RÉSUMÉ
Schizochytrium sp. hasreceived much attention for itsability to synthesize and accumulate high-level docosahexaenoic acid (DHA), which can reach nearly 40 % of total fatty acids. In this study, the titer of DHA in Schizochytrium sp. was successfully improved by enhancing DHA storage through overexpressing the diacylglycerol acyltransferase (ScDGAT2C) gene, as well as optimizing the supply of precursors and cofactors required for DHA synthesis by response surface methodology. Notably, malic acid, citric acid, and biotin showed synergistic and time-dependent effects on DHA accumulation. The maximum lipid and DHA titers of the engineered Schizochytrium sp. strain reached 84.28 ± 1.02 g/L and 42.23 ± 0.69 g/L, respectively, with the optimal concentration combination (1.62 g/L malic acid + 0.37 g/L citric acid + 8.28 mg/L biotin) were added 48 h after inoculation. This study provides an effective strategy for improving lipid and DHA production in Schizochytrium sp.
Sujet(s)
Acides gras , Malates , Straménopiles , Fermentation , Acide docosahexaénoïque , Biotine , Straménopiles/génétique , Acide citriqueRÉSUMÉ
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.
RÉSUMÉ
Due to the complexity of metabolic and regulatory networks in microorganisms, it is difficult to obtain robust phenotypes through artificial rational design and genetic perturbation. Adaptive laboratory evolution (ALE) engineering plays an important role in the construction of stable microbial cell factories by simulating the natural evolution process and rapidly obtaining strains with stable traits through screening. This review summarizes the application of ALE technology in microbial breeding, describes the commonly used methods for ALE, and highlights the important applications of ALE technology in the production of lipids and terpenoids in yeast and microalgae. Overall, ALE technology provides a powerful tool for the construction of microbial cell factories, and it has been widely used in improving the level of target product synthesis, expanding the range of substrate utilization, and enhancing the tolerance of chassis cells. In addition, in order to improve the production of target compounds, ALE also employs environmental or nutritional stress strategies corresponding to the characteristics of different terpenoids, lipids, and strains.
Sujet(s)
Microalgues , Terpènes , Terpènes/métabolisme , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , Microalgues/génétique , Lipides/génétique , Génie métabolique/méthodesRÉSUMÉ
It is well known that polyunsaturated fatty acids (PUFAs) in Schizochytrium sp. are mainly synthesized via the polyketide synthase (PKS) pathway. However, the specific mechanism of PKS in fatty acid synthesis is still unclear. In this work, the functions of ORFA, ORFB, ORFC, and their individual functional domain genes on fatty acid synthesis were investigated through heterologous expression in Yarrowia lipolytica. The results showed that the expression of ORFA, ORFB, ORFC, and their individual functional domains all led to the increase of the very long-chain PUFA content (mainly eicosapentaenoic acid). Furthermore, the transcriptomic analysis showed that except for the 3-ketoacyl-ACP synthase (KS) domain of ORFB, the expression of an individual functional domain, including malonyl-CoA: ACP acyltransferase, 3-hydroxyacyl-ACP dehydratase (DH), 3-ketoacyl-ACP reductase, and KS domains of ORFA, acyltransferase domains of ORFB, and two DH domains of ORFC resulted in upregulation of the tricarboxylic acid cycle and pentose phosphate pathway, downregulation of the triacylglycerol biosynthesis, fatty acid synthesis pathway, and ß-oxidation in Yarrowia lipolytica. These results provide a theoretical basis for revealing the function of PKS in fatty acid synthesis in Y. lipolytica and elucidate the possible mechanism for PUFA biosynthesis.
Sujet(s)
Polyketide synthases , Yarrowia , Polyketide synthases/métabolisme , Yarrowia/métabolisme , Acyltransferases/métabolisme , Acides gras insaturés/métabolisme , Acide eicosapentanoïque/métabolisme , Acides gras/métabolismeRÉSUMÉ
Eicosapentaenoic Acid (EPA) is an essential ω-3 polyunsaturated fatty acid for human health. Currently, high-quality EPA production is largely dependent on the extraction of fish oil, but this unsustainable approach cannot meet its rising market demand. Biotechnological approaches for EPA production from microorganisms have received increasing attention due to their suitability for large-scale production and independence of the seasonal or climate restrictions. This review summarizes recent research on different microorganisms capable of producing EPA, such as microalgae, bacteria, and fungi, and introduces the different EPA biosynthesis pathways. Notably, some novel engineering strategies have been applied to endow and improve the abilities of microorganisms to synthesize EPA, including the construction and optimization of the EPA biosynthesis pathway, an increase in the acetyl-CoA pool supply, the increase of NADPH and the inhibition of competing pathways. This review aims to provide an updated summary of EPA production.
Sujet(s)
Acides gras omega-3 , Microalgues , Voies de biosynthèse , Acide eicosapentanoïque/métabolisme , Acides gras omega-3/métabolisme , Génie métabolique , Microalgues/métabolismeRÉSUMÉ
Owing to the important physiological functions, polyunsaturated fatty acids (PUFAs) play a vital role in protecting human health, such as preventing cancer, cardiovascular disease, and diabetes. Specifically, Yarrowia lipolytica has been identified as the most popular non-conventional oleaginous yeast, which can accumulate the abundant intracellular lipids, indicating that has great potential as an industrial host for production of PUFAs. Notably, some novel engineering strategies have been applied to endow and improve the abilities of Y. lipolytica to synthesize PUFAs, including construction and optimization of PUFAs biosynthetic pathways, improvement of preucrsors acetyl-coA and NADPH supply, inhibition of competing pathways, knockout of ß-oxidation pathways, regulation of oxidative stress defense pathways, and regulation of genes involved in upstream lipid metabolism. Besides, some bypass approaches, such as strain mating, evolutionary engineering, and computational model based on omics, also have been proposed to improve the performance of engineering strains. Generally, in this review, we summarized the recent advances in engineering strategies and bypass approaches for improving PUFAs production by Y. lipolytica. In addition, we further summarized the latest efforts of CRISPR/Cas genome editing technology in Y. lipolytica, which is aimed to provide its potential applications in PUFAs production.