Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment.

BACKGROUND: Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. RESULTS: The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a ß-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. CONCLUSION: KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

[Isolation of Raoultella sp. sari01 and Its Heterotrophic Nitrification-Aerobic Denitrification Characteristics].

The genus of Raoultella belongs to the family of Enterobacteriaceae, and some strains of Raoultella sp. have the function of degrading chemical pollutants in the environment. A Raoultella sp. strain was isolated from activated sludge through enrichment cultured with heterotrophic nitrification medium. This strain was named Raoultella sp. sari01. Single-factor and response surface methodology experiments results showed that efficient heterotrophic nitrification of strain sari01 occurred with sodium citrate as the carbon source, at pH of 7.0-7.5, temperature of 30℃, C/N ratio of 15, inoculation volume of 7.5% and loading volume of 50 mL, while the removal rate of nitrogen was 99.9%, of which 33.7% was converted to gaseous product and escaped to air, and the residual nitrogen was fixed in cell biomass. Using nitrite and nitrate as the sole nitrogen source, the nitrogen degradation ratios were 98.4% and 65.2%, respectively. Hence, strain sari01 could remove nitrogen by heterotrophic nitrification-aerobic denitrification independently, quickly and effectively, which demonstrated that strain sari01 has the potential to be used in wastewater treatment.

[Effect of magnesium deficiency on photosynthetic physiology and triacylglyceride (TAG) accumulation of Chlorella vulgaris].

As an excellent biological resource, Chlorella has wide applications for production of biofuel, bioactive substances and water environment restoration. Therefore, it is very important to understand the photosynthetic physiology characteristics of Chlorella. Magnesium ions play an important role in the growth of microalgae, not only the central atom of chlorophyll, but also the cofactor of some key enzyme in the metabolic pathway. A laboratory study was conducted to evaluate the effects of magnesium deficiency on several photosynthetic and physiological parameters and the triacylglyceride (TAG) accumulation of the green alga, Chlorella vulgaris, in the photoautotrophic culture process. Chlorella vulgaris biomass, protein, chlorophyll a and chlorophyll b contents decreased by 20%, 43.96%, 27.52% and 28.07% in response to magnesium deficiency, while the total oil content increased by 19.60%. Moreover, magnesium deficiency decreased the maximal photochemical efficiency F(v)/F(m) by 22.54%, but increased the non-photochemical quenching parameters qN. Our results indicated the decline of chlorophyll caused by magnesium, which affected the photosynthesis efficiency, lead to the growth inhibition of Chlorella vulgaris and affected the protein synthesis and increased the triacylglyceride (TAG) accumulation.

Effect of zibu shenjing fang (see text) on growth and development of the mouse with kidney-essence insufficiency and study on the mechanism.

OBJECTIVE: To investigate effects of Zibu Shenjing Fang (see text) on growth and development of the mouse with insufficiency ofkidney-essence and the mechanism. METHODS: Total 50 mice were randomly divided into a normal group, a model group, a Jingui Shenqi Wan (see text) group, a Zibu Shenjing Fang high dose group and a Zibu Shenjing Fang low dose group, 10 mice in each group. The kidney-essence insufficiency mouse model was established by use of threat-injuring the kidney combined with over-fatigue. At the same time of modeling, the mice in the model group were intragastrically administrated with saline 20 mL x kg(-1) x d(-1), in the Jingui Shenqi Wan group with suspension of the Jingui Shenqi Wan 2.7 g x kg(-1) x d(-1), in the Zibu Shenjing Fang high dose group with Zibu Shenjing Fang 20 g x kg(-1) x d(-1) and in the Zibu Shenjing Fang low dose group with Zibu Shenjing Fang 10 g x kg(-1) x d(-1), for 21 consecutive days. The general state was observed, the body weight was weighted, and serum growth hormone (GH) and insulin-like growth factor-1 (IGF-1) contents were detected. RESULTS: Compared with model group, Zibu Shenjing Fang groups and Jingui Shenqi Wan group could improve manifestation of the mouse with kidney-essence insufficiency, increase body weight of the mouse and serum GH and IGF-1 contents, especially in the high dose group. CONCLUSION: Zibu Shenjing Fang gives play to the function of tonifying the kidney and replenishing essence through regulating GH and IGF-1 levels, so as to influence growth and development of the mouse.

Modeling human exposures to air pollution control (APC) residues released from landfills in England and Wales.

Human exposures to air pollution control (APC) residues released from 6 landfills were modeled and assessed. Following a qualitative risk characterisation, direct and indirect exposures were quantified. Site-specific air dispersion modeling was conducted for PM(10), PCDDs/PCDFs, Pb, Cd, As and Cr(VI) concentrations at the closest residential points of exposure for 4 landfill sites accepting, in total, 75% w/w of the APC residues disposed of in 2000-2001 (UK). Inhalation risks, assessed by reference to air quality standards at residential exposure points, were assessed as insignificant. Preliminary modeling suggested that indirect exposures from PCDDs/PCDFs at the 95th percentile level for the site where APC deposition rates were highest could potentially exceed the tolerable daily soil intake (TDSI) but this warrants further study given the model limitations. These results offer an initial screen of the significance of potential risks from APC disposal, which is of value in addressing concerns about the uncertainty of potential risks to human health from bulk APC disposal at strategic locations.

Cloning and Nucleotide Sequencing of Prolyl Endopeptidase Gene from Aeromonas punctata subsp. punctata.

Prolyl endopeptidase activity was found in Aeromonas punctata subsp. Punctata. The genomic DNA was partially digested with EcoRI and the recovered 8-16 kb DNA fragments were inserted into the EcoRI site of plasmid pUC19, and were transformated into Escherichia coli DH5alpha. The resulted clones were screened by using Benzyloxycarbonyl-Gly-Pro-beta-naphthylamide, the specific substrate of prolyl endopeptidase and a positive clone was obtained. The 12 kb insertion fragment of recombinant plasmid was digested with HincII and subcloned. The PEP gene was found in the 3.5 kb HincII/EcoRI fragment. Nucleotide sequence of the gene was completely sequenced by Auto Sequencer. The complete gene consisted of 2 073 bp corresponding to 690 amino acid residues with a calculated molecular weight of 76 467 Da. The amino acid sequence was 92.3% 53.2% 33.5% 33.2% and 20.5% homologous to those of Aeromonas hydrophila, Flavobacterium meningosepticum, porcine brain, human lymphocytes and Pyrococcus furiosus respectively. From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser(538) Asp(622) and His(657).