Screening And Metabolic Engineering Of Marine-derived Streptomyces Strains With Improved Xylanase Production

Agricultural residues such as corn stover and wheat straw are abundant and renewable bioresources, and the utilization of these residues has aroused the worldwide concern of researchers. Hemicellulose takes up one third of renewable organic carbon on the earth and xylan is the major component of hemicellulose. The degradation of xylan can not only be utilized by microbes, but also help the separation of cellulose and lignin, which is significantly important in the production of biofuel and paper pulp industry. Xylanase digestion is the major method of xylan degradation for its features of environmental friendly and high efficiency. Actinomycetes and filamentous fungi are the major xylanase producers, but the production level of wild type strains is usually very low. Therefore, it is important to enhance the production of xylanase by strain improvement.In this study, eight marine-derived actinomycetes strains with good xylanase production were isolated from the marine sediment sample collected from Xiaoping Island in Dalian. Two streptomycetes strains M8and Ml1with superior xylanase activity were further studied. The16S rRNA sequence analysis indicated that M8is the most close to Streptomyces xylophagus; and the16S rRNA sequence of M11is99%similar to that of S. viridochromogenes. Investigation of cell growth under different pH conditions indicated that strain M11is an alkalophilic Streptomyces and grows well at pH11.0, whereas M8showed good NaCl tolerance. Both strains showed high xylanase activity and reached the highest production of87.6U/mL and69.9U/mL after7days’culture. The optimum temperature and pH of xylanase from M8and M11are60℃,6.0and70℃,6.0, respectively. Xylanase from M11showed good stability under the condition of60℃, pH5.0to9.0, in addition, no cellulase activity was observed, which indicates that this xylanase has the potential to be applied in biobleaching of kraft pulp.Subsequently, ribosome engineering and artificial zinc-finger protein (ZFP) technique were used to regulate the production of xylanase in global level of cellular metabolisms. Twenty seven mutants with bigger transparent circles were obtained from370streptomycin-resistant strains. Xylanase production of a mutant M11-(10) increased by14%comparing with the wild type strain, and mutantion K88R on ribosomal S12protein was revealed. The mutation site locates in the core region of S12protein, which is associated with the stability of70S ribosomal complex and translation efficiency. It was hypothesized that the mutation in ribosomal S12protein leads to increased production of enzymes.Next, the artificial zinc-finger protein library containing zinc-finger domain and Ga14activation domain was constructed. Xylanase production of the conjugant M8-B32was17%higher than that of the wild-type strain. The zinc finger protein sequence from M8-B32was analyzed, and it was proved that the zinc finger protein was originated from human genome. We assumed that the zinc-finger protein binds with the promoter region of some target genes and regulates the production of xylanase.The results presented in this study demonstrated that ribosome engineering and ZFP technique can be successfully applied in Streptomyces for strain improvement. Currently only few studies are focused on the regulation of xylanase production from Streptomyces. The results of this work provide basis for further improving marine enzyme production.