Complete genome sequence of high-yield strain S. lincolnensis B48 and identification of crucial mutations contributing to lincomycin overproduction

The lincosamide family antibiotic lincomycin is a widely used antibacterial pharmaceutical generated by Streptomyces lincolnensis,and the high-yield strain B48 produces 2.5 g/L lincomycin,approximately 30-fold as the wild-type strain NRRL 2936.Here,the genome of S.lincolnensis B48 was completely sequenced,revealing a^10.0 Mb single chromosome with 71.03%G+C content.Based on the genomic information,lincomycinrelated primary metabolism network was constructed and the secondary metabolic potential was analyzed.In order to dissect the overproduction mechanism,a comparative genomic analysis with NRRL 2936 was performed.Three large deletions(LDI-III),one large inverted duplication(LID),one long inversion and 80 small variations(including 50 single nucleotide variations,13 insertions and 17 deletions)were found in B48 genome.Then several crucial mutants contributing to higher production phenotype were validated.Deleting of a MarRtype regulator-encoding gene slinc377 from LDI,and the whole 24.7 kb LDII in NRRL 2936 enhanced lincomycin titer by 244%and 284%,respectively.Besides,lincomycin production of NRRL 2936 was increased to 7.7-fold when a 71 kb supercluster BGC33 from LDIII was eliminated.As for the duplication region,overexpression of the cluster situated genes lmbB2 and lmbU,as well as two novel transcriptional regulator-encoding genes(slinc191 and slinc348)elevated lincomycin titer by 77%,75%,114%and 702%,respectively.Furthermore,three negative correlation genes(slinc6156,slinc4481 and slinc6011)on lincomycin biosynthesis,participating in regulation were found out.And surprisingly,inactivation of RNase J-encoding gene slinc6156 and TPR(tetratricopeptide repeat)domain-containing protein-encoding gene slinc4481 achieved lincomycin titer equivalent to 83%and 68%of B48,respectively,to 22.4 and 18.4-fold compared to NRRL 2936.Therefore,the comparative genomics approach combined with confirmatory experiments identified that large fragment deletion,long sequence duplication,along with several mutations of genes,especially regulator genes,are crucial for lincomycin overproduction.

Some ecological parameters of Artemia parthenogenetica Gahai and their use in resource exploitation

The effects of temperature on population characteristics of Artemia parthenogenetica Gahai from the Gahai Salt Lake,Qinghai Province,China,were studied in the laboratory at a salinity of 60‰.The major conclusions are as follows:(1)It was found that the adaptive temperature for the development of brine shrimp ranged from 10℃to 39℃.The threshold temperature of development(TD)and the effective accumulative temperature(TA)for hatching were 9.94℃·d and(22.91
2.08)℃·d,respectively.The TD and TA for the larva were 10.33℃·d and(261.26
24.1)℃·d,respectively,and for the whole generation were 10.28℃and(458.68
57.60)℃·d,respectively.(2)It showed that the population’s net reproduction rate(R_(o)),the intrinsic rate of natural increase(r_(m)),the mean generation time(T),the finite rate of increase(λ),and the days for population to double(t)of the brine shrimp were determined over temperatures ranging from 19℃to 34℃by analyzing the life table and numerical model.In the temperature range of 14.3℃to 37.3℃for R_(o)>1,the optimum temperature(℃)for R_(o),rm,λ,and t were 25.8℃,29.8℃,30.5℃,and 29.02℃,respectively.The maximum values of R_(o),r_(m) andλwere 54.86 ind.,0.106138/d,and 1.1070/d,respectively.The minimum value of t was 4.73 d.The value of T was in a range of 96.77 to 16.10 d.(3)Based on the 1993-1994 and 1997 data of the water temperature in the Gahai Salt Lake,Qinghai Province,it was estimated that the number of generation of A.parthenogenetica Gahai and the number of the reproductive peak value were 2.67
0.34 and 4.69
0.43 in a year,respectively.The peak of nauplii of the first generation was on April 20 to 28.The last whole generation began on August 10.The first reproductive peak was on June 18 to 25.The last reproductive peak was on September 12 to 17.The nauplii that hatched after September 1 cannot complete the development from nauplii to adults because of insufficient habitat effective accumulative temperature.During the period from July 11 to September 20,there was a relatively high productivity of the population.In this case,doubling the population would take less than 30 d,and the intrinsic rate of natural increase was over 0.02/d.Therefore,the value of resource exploitation would be maximal during that period annually.