Enhanced cold active lipase production by metagenomic library recombinant clone CALIP3 with a step-wise temperature and dissolved oxygen level control strategy

A metagenomic library recombinant clone CAPL3, an Escherichia coli strain generated by transformed with metagenomic library from deep-sea sediments, can efficiently produce cold active lipase. The effects of both temperature and dissolved oxygen（DO） on cold active lipase production by batch culture of metagenomic library recombinant clone（CAPL3） from deep-sea sediment were investigated. First, a two-stage temperature control strategy was developed, in which the temperature was kept at 34 ℃ for the first 15 h, and then switched to30 ℃. The cold active lipase activity and productivity reached 315.2 U·ml^-1and 8.08 U·ml^-1·h^-1, respectively,increased by both 14.5% compared to the results obtained with temperature controlled at 30℃. In addition, different DO control modes were conducted, based on the data obtained from the different DO control strategies and analysis of kinetics parameters at different DO levels. A step-wise temperature and DO control strategy were developed to improve lipase production, i.e., temperature and DO level were controlled at 34℃, 30% during 0–15 h;30 ℃, 30% during 15–18 h, and 30 ℃, 20% during 18–39 h. With this strategy, the maximum lipase activity reached 354.6 U·ml^-1at 39 h, which was 28.8% higher than that achieved without temperature and DO control（275.3 U·ml^-1）.

Construction of a Metagenomic DNA Library of Sponge Symbionts and Screening of Antibacterial Metabolites

To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the library was 20 kb. This library was screened for antibiotic activity using paper disc assaying. Two clones displayed the antibacterial activity against Micrococcus tetragenus. The metabolites of these two clones were analyzed through HPLC. The result showed that their metabolites were quite different from those of the host E. coli DNA and the host containing vector pHZ132. This study may present a new approach to exploring bioactive metabolites of sponge symbionts.

Screening an Na^+/H^+ Antiporter Gene from the Halophiles Colonizing in the Dagong Ancient Brine Well of Zigong City,China

[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.

Distribution and diversity of endophytic fungi in Gentiana rigescens and cytotoxic activities

Objective:In the present study,Gentiana rigescens was screened for fungi communities to clarify their diversity and community assemblage in hosts.Meanwhile,the identification and activity assays of the strains were also conducted.Methods:By culture-dependent(endophytic fungi isolations from plant sections)and culture-independent(metagenomic library and cloning from plant sections)techniques,fungi communities were studied.The metagenomic library was generated using direct DNA isolation of whole plants,plant radixes,plant stems,plant leaves,plant flowers and soils around the plant.Meanwhile,endophytes were isolated from all parts ofG.rigescens plants.After fermentation of the fungi isolations,all the isolates were evaluated for their cytotoxicity against four kinds of human cancer cell lines(HCT116,BEL7404,A549,MDA-MB-231).Results:Eventually,200 strains were isolated and 103 strains were further identified through the internal transcribed spacer(ITS,ITS1 and ITS2 regions)sequence by using the universal primers ITS5 and ITS4.A total of 59,106 fungal sequences corresponding to 374 putative operational taxonomic units(OTU)were identified by 454 pyrosequencing.Through 454 pyrosequencing,the main fungal genera were Sebacina,Botrytis,Mycosphaerella,Boletus and Gibberella,and the major fungal genera which were directly isolated were Aspergillus,Fusarium,Penicillium and Alternaria.Activity assays showed strains 5-26(Aspergillus sp.)and 6-2(Fusarium avenaceum)had the outstanding cytotoxicity to all the tested cell lines with IC5o values<5μg/mL.Conclusion:This study revealed the abundance of endogenetic fungal resources and a variety of genetic information in G.rigescens by high-throughput 454 sequencing technology and fungi isolation methods.Activity assays indicated that endophytes were a promising natural source of potential anticancer agents.