Heterologous biosynthesis of taraxerol by engineered Saccharomyces cerevisiae.

Taraxerol is an oleanane-type pentacyclic triterpenoid compound distributed in many plant species that has good effects on the treatment of inflammation and tumors. However, the taraxerol content in medicinal plants is low, and chemical extraction requires considerable energy and time, so taraxerol production is a problem. It is a promising strategy to produce taraxerol by applying recombinant microorganisms. In this study, a Saccharomyces cerevisiae strain WKde2 was constructed to produce taraxerol with a titer of 1.85 mg·l-1, and the taraxerol titer was further increased to 12.51 mg·l-1 through multiple metabolic engineering strategies. The endoplasmic reticulum (ER) size regulatory factor INO2, which was reported to increase squalene and cytochrome P450-mediated 2,3-oxidosqualene production, was overexpressed in this study, and the resultant strain WTK11 showed a taraxerol titer of 17.35 mg·l-1. Eventually, the highest reported titer of 59.55 mg·l-1 taraxerol was achieved in a 5 l bioreactor. These results will serve as a general strategy for the production of other triterpenoids in yeast.

Biosynthesis of valerenic acid by engineered Saccharomyces cerevisiae.

OBJECTIVE: To produce valerenic acid (VA) in Saccharomyces cerevisiae by engineering a heterologous synthetic pathway. RESULT: Valerena-4,7(11)-diene synthase (VDS) derived from Valeriana officinalis (valerian) was expressed in S. cerevisiae to generate valerena-4,7(11)-diene as the precursor of VA. By overexpressing the key genes of the mevalonate pathway ERG8, ERG12 and ERG19, and integrating 4 copies of MBP (maltose-binding protein)-VDS-ERG20 gene expression caskets into the genome, the production of valerena-4,7(11)-diene was improved to 75 mg/L. On this basis, the cytochrome P450 monooxygenase LsGAO2 derived from Lactuca sativa was expressed to oxidize valerena-4,7(11)-diene to produce VA, and the most effective VA production strain was used for fermentation. The yield of VA reached 2.8 mg/L in the flask and 6.8 mg/L in a 5-L bioreactor fed glucose. CONCLUSIONS: An S. cerevisiae strain was constructed and optimized to produce VA, but the valerena-4,7(11)-diene oxidation by LsGAO2 is still the rate-limiting step for VA synthesis that needs to be further optimized in future studies.

[Effect of electroacupuncture combined with caudal epidural injection on functional rehabilitation of patients with lumbar hernia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with caudal epidural injection on subjective pain, walking capability, lumbar flexibility and muscle strength in patients with lumbar disc hernia (LDH). METHODS: Sixty LDH patients were randomly allocated to the control group and the research group. The patients of the control group received ultrasound guided caudal epidural injection, and those of the research group received EA combined with ultrasound guided caudal epidural injection. Bilateral Jiaji (EX-B2) and adjunct points Guanyuanshu (BL26), Shenshu (BL23), Chengfu (BL36), Huantiao (GB30), Zhibian (BL54), etc. on the affected side were stimulated with EA (2 Hz/16 Hz, 5-8 mA) for 30 min each time, once every other day for 4 weeks, with 2 days' rest between every two weeks. The patients' pain was evaluated by using visual analogue scale (VAS), walking capability assessed by timed-up and go (TUG) test (time of walking back and forth in 3 m distance), lumbar flexibility (range of motion, ROM) detected by using an inclinometer and the strength of the lumbar flexor and extensor determined by using a push-pull dynamometer. RESULTS: After the treatment, self-comparison showed that the VAS score and TUG-measured time in both groups were significantly decreased (P<0.01, P<0.05), and the post-bucking ROM and extension ROM in the research group, and the lumbar flexor and extensor muscle strength in both groups were obviously increased compared with their own pre-treatment (P<0.05). Comparison between two groups showed that the VAS score and TUG-measured time of the research group were significantly lower than those of the control group (P<0.01), while the lumbar flexor's ROM as well as the extensor's strength were significantly higher in the research group than in the control group (P<0.05). CONCLUSION: For patients with LDH, EA combined with caudal epidural injection can alleviate pain, improve the walking capability, lumbar flexibility and strength of the lumbar extensor, and the therapeutic effect of the combined treatment is significantly better than that of simple caudal epidural injection.

Research progress in bioremediation of petroleum pollution.

With the enhancement of environmental protection awareness, research on the bioremediation of petroleum hydrocarbon environmental pollution has intensified. Bioremediation has received more attention due to its high efficiency, environmentally friendly by-products, and low cost compared with the commonly used physical and chemical restoration methods. In recent years, bacterium engineered by systems biology strategies have achieved biodegrading of many types of petroleum pollutants. Those successful cases show that systems biology has great potential in strengthening petroleum pollutant degradation bacterium and accelerating bioremediation. Systems biology represented by metabolic engineering, enzyme engineering, omics technology, etc., developed rapidly in the twentieth century. Optimizing the metabolic network of petroleum hydrocarbon degrading bacterium could achieve more concise and precise bioremediation by metabolic engineering strategies; biocatalysts with more stable and excellent catalytic activity could accelerate the process of biodegradation by enzyme engineering; omics technology not only could provide more optional components for constructions of engineered bacterium, but also could obtain the structure and composition of the microbial community in polluted environments. Comprehensive microbial community information lays a certain theoretical foundation for the construction of artificial mixed microbial communities for bioremediation of petroleum pollution. This article reviews the application of systems biology in the enforce of petroleum hydrocarbon degradation bacteria and the construction of a hybrid-microbial degradation system. Then the challenges encountered in the process and the application prospects of bioremediation are discussed. Finally, we provide certain guidance for the bioremediation of petroleum hydrocarbon-polluted environment.

Structural elucidation and antidiabetic activity of fucosylated chondroitin sulfate from sea cucumber Stichopus japonicas.

A fucosylated chondroitin sulfate was isolated from the body wall of sea cucumber Stichopus japonicus (FCSsj), whose structure was characterized by NMR spectroscopy and HILIC-FTMS. At the ratio of 1.00:0.26:0.65, three fucosyl residues were found: 2,4-disulfated-fucose (Fuc2,4S), 4-sulfated-fucose (Fuc4S) and 3,4-disulfated-fucose (Fuc3,4S), which were only linked to the O-3 of glucuronic acid residues (GlcA). Besides mono-fucosyl moieties, di-fucosyl branches, namely Fuc2,4Sα(1→3)Fuc4S, were also found to be attached to the O-3 of GlcA. The antidiabetic activity of FCSsj was evaluated using glucosamine induced insulin resistant (IR) Hep G2 cells in vitro. It was found that FCSsj significantly promoted the glucose uptake and glucose consumption of IR-Hep G2 cells in a dose-dependent manner, and could alleviate the cell damage. Furthermore, FCSsj could promote the glycogen synthesis in the glucosamine-induced IR-Hep G2 cells. These results provided a supplement for studying the antidiabetic activity of FCSsj.