Unlocking a new target for streptomycetes strain improvement

Bacteria of the Streptomyces genus are well-known producers of secondary metabolites of high medical value.They contributed nearly 60%of current antibiotics(i.e.,vancomycin,daptomycin and tetracycline),as well as antifungals,antiparasitics(avermectins),anticancer drugs(doxorubicin),immunosuppressants and others[1].Therefore,in the past several decades,Streptomyces bacteria or the therein involved biosynthetic pathways have long been the central topic of strain improvement,metabolic engineering,and bioengineering.Much of this research has focused on regulatory elements;however,it has been a challenge due to the complex regulatory network that controls Streptomyces secondary metabolism and its complex fungus-like morphological differentiation[2].On agar plates,production of secondary metabolites coincides with the switch from vegetative growth(substrate mycelium)to aerial mycelium hyphae and subsequent spore formation,whereas in a liquid culture,early stationary phase when cells stop growing marks the initiation of secondary metabolism.

Improved production of spiramycin by mutant Streptomyces ambofaciens

Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S. ambofaciens XC 2-37 were studied. The potency of S. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation. The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.

Microsatellite analysis of variation among wild, domesticated, and genetically improved populations of blunt snout bream （Megalobrama amblycephala）

In the present study, the genetic diversity of one selected strain （Pujiang No. 1）, two domesticated populations （GA and HX） and four wild populations （LZ, YN, SS and JL） of blunt snout bream （Megalobrama amblycephala） was analyzed using 17 microsatellite markers. The results showed that an average of 4.88-7.65 number of alleles （A）; an average of 3.20-5.33 effective alleles （Ne）; average observed beterozygosity （Ho） of 0.6985-0.9044; average expected beterozygosity （He） of 0.6501--0.7805; and the average polymorphism information content （PIC） at 0.5706-0.7226. Pairwise FST value between populations ranged from 0.0307-0.1451, and Nei＇s standard genetic distance between populations was 0.0938-0.4524. The expected heterozygosities in the domesticated populations （GA and HX） were significantly lower than those found in three wild populations （LZ, SS and JL）, but no difference was detected when compared with the wild YN population. Likewise, no difference was found between the four wild populations or two domesticated populations. The expected heterozygosity in Pujiang No. 1 was higher than the two domesticated populations and lower than the four wild populations. Regarding pairwise Fsr value between populations, permutation test P-values were significant between the GA, HX and PJ populations, but not between the four wild populations. These results showed that the expected beterozygosity in the selected strain of blunt snout bream, after seven generations of selective breeding, was lower than that of wild populations, but this strain retains higher levels of genetic diversity than domesticated populations. The genetic differences and differentiation amongst wild populations, domesticated populations and the genetically improved strain of blunt snout bream will provide important conservation criteria and guide the utilization of germplasm resources.

Evolutionary engineering of Phaffia rhodozyma for astaxanthin-overproducing strain

Evolutionary engineering is a novel whole- genome wide engineering strategy inspired by natural evolution for strain improvement. Astaxanthin has been widely used in cosmetics, pharmaceutical and health care food due to its capability of quenching active oxygen. Strain improvement ofPhaffia rhodozyma, one of the main sources for natural astaxanthin, is of commercial interest for astaxanthin production. In this study a selection procedure was developed for adaptive evolution of P. rhodozyma strains under endogenetic selective pressure induced by additive in environmental niches. Six agents, which can induce active oxygen in cells, were added to the culture medium respectively to produce selective pressure in process of evolution. The initial strain, P. rhodozyma AS2-1557, was mutagenized to acquire the initial strain population, which was then cultivated for 550 h at selective pressure and the culture was transferred every 48h. Finally, six evolved strains were selected after 150 generations of evolution. The evolved strains produced up to 48.2% more astaxanthin than the initial strain. Our procedure may provide a promising alternative for improvement of highproduction strain.

Regulation of antibiotic biosynthesis in actinomycetes:Perspectives and challenges

Actinomycetes are the main sources of antibiotics.The onset and level of production of each antibiotic is subject to complex control by multi-level regulators.These regulators exert their functions at hierarchical levels.At the lower level,cluster-situated regulators(CSRs)directly control the transcription of neighboring genes within the gene cluster.Higher-level pleiotropic and global regulators exert their functions mainly through modulating the transcription of CSRs.Advances in understanding of the regulation of antibiotic biosynthesis in actinomycetes have inspired us to engineer these regulators for strain improvement and antibiotic discovery.

Remarkable enhancement of bleomycin production through precise amplification of its biosynthetic gene cluster in Streptomyces verticillus

Amplification of biosynthetic gene clusters is important to increase secondary metabolite production.However,the copy number of amplified gene clusters is difficult to control precisely.In this study,the tandem amplification of a 70 kb bleomycin biosynthetic gene cluster was precisely regulated through the combined strategy of a Zou A-dependent DNA amplification system and double-reporter-guided recombinant selection in Streptomyces verticillus ATCC15003.The production of bleomycin in the recombinant strain containing six copies of the bleomycin gene cluster was 9.59-fold higher than that in the wild-type strain.The combined strategy used in this study is powerful and applicable for precisely regulating the amplification of gene clusters and improving the corresponding secondary metabolite production.