Enhancement of doxorubicin production in Streptomyces peucetius by genetic engineering and process optimization.

Doxorubicin is an important class of anthracycline antitumor antibiotics produced by Streptomyces peucetius. The doxorubicin fermentation yield of the wild-type strain was very low, so it could not be produced directly by fermentation at an industrial scale due to the high cost. In the present study, S. peucetius SIPI-7-14 was obtained from SIPI-14 through several rounds of doxorubicin resistance screening. Then, the ketoreductase gene dnrU was knocked out to reduce (13S)-13-dihydrodaunorubicin production, and the resistance gene drrC was overexpressed to further enhance resistance to doxorubicin. The resulting engineered strain S. peucetius △U1/drrC produced 1128 mg/L doxorubicin, a 102.1% increase compared to that of SIPI-14. Then, fermentation medium was optimized using the response surface method. In the optimized fermentation medium, the yield of doxorubicin was increased to 1406 mg/L in shake flask on the 7th day. Furthermore, batch culture was carried out in a 10 L fermenter, and the concentration of doxorubicin reached 1461 mg/L after 7 days of culture, which was the highest yield reported to date, indicating the potential for industrial production of doxorubicin by fermentation.

Nutrient Deficiency and an Algicidal Bacterium Improved the Lipid Profiles of a Novel Promising Oleaginous Dinoflagellate, Prorocentrum donghaiense, for Biodiesel Production.

The lipid production potentials of 8 microalgal species were investigated. Among these 8 species, the best strain was a dominant bloom-causing dinoflagellate, Prorocentrum donghaiense; this species had a lipid content of 49.32% ± 1.99% and exhibited a lipid productivity of 95.47 ± 0.99 mg liter-1 day-1, which was 2-fold higher than the corresponding values obtained for the oleaginous microalgae Nannochloropsis gaditana and Phaeodactylum tricornutum. P. donghaiense, which is enriched in C16:0 and C22:6, is appropriate for commercial docosahexaenoic acid (DHA) production. Nitrogen or phosphorus stress markedly induced lipid accumulation to levels surpassing 75% of the dry weight, increased the C18:0 and C17:1 contents, and decreased the C18:5 and C22:6 contents, and these effects resulted in decreases in the unsaturated fatty acid levels and changes in the lipid properties of P. donghaiense such that the species met the biodiesel specification standards. Compared with the results obtained under N-deficient conditions, the enhancement in the activity of alkaline phosphatase of P. donghaiense observed under P-deficient conditions partly alleviated the adverse effects on the photosynthetic system exerted by P deficiency to induce the production of more carbohydrates for lipogenesis. The supernatant of the algicidal bacterium Paracoccus sp. strain Y42 culture lysed P. donghaiense without decreasing its lipid content, which resulted in facilitation of the downstream oil extraction process and energy savings through the lysis of algal cells. The Y42 supernatant treatment improved the lipid profiles of algal cells by increasing their C16:0, C18:0, and C18:1 contents and decreasing their C18:5 and C22:6 contents, which is favorable for biodiesel production. IMPORTANCE This study demonstrates the high potential of Prorocentrum donghaiense, a dominant bloom-causing dinoflagellate, for lipid production. Compared with previously studied oleaginous microalgae, P. donghaiense exhibit greater potential for practical application due to its higher biomass and lipid contents. Nutrient deficiency and the algicidal bacterium Paracoccus sp. strain Y42 improved the suitability of the lipid profile of P. donghaiense for biodiesel production. Furthermore, Paracoccus sp. Y42 effectively lysed algal cells, which facilitates the downstream oil extraction process for biodiesel production and results in energy savings through the lysing of algal cells. This study provides a more promising candidate for the production of docosahexaenoic acid (DHA) for human nutritional products and of microalgal biofuel as well as a more cost-effective method for breaking algal cells. The high lipid productivity of P. donghaiense and algal cell lysis by algicidal bacteria contribute to reductions in the production cost of microalgal oil.

Isolation of Thalassobius mangrovi sp. nov., a novel bacterium in the family Rhodobacteraceae, from marine mangrove sediment.

A Gram-stain-negative, strictly aerobic and oval-shaped bacterial strain with a flagellum, designated GS-10T, was isolated from mangrove wetland sediment. GS-10T grew at 20-40 °C (optimum, 37 °C), in the pH range of 5.0-11.0 (optimum, 6.0-8.0) and under various NaCl concentrations from 1 to 11 % (w/v) (optimum, 5-6 %). The respiratory quinone was ubiquinone-10, and the predominant polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids (>10 % of the total fatty acids) were summed feature 4 (C17 : 1iso I/anteiso B) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The G+C content of the genomic DNA was 63.71 %. On the basis of the results from comparative analysis of the 16S rRNA gene sequence, GS-10T represents a member of the family Rhodobacteraceae and had the highest sequence similarity to Thalassobius gelatinovorus CECT 4357T (97.47 %), followed by Lutimaribacter pacificus W11-2BT (97.03 %), Marivita cryptomonadis CL-SK44T (96.83 %), Thalassobius autumnalis CECT 5118T (96.75 %) and Thalassobius mediterraneus CECT 5383T (96.68 %). Phylogenetic trees based on 16S rRNA gene sequences, multilocus sequence analysis (MLSA) and whole genome sequences revealed that GS-10T clustered with species within the genus Thalassobius. The average nucleotide identity (ANI) and the average amino acid identity (AAI) values were calculated from complete genome sequences and indicated that GS-10T represented a novel species of the genus Thalassobius, and the name Thalassobius mangrovi sp. nov. is proposed for this species. The type strain of Thalassobius mangrovi is GS-10T (=MCCC 1K03624T=KCTC 82131T).

Marisediminitalea mangrovi gen. nov., sp. nov., isolated from marine mangrove sediment, and reclassification of Aestuariibacter aggregatus as Marisediminitalea aggregata comb. nov.

Strain GS-14T was isolated from a mangrove sediment sample collected at Beilun Estuary National Nature Reserve, Guangxi Province, PR China. Cells were Gram-stain-negative, strictly aerobic and rod-shaped with a polar flagellum. Optimal growth occurred in the presence of 3-6 % (w/v) NaCl, at pH 6-8 and at a temperature of 37 °C. The predominant polar lipids were phosphatidylglycerol and phosphatidylethanolamine. Ubiquinone 8 (Q-8) was the sole respiratory quinone. The major fatty acids (>10 % of the total fatty acids) were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The DNA G+C content was 47.6 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain GS-14T had the highest sequence similarity to Aestuariibacter aggregatus WH169T (96.63 %), Aliiglaciecola coringensis AK49T (96.56 %) and Alteromonas lipolytica JW12T (96.22 %). In addition, the OrthoANIu value and dDDH values calculated from the genomes of strain GS-14T and A. aggregatus WH169T were 79.5 and 21.9 %, respectively. Based on the polyphasic taxonomic results, strain GS-14T is considered to represent a novel species in a new genus, for which the name Marisediminitalea mangrovi gen. nov., sp. nov. is proposed. The type strain of Marisediminitalea mangrovi is GS-14T (=KCTC 72401T=MCCC 1K03622T). Because Aestuariibacter aggregatus WH169T clustered with strain GS-14T in the phylogenetic trees and was clearly separated from the two species within the genus Aestuariibacter, it is reclassified as a member of the genus Marisediminitalea as Marisediminitalea aggregata comb. nov. (type strain WH169T=CGMCC 1.8995T=LMG 25283T). The type species of the genus Marisediminitalea is Marisediminitalea aggregata gen. nov., comb. nov.

Thalassotalea mangrovi sp. nov., a bacterium isolated from marine mangrove sediment.

A novel Gram-stain-negative, strictly aerobic bacterium that has a rod-like shape with a single polar flagellum in the exponential phase of growth and a spherical or ovoid shape without a flagellum in the stationary phase was isolated from a mangrove wetland sediment sample collected at Beilun Estuary National Nature Reserve, Guangxi Province, PR China and designated strain ZS-4T. This strain grew optimally at pH 6.0-8.0, at a temperature of 37 °C and in the presence of 3-4 % (w/v) NaCl. Its polar lipid profile included phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid and two uncharacterized lipids. Ubiquinone 8 (Q-8) was the sole respiratory quinone and the cellular fatty acids were dominated by C17 : 1ω8c and C16 : 0. A phylogenetic analysis based on the 16S rRNA gene sequence showed that strain ZS-4T exhibited its highest similarities to the type strains Thalassotalea litorea HMF4135T (97.8 %) and Thalassotalea ponticola GJSW-36T (95.9 %). A whole genome-level comparison of strain ZS-4T with T. litorea MCCC 1K03283T revealed an average nucleotide identity value of 75.6 % and a calculated DNA-DNA hybridization value of 19.6 %. In addition, the genomic DNA G+C content of strain ZS-4T was 45.9 mol%. Thus, based on analyses of its morphology, physiology, fatty acid composition and 16S rRNA gene sequence, strain ZS-4T should be considered a novel species of the genus Thalassotalea, with the proposed name Thalassotaleamangrovi sp. nov. The type strain is ZS-4T (=KCTC 72399T=MCCC 1K03630T).