Engineering Of Streptomyces Sp.FR-008 For Heterologous Biosynthesis Of Natural Products

Heterologous expression strategy is widely used in the research of biosynthesis of natural products for the reason that some native producers have the features of unculturability,difficult genetic manipulation,slow growth,low yield of antibiotic,etc.Heterologous expression are commonly used in cloning of biosynthetic gene clusters,functional characterization of related genes,and regulation of metabolism in the heterologous hosts.Heterologous expression usually requires a proficient expression host.Streptomyces is commonly used as a heterologous host for the reasons that it encodes varieties of secondary metabolic biosynthetic gene clusters,and produces a variety of intermediates which can be used as precursors for the biosynthesis of different types of natural products,and its biosynthetic modules are more compatible with the primitive bacteria than other bacterias.In this study,Streptomyces sp.FR-008 was selected as the starting strain for the heterologous expression host for the reason that it grows rapidly,its aerial mycelium can produce mature spores in 3 days;the genetic manipulation is easy;the fermentation conditions are relatively lenient and itself can produces10 g/L candicidin;its whole genome sequencing was completed,laying the theoretical foundation for the molecular genetic experiment.Streptomyces sp.FR-008 contains two linear plasmids?142 kb,pHZ227 encodes arsenite resistance gene clusters;24 kb,pHZ228?,the genome of FR-008 was simplified by eliminating the linear plasmid with the method of nitroguanidine mutagenesis?NTG?.Three arsenite sensitive mutant strains?10#,59#,115#?were screened from 103 mutant strains,and the results of Pulsed Field Gel Electrophoresis showed that the large linear plasmids pHZ227 in these three mutants are all eliminated.In addition,the small linear plasmid pHZ228 was eliminated in the mutant strain of 42#.Based on the mutant strain of 42#,a second round of nitroguanidine mutagenesis resulted in a double plasmids deletion mutant XYS1.The elimination rates of large linear plasmid pHZ227 and small linear plasimid pHZ228 are about 3% and 1%,respectively.The yeild of candicidin III of two large linear plasmid elimination of mutant strains?10 # and 115 #?increased by 40% and 30%,respectively.To investigate whether there is a correlation between the elimination of large linear plasmid and the increase of candicidin production,we use the CRISPR/Cas9-CodA?sm?system to eliminate the linear plasmid.Fermentation results showed that loss of linear plasmid had no effect on the yield of the candicidin,and we inferred that the increase in the yield of mutant strains in 10# and 115# may be due to the mutation in chromosomal DNA.Futhermore,the mutant strains of 115# and 10# obtained by nitroguanidine mutagenesis were genetically modified to make them as efficient heterologous expression hosts.The matAB governing mycelium aggregation was deleted and concomitantly replaced with attachment site attB as well as cognate T7 polymerase gene in 115# and 10# to generate respective 115#1 and 10#1.The mycelia of the mutant strains 115#1 and 10#1 were no longer aggregated in the liquid medium,and the biomass was enhanced by about 40% compared with the original strain.The results of fermentation showed that the candicidin yield of 115#1 and 10#1 had increased 46%,18%,respectively.115#1 had the highest yield among all mutants.In addition,the introduction of attB enhanced the conjugation efficiency of integrative plasmid;the introduction of T7 polymerase contributed to the expression of the gene regulated by T7 promoter in the mutant strain.Above all,we obtained the genetically modified strain 115#1 which drivated from Streptomyces sp.FR-008.Finally,we selected the mutant strain 115#1,which has the highest yield of candicidin,as a heterologous expression host,and studied the heterologous expression of different types of antibiotics in this host.The nucleoside antibiotic mildiomycin biosynthesis gene cluster 14A6 was transferred to 115#1 by conjugation.Mildiomycin was successfully expressed in 115#1,which was confirmed by HPLC and MS assay.The yield of mildiomycin in 115#1::14A6 is 3770 mg/L,much more than the the yield of mildiomycin in native strain Streptoverticillium rimofaciens ZJU5119?1015 mg/L?;however,the yield of mildiomycin in S.lividans 1326::14A6 is less than 10 mg/L,suggesting that 115#1 has a great advantage over S.lividans 1326.Meanwhile,the aminoglycoside streptothricin biosynthesis gene cluster 8E6 was transferred to 115#1 by conjugation,and the bioassay showed that 115 # 1::8E6 was able to produce streptothricin.The yields of streptothricin 115#1 and S.ceolicolor M1154 were compared by bioactivity assay,which showed that the yield in 115#1 was slightly higher than S.ceolicolor M1154.In conclusion,we obtained two genome-reduced strains?10#,115#?with linear plasmid was deleted by nitroguanidine mutagenesis,and led to enhanced the yield of candicidin.Gene replacement of ORF₀6931 coding for aggregation of mycelia with attB site and T7 polymerase gene increases the production of candicidin as well as the biomass of the resultant mutant strains 115#1 and 10#1.Mutant strain 115#1 with the highest yield of candicidin was chosen to express nucleoside mildiomycin gene cluster.its yield in 115#1 is 3.5-folds to the wild type strain,and 500-folds to that in S.lividans 1326,demonstrating 115#1 a potential advantageous host to produce nucleoside.Also,streptothricin biosynthetic gene cluster are successfully achieved heterologous expression in 115#1.Therefore,we developed Streptomyces sp.FR-008 into a heterologous expression host.