The mechanisms of how zinc protects the cells against acetic acid toxicity and acts as an antioxidant are still not clear. Here we present results of the metabolic profiling of the eukaryotic model yeast species Saccharomyces cerevisiae subjected to long term high concentration acetic acid stress treatment in the presence and absence of zinc supplementation. Zinc addition decreased the release of reactive oxygen species (ROS) in the presence of chronic acetic acid stress. The dynamic changes in the accumulation of intermediates in central carbon metabolism were observed, and higher contents of intracellular alanine, valine and serine were observed by zinc supplementation. The most significant change was observed in alanine content, which is 3.51-fold of that of the control culture in cells in the stationary phase. Subsequently, it was found that 0.5 g L−1 alanine addition resulted in faster glucose consumption in the presence of 5 g L−1 acetic acid, and apparently decreased ROS accumulation in zinc-supplemented cells. This indicates that alanine exerted its antioxidant effect at least partially through the detoxification of acetic acid. In addition, intracellular glutathione (GSH) accumulation was enhanced by zinc addition, which is related to the protection of yeast cells from the oxidative injury caused by acetic acid. Our studies revealed for the first time that zinc modulates cellular amino acid metabolism and redox balance, especially biosynthesis of alanine and glutathione to exert its antioxidant effect.

Passalora fulva (or Fulvia fulva) is the causal microorganism of tomato leaf mold, the outbreak of which occurs worldwide in greenhouse especially when humidity is high. However, studies on antifungal agents of P. fulva are still very limited. In this study, a marine-derived Streptomycesalbidoflavus strain L131 showing potent inhibitory activities against P. fulva was identified and characterized. The active antifungal components were obtained, and studies on the antifungal mechanisms of the crude extract showed that the antifungal metabolites of L131 caused damage of hyphae and spore development, as well as plasma membrane of P. fulva. In addition, accumulation of endogenous reactive oxygen species of the leaf pathogen was also observed after treatment by culture extracts of L131. To our knowledge, this is the first report on the studies of the antifungal mechanisms against P. fulva, which benefit further development of biocontrol agent against tomato leaf mold disease.

A novel streptomycete, designated as strain DUT 180T, was isolated from a marine sediment sample collected from a sea cucumber farm in Dalian, northeast China. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DUT 180T is phylogenetically affiliated to the genus Streptomyces where it formed a distinct phyletic line with recognized Streptomyces species. Morphological and chemotaxonomic data also supported the affiliation of this isolate to the genus Streptomyces. Strain DUT 180T was found to exhibit highest sequence similarities of 99.52 and 99.36 % to Streptomyces halophytocola KLBMP 1284T and Streptomyces sulphureus NRRL B-1627T, respectively. However, strain DUT 180T could be distinguished from these two closest neighbours by a range of phenotypic properties. The DNA–DNA hybridization analyses between strain DUT 180T and the type strains of the phylogenetic neighbours revealed 54.8 ± 1.4 and 52.4 ± 2.8 % relatedness. Based on the phenotypic, chemotaxonomic and phylogenetic evidence, we suggest that the isolate DUT 180T represents a novel species of the genus Streptomyces, for which the name Streptomyces xiaopingdaonensis sp. nov. is proposed, with the type strain DUT 180T (= KCTC 29679T = CGMCC 4.7208T).

Xylanase is the enzyme complex that is responsible for the degradation of xylan; however, novel xylanase producers remain to be explored in marine environment. In this study, a Streptomyces strain M11 which exhibited xylanase activity was isolated from marine sediment. The 16S rDNA sequence of M11 showed the highest identity (99 %) to that of Streptomyces viridochromogenes. The xylanase produced from M11 exhibited optimum activity at pH 6.0, and the optimum temperature was 70 °C. M11 xylanase activity was stable in the pH range of 6.0–9.0 and at 60 °C for 60 min. Xylanase activity was observed to be stable in the presence of up to 5 M NaCl. Antibiotic-resistant mutants of M11 were isolated, and among the various antibiotics tested, streptomycin showed the best effect on obtaining xylanase overproducer. Mutant M11-1(10) isolated from 10 μg/ml streptomycin-containing plate showed 14 % higher xylanase activities than that of the wild-type strain. An analysis of gene rpsL (encoding ribosomal protein S12) showed that rpsL from M11-1(10) contains a K88R mutation. This is the first report to show that marine-derived S. viridochromogenes strain can be used as a xylanase producer, and utilization of ribosome engineering for the improvement of xylanase production in Streptomyces was also first successfully demonstrated.

Marine actinobacteria were isolated from the sediment samples collected in Xinghai Bay, Xiaoping Island and Changhai in Dalian, China. Fifteen selective media were employed, of which Humic acid-Vitamin medium recovered the highest number of isolates. Eleven of the 239 isolates obtained from the selective media were selected for further investigations based on colony morphology and pigment formation. Phylogenetic analysis of their 16S rRNA sequences showed that these strains belong to the genera of Streptomyces and Micromonospora. One of these strains identified is a new species of Streptomyces. Type I polyketide synthase (PKSI) gene fragments were amplified from three strains. The PKSI sequence of one of these strains (S187) showed high homology to the KS gene involved in meridamycin biosynthesis. Based on this result, the neurotrophic activity of S187 was further investigated. Culture broth of S187 was applied to rat pheochromocytoma (PC12) cells, and a 2.3-fold increase in growth over control cells was observed by the 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay. These results indicated the importance of further exploration of the marine actinobacteria in Dalian sea area for antimicrobial agents and type I polyketides.

High ethanol tolerance is a desired property of industrial yeast strains for efficient ethanol fermentation. In this study, the impact of medium composition on ethanol tolerance of the self-flocculating yeast SPSC01 was investigated using a chemically defined medium. Single-factor experiments revealed that besides magnesium and calcium, zinc also exhibited significant protective effect against ethanol toxicity; addition of 0.02 g/l zinc sulfate significantly increased cell viability in the ethanol shock treatment. Metal ions of manganese, cobalt, and ferrous failed to promote ethanol tolerance, although addition of 0.02 g/l cobalt increased ethanol production without apparent influence on ethanol tolerance. Furthermore, Uniform Design method was employed to obtain the medium with high cell viability, and the key nutrient factors in the medium composition were revealed to be (NH4)2SO4, K2HPO4, vitamin mixtures, and the metal ions of magnesium, calcium and zinc. The optimized combination of metal ions addition was (g/l): MgSO4 0.4, CaCl2 0.2, ZnSO4 0.01. The highest cell viability (90.2%) of SPSC01 against ethanol shock treatment was observed in the optimized medium, which demonstrated significant improvement of ethanol tolerance of the self-flocculating yeast.

•Designer artificial DNA fragments were used to construct BAC expression vectors.•In-Fusion cloning and λ-RED recombination were used for rapid vector construction.•The upstream unwanted sequences of the gene cluster can be deleted simultaneously.Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates heterologous expression of large gene clusters for drug discovery.

High cost of biomass recovery is one of the bottlenecks for developing cost-effective processes with microalgae, particularly for the production of biofuels and bio-based chemicals through biorefinery, and microalgal biomass recovery through cell flocculation is a promising strategy. Some microalgae are naturally flocculated whose cells can be harvested by simple sedimentation. However, studies on the flocculating agents synthesized by microalgae cells are still very limited. In this work, the cell flocculation of a spontaneously flocculating microalga Chlorella vulgaris JSC-7 was studied, and the flocculating agent was identified to be cell wall polysaccharides whose crude extract supplemented at low dosage of 0.5 mg/L initiated the more than 80% flocculating rate of freely suspended microalgae C. vulgaris CNW11 and Scenedesmus obliquus FSP. Fourier transform infrared (FTIR) analysis revealed a characteristic absorption band at 1238 cm−1, which might arise from PO asymmetric stretching vibration of PO2− phosphodiester. The unique cell wall-associated polysaccharide with molecular weight of 9.86×103 g/mol, and the monomers consist of glucose, mannose and galactose with a molecular ratio of 5:5:2. This is the first time to our knowledge that the flocculating agent from C. vulgaris has been characterized, which could provide basis for understanding the cell flocculation of microalgae and breeding of novel flocculating microalgae for cost-effective biomass harvest.

An addition of 3% (w/v) adsorbent resin HP-20 at 3 days post-inoculation significantly improved xinghaiamine A production from 0.35 to 3.4 mg/L and shortened fermentation time from 18 to 9 days. Self-cytotoxicity of xinghaiamine A was verified and the minimal inhibitor concentration was estimated to be 8 μg/mL.

•Heavy metal ions were removed efficiently by the self-flocculating Chlorella vulgaris JSC-7.•Surface adsorption played a major role in the heavy metal ion removal process.•The underlying mechanism might be with the unique cell wall component.Microalgae are attracting attention due to their potentials in mitigating CO2 emissions and removing environmental pollutants. However, harvesting microalgal biomass from diluted cultures is one of the bottlenecks for developing economically viable processes for this purpose. Microalgal cells can be harvested by cost-effective sedimentation when flocculating strains are used. In this study, the removal of Zn2+ and Cd2+ by the flocculating Chlorella vulgaris JSC-7 was studied. The experimental results indicated that more than 80% Zn2+ and 60% Cd2+ were removed by the microalgal culture within 3 days in the presence up to 20.0 mg/L Zn2+ and 4.0 mg/L Cd2+, respectively, which were much higher than that observed with the culture of the non-flocculating C. vulgaris CNW11. Furthermore, the mechanism underlying this phenomenon was explored by investigating the effect of Zn2+ and Cd2+ on the growth and metabolic activities of the microalgal strains. It was found that the flocculation of the microalga improved its growth, synthesis of photosynthetic pigments and antioxidation activity under the stressful conditions, indicating a better tolerance to the heavy metal ions for a potential in removing them more efficiently from contaminated wastewaters, together with a bioremediation of other nutritional components contributed to the eutrophication of aquatic ecosystems.

Scenedesmus obliquus belongs to green microalgae, which is attracting attention as a feedstock for biofuels production and biorefinery as well as in bioremediation of environmental pollutants, making its genetic modifications for more efficient growth and accumulation of aimed metabolites significant. However, the genetic transformation system of S. obliquus is still not well established. In the current work, S. obliquus was transformed via electroporation using a plasmid containing chloramphenicol resistance gene (CAT) as a selectable marker and the green fluorescent protein gene (gfp) as a reporter. Using the optimized transformation conditions, the transformation efficiency was 494 ± 48 positive transgenic clones per 106 recipient cells, which is more efficient comparing with those reported in other microalgal transformation studies. Green fluorescence was observed after six months of cultivation, and CAT-specific products were also detected in the transformants by PCR, Southern blot and RT-PCR analysis. This is the first report on establishing such an efficient and stable transformation system for S. obliquus, a prerequisite for both functional genomic studies and strain improvement for other biotechnology applications of this important microalgal species.Highlights► Genetic transformation method of Scenedesmus obliquus was established and high transformation efficiency was obtained. ► Stable transformants maintaining the plasmid for more than 6 months were obtained. ► It was proved that GFP and CaMV35S promoter are usable marker and promoter for genetic engineering of S. obliquus.