Cytochrome P450 Mining for Bufadienolide Diversification.

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1ß-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

[Advances in the bioaugmentation-assisted remediation of petroleum contaminated soil].

Bioremediation is considered as a cost-effective, efficient and free-of-secondary-pollution technology for petroleum pollution remediation. Due to the limitation of soil environmental conditions and the nature of petroleum pollutants, the insufficient number and the low growth rate of indigenous petroleum-degrading microorganisms in soil lead to long remediation cycle and poor remediation efficiency. Bioaugmentation can effectively improve the biodegradation efficiency. By supplying functional microbes or microbial consortia, immobilized microbes, surfactants and growth substrates, the remediation effect of indigenous microorganisms on petroleum pollutants in soil can be boosted. This article summarizes the reported petroleum-degrading microbes and the main factors influencing microbial remediation of petroleum contaminated soil. Moreover, this article discusses a variety of effective strategies to enhance the bioremediation efficiency, as well as future directions of bioaugmentation strategies.

Characterization of an efficient estrogen-degrading bacterium Stenotrophomonas maltophilia SJTH1 in saline-, alkaline-, heavy metal-contained environments or solid soil and identification of four 17ß-estradiol-oxidizing dehydrogenases.

The efficient bioremediation of estrogen contamination in complex environments is of great concern. Here the strain Stenotrophomonas maltophilia SJTH1 was found with great and stable estrogen-degradation efficiency even under stress environments. The strain could utilize 17ß-estradiol (E2) as a carbon source and degrade 90% of 10 mg/L E2 in a week; estrone (E1) was the first degrading intermediate of E2. Notably, diverse pH conditions (3.0-11.0) and supplements of 4% salinity, 6.25 mg/L of heavy metal (Cd2+ or Cu2+), or 1 CMC of surfactant (Tween 80/ Triton X-100) had little effect on its cell growth and estrogen degradation. The addition of low concentrations of copper and Tween 80 even promoted its E2 degradation. Bioaugmentation of strain SJTH1 into solid clay soil achieved over 80% removal of E2 contamination (10 mg/kg) within two weeks. Further, the whole genome sequence of S. maltophilia SJTH1 was obtained, and a series of potential genes participating in stress-tolerance and estrogen-degradation were predicted. Four dehydrogenases similar to 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) were found to be induced by E2, and the four heterogenous-expressed enzymes could oxidize E2 into E1 efficiently. This work could promote bioremediation appliance potential with microorganisms and biodegradation mechanism study of estrogens in complex real environments.

Isolation and characterization of Pseudomonas aeruginosa strain SJTD-2 for degrading long-chain n-alkanes and crude oil.

OBJECTIVE: Oil pollution poses a severe threat to ecosystems, and bioremediation is considered as a safe and efficient alternative to physicochemical. METHODS: for eliminating this contaminant. In this study, a gram-negative bacteria strain SJTD-2 isolated from oil-contaminated soil was found capable of utilizing n-alkanes and crude oil as sole energy sources. The efficiency of this strain in degrading these pollutants was analyzed. METHODS: Strain SJTD-2 was identified on the basis of its phenotype, its physiological features, and a comparative genetic analysis using 16S rRNA sequence. Growth of strain SJTD-2 with different carbon sources (n-alkanes of different lengths and crude oil) was assessed, and the gas chromatography-mass spectrometry method was used to analyze the degradation efficiency of strain SJTD-2 for n-alkanes and petroleum by detecting the residual n-alkane concentrations. RESULTS: Strain SJTD-2 was identified as Pseudomonas aeruginosa based on the phenotype, physiological features, and 16S rRNA sequence analysis. This strain can efficiently decompose medium-chain and long-chain n-alkanes (C10-C26), and petroleum as its sole carbon sources. It preferred the long-chain n-alkanes (C18-C22), and n-docosane was considered as the best carbon source for its growth. In 48 h, 500 mg/L n-docosane could be degraded completely, and 2 g/L n-docosane was decomposed to undetectable levels within 72 h. Moreover, strain SJTD-2 could utilize about 88% of 2 g/L crude oil in 7days. Compared with other alkane-utilizing strains, strain SJTD-2 showed outstanding degradation efficiency for long-chain n-alkanes and high tolerance to petroleum at elevated concentrations. CONCLUSION: The isolation and characterization of strain SJTD-2 would help researchers study the mechanisms underlying the biodegradation of n-alkanes, and this strain could be used as a potential strain for environmental governance and soil bioremediation.

Genome sequence of Pseudomonas aeruginosa strain SJTD-1, a bacterium capable of degrading long-chain alkanes and crude oil.

Pseudomonas aeruginosa strain SJTD-1 can utilize long-chain alkanes, diesel oil, and crude oil as sole carbon sources. We report the draft genome sequence of strain SJTD-1 (6,074,058 bp, with a GC content of 66.83%) and major findings from its annotation, which could provide insights into its petroleum biodegradation mechanism.