs (Fig. S4). The enhanced MLM synthesis upon glutamate addition may be attributed to the following mechanisms: (i) glutamate promotes fatty acid biosynthesis (Hung et al. 2024; Kaya et al. 2020); and (ii) the abundant intracellular pool of glutamate supports various bioprocesses, including amino acid biosynthesis, nitrogen assimilation, and cofactor production (Walker and van der Donk 2016). Based on these findings, glutamate was selected for further investigation, with three supplementation levels (0.5, 1, and 1.5 g/L) evaluated. As shown in Fig. 5A, glutamate supplemention with different concentrations did not significantly affect cell growth, although a slightly faster growth rate was observed at 0.5 g/L. Further analysis revealed that MLM accounted for 34.4% of total TAGs when 0.5 g/L glutamate was supplemented with, 104-fold higher than that produced by the starting strain (Fig. 5B). However, higher concentrations of glutamate reduced MLM content under the current experimental conditions. At the optimal glutamate dosage, the C12:0 content at the sn-1,3 positions of TAGs reached 58.8% (v/v), while the levels of unsaturated fatty acids such as C16:1 and C18:1 dropped to 3.8% and 1.6% (v/v), respectively (Fig. 5C). In addition, glutamate supplementation differentially influenced acyl composition at the sn-2 position (Fig. 5D). Notably, high-dose glutamate (1.5 g/L) increased the C18:0 content at both the sn-1,3 and sn-2 positions. Collectively, these results demonstrated that appropriate supplementation with specific amino acid could effectively augment the synthesis of MLM.
Fig. 5. Enhanced MLM production by adding glutamate. A Growth curves and B MLM content of recombinants cultivated in medium supplemented with 0.5, 1, and 1.5 g/L glutamate. C, D Acyl composition at sn-1,3 positions and sn-2 position of TAGs. “Others” here refers to all fatty acids except fatty acid C12:0, C16:0, C16:1, C18:0 and C18:1. Recombinant ACSM4-ΔLRO1 was cultivated in a shake flask and supplemented with 0.8 mM sodium laurate after 24 h of cultivation at 30 °C, and then transferred to 23 °C for 72 h. Samples were taken at 96 h of fermentation
MLM production in 5 L fermentation
To achieve higher MLM production, the recombinant was cultivated in optimized media containing 20 g/L glucose, 5 g/L (NH_4_)2_SO_4, 1.7 g/L YNB, dropout-Leu supplement 1.29 g/L, and 0.5 g/L glutamate. The culture was incubated at 30 °C and 250 rpm for 24 h, followed by supplementation with 0.8 mM SL and transfer to 23 °C with continued agitation at 250 rpm for an additional 72 h. Under the optimized conditions, the recombinant exhibited cell growth and sugar consumption profiles comparable to those of the control during fermentation in a 5 L bioreactor (Fig. 6A). The total TAGs yield reached 58.4 mg/g DCW, representing an increase of 28.8% compared to the control (Fig. 6B). Notably, an MLM yield of 18.5 mg/g DCW was achieved, 135-fold higher than that of the control strain. These findings demonstrated that the engineered S. cerevisiae holds promise as a biocatalyst for scalable MLM production.
Fig. 6MLM production in a 5-L bioreactor. A Cell growth and residual glucose of recombinants cultured in the optimized media containing 5 g/L NH_4_Cl, 20 g/L glucose, 1.7 g/L YNB, dropout-Leu supplement 1.29 g/L, and 0.5 g/L glutamate. B MLM production after 96 h of fermentation
Although MLM production was significantly improved through exploitation of the substrate preferences of endogenous acyltransferases and optimization of culture conditions, the overall yield of MLM remains relatively low. This limitation can be primarily attributed to the diversity and dynamic fluctuations of intracellular fatty acids, the broad substrate specificity of acyltransferases, and the rapid turnover and remodeling of intracellular TAGs, all of which contribute to temporal variations in TAG composition and fatty acid profiles throughout cultivation (Peng et al. 2025; Wunderling et al. 2023). Therefore, a systematic and in-depth investigation into the regulation of TAG metabolism is essential for the efficient synthesis of MLM. In particular, current knowledge gaps exist regarding the functional interactions among the acyltransferases involved in TAG assembly, the signaling roles of metabolic intermediates, and the mechanisms governing the rapid turnover of TAGs. In summary, further research is required to fully elucidate the regulatory network of TAG biosynthesis to enable targeted strategies for enhancing MLM production.
Conclusions
In this work, we developed a sustainable approach for MLM-TAGs production using engineered yeast. Supplementation with sodium laurate resulted in higher MLM-TAGs levels compared to de novo fatty acid synthesis. Through metabolic engineering and optimization of culture conditions, a maximum MLM-TAGs yield of 18.5 mg/g DCW was achieved in a 5 L bioreactor. This study not only presents a practicable method for sustainable production of MLM-TAGs but also deepens our understanding of key factors influencing their biosynthesis.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1.