No abstract

Terpenoids are an important class of secondary metabolites that play an important role in food, agriculture, and other fields. Microorganisms are rapidly emerging as a promising source for the production of terpenoids. As an oleaginous yeast, Yarrowia lipolytica contains a high lipid content which indicates that it must produce high amounts of acetyl-CoA, a necessary precursor for the biosynthesis of terpenoids. Y. lipolytica has a complete eukaryotic mevalonic acid (MVA) pathway but it has not yet seen commercial use due to its low productivity. Several metabolic engineering strategies have been developed to improve the terpenoids production of Y. lipolytica, including developing the orthogonal pathway for terpenoid synthesis, increasing the catalytic efficiency of terpenoids synthases, enhancing the supply of acetyl-CoA and NADPH, expressing rate-limiting genes, and modifying the branched pathway. Moreover, most of the acetyl-CoA is used to produce lipid, so it is an effective strategy to strike a balance of precursor distribution by rewiring the lipid biosynthesis pathway. Lastly, the latest developed non-homologous end-joining strategy for improving terpenoid production is introduced. This review summarizes the status and metabolic engineering strategies of terpenoids biosynthesis in Y. lipolytica and proposes new insights to move the field forward.

Lung cancer is the leading cause of cancer‑related mortality worldwide. Over half of lung cancer cases are diagnosed after metastasis, for which the median survival time is approximately 8 months. microRNAs (miRNAs), which are a class of single‑stranded endogenous non‑coding RNAs, are likely to be involved in most biological processes. miR‑133 plays roles in cardiac development and disease, and recent studies showed that miR‑133 is downregulated in various human malignancies, such as bladder and lung cancer. However, its detailed role in the processes of cancer remains to be determined. In the present study, we found that in the lung cancer cell lines A549 and NCI‑H1299 overexpression of miR‑133a suppressed cell proliferation, migration and invasion. The miR‑133a‑induced suppression of cell migration and invasion can be reversed by miR‑133a‑specific inhibitor. According to the mRNA sequence, matrix metalloproteinase (MMP)‑14, which is an important regulator of metastasis, is a predicted target of miR‑133a. This was confirmed by dual luciferase reporter assay. Moreover, miR‑133a overexpression decreases the mRNA and protein levels of MMP‑14. Collectively, these results suggest that miR‑133a may inhibit lung cancer metastasis by targeting MMP‑14 and may be used as an anti‑metastatic therapy in lung cancer patients.

Background The oleaginous microorganism Schizochytrium sp. is widely used in scientific research and commercial lipid production processes. However, low glucose-to-lipid conversion rate (GLCR) and low lipid productivity of Schizochytrium sp. restrict the feasibility of its use. Results Orlistat is a lipase inhibitor, which avoids triacylglycerols (TAGs) from hydrolysis by lipase. TAGs are the main storage forms of fatty acids in Schizochytrium sp. In this study, the usage of orlistat increased the GLCR by 21.88% in the middle stage of fermentation. Whereas the productivity of lipid increased 1.34 times reaching 0.73 g/L/h, the saturated fatty acid and polyunsaturated fatty acid yield increased from 21.2 and 39.1 to 34.9 and 48.5 g/L, respectively, indicating the advantages of using a lipase inhibitor in microbial lipids fermentation. Similarly, the system was also successful in Thraustochytrid Aurantiochytrium. The metabolic regulatory mechanisms stimulated by orlistat in Schizochytrium sp. were further investigated using transcriptomics and metabolomics. The results showed that orlistat redistributed carbon allocation and enhanced the energy supply when inhibiting the TAGs’ degradation pathway. Therefore, lipase in Schizochytrium sp. prefers to hydrolyze saturated fatty acid TAGs into the β-oxidation pathway. Conclusions This study provides a simple and effective approach to improve lipid production, and makes us understand the mechanism of lipid accumulation and decomposition in Schizochytrium sp., offering new guidance for the exploitation of oleaginous microorganisms.

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.