Polyunsaturated fatty acids (PUFAs) have been widely applied in the food and medical industry. In this study, malonyl-CoA: ACP transacylase (MAT) was overexpressed through homologous recombination to improve PUFA production in Schizochytrium. The results showed that the lipid and PUFA concentration were increased by 10.1 and 24.5% with MAT overexpression, respectively. Metabolomics analysis revealed that the intracellular tricarboxylic acid cycle was weakened and glucose absorption was accelerated in the engineered strain. In the mevalonate pathway, intracellular carotene content was decreased, and the carbon flux was then redirected toward PUFA synthesis. Furthermore, a glucose fed-batch fermentation was finally performed with the engineered Schizochytrium. The total lipid yield was further increased to 110.5 g/L, 39.6% higher than the wild strain. Docosahexaenoic acid and eicosapentaenoic acid yield were enhanced to 47.39 g/L and 1.65 g/L with an increase of 81.5 and 172.5%, respectively. This study provided an effective metabolic engineering strategy for industrial PUFA production.

The mechanisms underlying gut development, especially peristalsis, are widely studied topics. However, the causes of gut peristalsis-related diseases, especially Opioid-Induced Bowel Dysfunction (OIBD) disorder, have not been well defined. Therefore, our study used zebrafish, a popular model for studying both gut development and peristalsis, and DCFH-DA, a dye that clearly labels the live fish gut lumen, to characterize the formation process of gut lumen as well as the gut movement style in vivo. By applying Loperamide Hydrochloride (LH), the μ-opioid receptor-specific agonist, we established an OIBD-like zebrafish model. Our study found that acetylcholine (ACh) was a key transmitter that derepressed the phenotype induced by LH. Overall, the study showed that the antagonistic role of ACh in the LH-mediated opioid pathway was evolutionarily conserved; moreover, the OIBD-like zebrafish model will be helpful in the future dissection of the molecular pathways involved in gut lumen development and pathology.

Schizochytrium sp. is a kind of marine microalgae with great potential as promising sustainable source of polyunsaturated fatty acids (PUFAs). Polyketide synthase-like (PKS synthase) is supposed to be one of the main ways to synthesize PUFAs in Schizochytrium sp. In order to study the exact relationship between PKS and PUFA biosynthesis, chain length factor (CLF) and dehydrogenase (DH) were cloned from the PKS gene cluster in Schizochytrium sp., then disrupted by homologous recombination. The results showed that DH- and CLF-disrupted strains had significant decreases (65.85 and 84.24%) in PUFA yield, while the saturated fatty acid (SFA) proportion in lipids was slightly increased. Meanwhile, the disruption of CLF decreased the C-22 PUFA proportion by 57.51% without effect on C-20 PUFA accumulation while DH-disrupted mutant decreased the production of each PUFA. Combined with analysis of protein prediction, it indicated that CLF gene exerted an enormous function on the carbon chain elongation in PUFA synthesis, especially for the final elongation from C-20 to C-22 PUFAs. Metabolomics analysis also suggested that the disruption of both genes resulted in the decrease of PUFAs but increase of SFAs, thus weakening glycolysis and tricarboxylic acid (TCA) cycle pathways. This study offers a broad new vision to research the mechanism of PUFA synthesis in Schizochytrium sp.

Background As a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear. Results In this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain. Conclusion Therefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.

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