Cocktail biosynthesis of triacylglycerol by rational modulation of diacylglycerol acyltransferases in industrial oleaginous Aurantiochytrium

Lan, Chuanzeng; Wang, Sen; Zhang, Huidan; Wang, Zhuojun; Wan, Weijian; Liu, Huan; Hu, Yang; Cui, Qiu; Song, Xiaojin

doi:10.1186/s13068-021-02096-5

Cited by 12 publications

(14 citation statements)

References 57 publications

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“…Although DGAT s plays an important role in promoting TG synthesis, there are some functional differences among different sources and types of DGAT s. For example, the functions of four DGAT2 s ( DGAT2A , DGAT2B , DGAT2C , and DGAT2D ) in Aurantiochytrium sp. SD116 were investigated, and it was found that DGAT2C was mainly responsible for connecting PUFAs to the sn-3 position of the TG molecules, while DGAT2A and DGAT2D mediated the SFA-TGs production [ 5 ]. Comparative analysis of the differential metabolites between the two groups showed that 17 kinds of EPA-related TG were significantly increased compared to the yl-EPA strain, including TG (16:0/17:1/20:5), TG (20:5/17:1/20:5), TG (20:5/18:2/18:2), TG (20:5/20:5/22:5), TG (20:5/10:1/11:1), TG (20:5/18:2/20:5), TG (10:0/18:2/20:5), TG (8:0/18:2/20:5), TG (16:0/18:1/20:5), TG (20:5/14:1/18:2), TG (16:1/18:2/20:5), TG (16:0/16:0/20:5), TG (20:3/11:3/20:5), TG (20:5/11:4/22:3), TG (6:0/18:1/20:5), TG (18:4/16:0/20:5), and TG (20:5/18:2/22:6) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids because they can accumulate lipids exceeding 20% of their dry cell weight (DCW) [ 4 ]. In addition, they have several advantages such as ease of culture, multiple lipid composition, and extensive carbon source substrate utilization [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

“…The functions of four DGAT2 s ( DGAT2A , DGAT2B , DGAT2C , and DGAT2D ) in Aurantiochytrium sp. SD116 were investigated, and it was found that DGAT2C was mainly responsible for connecting PUFAs to the sn-3 position of the TG molecules, while DGAT2A and DGAT2D mediated the SFA-TGs production [ 5 ]. Overexpression of DGAT2 in Nannochloropsis oceanica resulted in a 69% increase in TG content.…”

Section: Introductionmentioning

confidence: 99%

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Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica

Jia,

Zhang,

et al. 2024

Biotechnol Biofuels

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Background Eicosapentaenoic acid (EPA) is widely used in the functional food and nutraceutical industries due to its important benefits to human health. Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids. However, the storage of EPA in triglyceride (TG) becomes a key factor limiting its level. Results This study aimed to incorporate more EPA into TG storage through metabolic engineering. Firstly, key enzymes for TG synthesis, the diacylglycerol acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (GPAT) genes from Schizochytrium sp. HX-308 were expressed in Yarrowia lipolytica to enhance lipid and EPA accumulation. In addition, engineering the enzyme activity of DGATs through protein engineering was found to be effective in enhancing lipid synthesis by replacing the conserved motifs “HFS” in ScDGAT2A and “FFG” in ScDGAT2B with the motif “YFP”. Notably, combined with lipidomic analysis, the expression of ScDGAT2C and GPAT2 enhanced the storage of EPA in TG. Finally, the accumulation of lipid and EPA was further promoted by identifying and continuing to introduce the ScACC, ScACS, ScPDC, and ScG6PD genes from Schizochytrium sp., and the lipid and EPA titer of the final engineered strain reached 2.25 ± 0.03 g/L and 266.44 ± 5.74 mg/L, respectively, which increased by 174.39% (0.82 ± 0.02 g/L) and 282.27% (69.70 ± 0.80 mg/L) compared to the initial strain, respectively. Conclusion This study shows that the expression of lipid synthesis genes from Schizochytrium sp. in Y. lipolytica effectively improves the synthesis of lipids and EPA, which provided a promising target for EPA-enriched microbial oil production.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica

Jia,

Zhang,

et al. 2024

Biotechnol Biofuels

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“…Metabolism of aspartate and glycine produces serine that is involved in the synthesis of phospholipids, a part of the total lipids. Phospholipids in turn can be used to produce tag in diverse species including thraustochytrids (Dahlqvist et al, 2000; Zhang et al, 2023). In the yeast S. cerevisiae , serine supplementation of culture media has enhanced serine conversion to pyruvate (Lee et al, 2013), an important metabolite contributing to general metabolic enhancement.…”

Section: Discussionmentioning

confidence: 99%

Genome‐scale metabolic modeling of Thraustochytrium sp. RT2316‐16: Effects of nutrients on metabolism

Shene,

Leyton,

Flores

et al. 2024

Biotech & Bioengineering

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Marine thraustochytrids produce metabolically important lipids such as the long‐chain omega‐3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316‐16. A reconstructed metabolic network based on the annotated genome of RT2316‐16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol‐free biomass in the genome‐scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316‐16 in different experimental conditions.

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“…DGAT is divided into the DGAT1, 2, and 3 families based on their sequence similarities. Lan et al found that four DGAT2 homologs are present in Aurantiochytrium , tentatively named DGAT2A, B, C, and D ( 16 ). They showed that DGAT2A, C, and D are TG synthases by heterologous expression in budding yeast.…”

Section: Introductionmentioning

confidence: 99%

Characterization of thraustochytrid-specific sterol O -acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186

Ishibashi,

Sadamitsu,

Fukahori

et al. 2023

Appl Environ Microbiol

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Thraustochytrids are marine microorganisms expected to produce useful lipids. They synthesize polyunsaturated fatty acids and sterols and store them in lipid droplets as a form of triacylglycerol (TG) and sterol ester (SE), respectively. TG is synthesized by diacylglycerol O -acyltransferase (DGAT). There are several DGAT2 homologs in Aurantiochytrium limacinum . This study indicated that DGAT2C and DGAT2D are SE synthase and TG synthase, respectively, by disrupting their corresponding genes in A. limacinum mh0186. DGAT2C is revealed as thraustochytrid-specific acyl-CoA:sterol- O -acyltransferase by performing in vivo and in vitro assays after heterologous expression in Saccharomyces cerevisiae . DGAT2C and DGAT2D localized mainly to the endoplasmic reticulum (ER) and the lipid droplet, respectively, and the two of the N-terminal domains unique to DGAT2C were essential for ER localization and SE synthesis in A. limacinum mh0186. Interestingly, the study also found that deletion of the first eight transmembrane domains in the unique N-terminal region of DGAT2C increased SE and total sterol productivity when expressed in DGAT2C-deficient A. limacinum mh0186. In addition, intermediates such as Δ7-cholesterol (provitamin D3) also accumulated in SE by expression of the N-terminal-truncated DGAT2C, along with cholesterol and Δ7-stigmasterol, a major phytosterol in A. limacinum . This study proves that DGAT2C is an ER-localized SE synthase and that its suitable N-terminal deletion can increase sterol production in thraustochytrids. IMPORTANCE Since the global market for sterols and vitamin D are grown with a high compound annual growth rate, a sustainable source of these compounds is required to keep up with the increasing demand. Thraustochytrid is a marine oleaginous microorganism that can synthesize several sterols, which are stored as SE in lipid droplets. DGAT2C is an unconventional SE synthase specific to thraustochytrids. Although the primary structure of DGAT2C shows high similarities with that of DGAT, DGAT2C utilizes sterol as an acceptor substrate instead of diacylglycerol. In this study, we examined more detailed enzymatic properties, intracellular localization, and structure-activity relationship of DGAT2C. Furthermore, we successfully developed a method to increase sterol and provitamin D3 productivity of thraustochytrid by more than threefold in the process of elucidating the function of the DGAT2C-specific N-terminal region. Our findings could lead to sustainable sterol and vitamin D production using thraustochytrid.

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Cocktail biosynthesis of triacylglycerol by rational modulation of diacylglycerol acyltransferases in industrial oleaginous Aurantiochytrium

Cited by 12 publications

References 57 publications

Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica

Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica

Genome‐scale metabolic modeling of Thraustochytrium sp. RT2316‐16: Effects of nutrients on metabolism

Characterization of thraustochytrid-specific sterol O -acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186

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