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In this study, six lipase-producing strains from food sources were screened for their ability to produce lipase. Among them, Staphylococcus epidermidis N30 and Yarrowia lipolytica C11 demonstrated high enzyme activities of 1.07 and 0.84 U/mL, respectively. They had the following enzymatic properties: optimum reaction temperature of 50 ℃ and pH of 8.0. The optimum substrate for S. epidermidis N30 was C4 ester, while that for Y. lipolytica C11 was C8 ester. The inoculation of sour meat with S. epidermidis N30 or Y. lipolytica C11 effectively enhanced the production of flavor substances, especially ethyl butyrate, ethyl caproate, ethyl caprylate, ethyl caprate, nonanal and 1-octen-3-ol, thus promoting the production of fruity, floral, wine-like, mushroom-like and fatty aromas in sour meat.

[Purpose/Significance] Combing the relevant influencing factors of the citation frequency of a single paper and the research status of the prediction of the citation frequency, this paper provides a comprehensive and systematic cognitive framework from the perspective of the involvement of scientific researchers and scientific research institutions in such research. [Method/Process] Using the literature research method, through the systematic combing of the existing literature, this paper summarized the relevant contents and characteristics of the influencing factors, research objects and research methods of citation prediction, compared and analyzed different methods by means of list, and summarized the common problems and some innovative solutions of the existing research. [Result/Conclusion] In the process of systematic combing and summarizing, it is found that the causal relationship between influencing factors and prediction results is not clear, the research sample data is lack of diversity, the relationship between the applicability of research results and prediction cycle is not clear, and the interpretability of model evaluation is weak. Therefore, we should improve the follow-up research quality from the aspects of solving the preconditions of the problem, selecting targeted samples, improving the extraction methods of influencing factors, and using mathematical thinking mode for modeling.

Objective: To construct a phosphate starvation derepressed expression vector for functional integration and expression of exogenous genes in Rhodosporidium toruloides., Methods: Sodium: inorganic phosphate symporter promoter pPHO89 and heat shock protein terminator tHSP were predicted by bioinformatics assay. DNA fragments of the two elements were amplified from the cDNA of the oleaginous yeast R. toruloides NP11. Then the lyceric acid kinase promoter pPGK and terminator tNOS in vector pZPK-pPGK-hyg-tNOS were replaced by pPHO89 and tHSP, respectively, using restriction free (RF) cloning method. Hygromycin phosphotransferase gene hyg was retained and as a report gene. The resulting vector pZPK-pPHO89-hyg-tHSP was transformed into R. toruloides using agrobacterium-mediated transformation (ATMT). The function of the pPHO89 promoter and the tHSP terminator can be confirmed by the transformant harboring the hygromycin resistance. Furthermore, we developed new inducible vectors containing two expression cassettes driven by the constitutive pPGK promoter and inducible pPHO89 promoter., Results: pPHO89 was regulated by phosphate and activated when the recombinant R. toruloides strains were grown under phosphate-limited condition., Conclusion: pPHO89 is an ideal promoter in terms of regulation by phosphate, comparative response strength, simplicity, and cost effectiveness. These ATMT vectors can provide useful tools for rational engineering of oleaginous yeast to produce fatty acid derived biofuels and biochemicals.

Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids. It is one of the most important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21 (DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.