The Microbial Synthesis Of Amino-acid Homopolymers And Its Relevant Molecular Biology Study

With the characteristics of water-soluble, biodegrable and various biological activities, the amino acid homopolymers can be widely used, such as food, medical and industrial fields.In this study, the complete genome of y-PGA producing strain Bacillus amyloliquefaciens LL3was sequenced and analysised systemically. The IS sequence, prophage revelant sequence, and transmembrane protein in Bacillus amyloliquefaciens LL3were studied, which would help to explain the change in physiological activities in the construction of minimal genome strain. The genes revelant in biosynthsis and metabolic regulation was analyzed by studying the metabolic pathway of L/D glutamatic acid in Bacillus amyloliquefaciens LL3. These studies on the genome would contribute to explaining the biosynthetic mechanism of amino acid homopolymers.In this study, by methylene blue screening plates and Dragendorff reagent, two strains named NK660and NK49capable of producing ε-poly-L-lysine were isolated from the soil samples collected from Fujian Provience and Liaoning Provience. Based on the16S rDNA phylogenetic similarity, the strains were identified as Streptomyces. According to the keys on the species of Streptomyces genus in Bergey’s manual, the morphological and biochemical features of NK660and NK49were observed. The results showed that both NK.660and NK49belonged to Streptomyces albulus.The two strains were named Streptomyces albulus NK660and Streptomyces albulus NK49, respectively.Single factor experiments and orthogonal experiments were carried out to optimize the culture conditions of Streptomyces albulus NK660. Results of single factor experiments showed that the best carbon source and nitrogen source were glycerol, peptone and (NH4)2SO4, respectively. The range analysis of orthogonal experiment indicated glycerol was the most significant factor. The best combination of carbon and nitrogen source was glycerol30g/L, peptone7.0g/L and (NH4)SO44.0g/L. The analysis of variance showed that the results were significant in99% confidence interval. Through30L scale fed-batch fermentation of Streptomyces albulus NK660using pH control, the s-PL yield finally reached a concentration of4.19g/L after210h fermentation.The s-PL extraction method using weak acid resin D152was explored by studying the D152adsorption time curve, the maximum adsorption capacity and the effection of fermentation supernatant pH. The structure and feature of ε-PL produced by the two strains were identified and analyzed by nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MOLDI-TOF-MS) and fourier transform infrared spectroscopy (FTIR). The results showed that, the1H-NMR and the13C-NMR spectra of the ε-PL product from strain Streptomyces albulus NK660and Streptomyces albulus NK49are consistent with that of the ε-PL standard. The relative molecular mass distribution of ε-PL products from ε-PL producing strains were determined by MALDI-TOF MS. These data showed that the molecular weight range of the ε-PL produced by Streptomyces albulus NK660and Streptomyces albulus NK49was2453-4248Da (19-33L-lysine monomers) and2710-4376Da (22-34L-lyisine monomers), respectively. Since the polymerization degree and the molecular weight of ε-PL are closely related to its antibacterial activity, differrent polymerization degrees of ε-PL would exert different antimicrobial activities on different groups of bacteria in food preservation.In this study, ε-poly-L-lysine synthetase gene (pls) of Streptomyces albulus NK660was cloned and heterologous expressed. Firstly, the pls gene was cloned from Streptomyces albulus NK660by genome walking. Then a high copy clone vector pHZ1358was used as the vector for heterologous expression. The pls fragment was inserted into the vector and transformed into Streptomyces lividans ZX7. The pls would expressed under the control of its native promoter Ppls, and ε-PL was detected by Dragendorff reagent in the culture supernatant after72h incubation. The heterologous expression of pls gene had significant implication for the study of ε-PL molecular synthetic mechanism and the simplification of culture condition.Dissolve oxygen was the primary limiting factor in the process of ε-PL fermentation. In this study, the Vitreoscilla hemoglobin (VHb) was expressed in the ε-PL producing strain to solve the issue of dissolve oxygen deficiency. To construct the engineering strain, the site specific integration vector pSET152was used. Vitreoscilla hemoglobin gene (vgb) was integrated stablely into the attB site in Streptomyces albulus NK660genome to form the engineering strain Streptomyces albulus NK660-VHb. The expression of VHb was confirmed by the CO-spectral analysis. The shake flask experiment showed that the engineering strain Streptomyces albulus NK660-VHb grew faster due to its high level of oxygen utilization, and compared with the wild-type strain Streptomyces albulus NK660, the ε-PL yield increased by27%.