Study On The Secondary Metabolites Of ?-poly-L-lysine Synthase Gene Deletion Mutant Of Streptomyces Albulus M-Z18

The preservative ?-poly-L-lysine which has excellent properties such as antibacterial,safe,non-toxic,and biodegradable,is widely used in the pharmaceutical,food,and cosmetic industries.Streptomyces albulus is one of the ?-poly-L-lysine producing bacteria.There have been many researches to improve its yield through biosynthetic pathways,screening of high-yielding strains,optimization of medium conditions,and regulation of fermentation processes.This paper analyzed and compared the differences in secondary metabolites between the wild-type S.albulus M-Z18 strain and the?-poly-L-lysine synthase gene deletion mutant strain by high performance liquid chromatography.It is hoped to provide the basis for increasing the yield of?-poly-L-lysine from the level of metabolic flow.Firstly,the crude extract of 2.48 g was extracted from the fermentation broth of16 L S.albulus M-Z18 ?-poly-L-lysine synthase gene deletion mutant strain by extraction and evaporation concentration.Nine compounds(T01,T04,T05,T09,T10,T12,T13,T16 and T18)were separated from the crude extract using the column chromatograph techniques over sephadex LH-20,reverse phase silica gel and normal phase silica gel.According to the data of nuclear magnetic resonance spectrum and mass spectrum,the structure of compounds were respectively identified as methyl-5,10-dihydroxy-10-methyldodecanoate?4,10-dihydroxy-10-methyldodecanoica acid?methyl-3a-bromo-3,6-dioxohexahydro-1H-cyclopenta[c]furan-1-carboxylate?2-phenylacetic acid?4,10-dihydroxy-10-methyldodecan-4-olide?(2E,4E)-6-ethyl-7-hydroxyocta-2,4-dienoic acid?(2E,4E)-7,9-dihydroxy-6,8-dimethyldeca-2,4-dienoic acid?(E)-2,5,8,11,13 a,17,17-heptamethyl-1,5,8,12,13,13 a,14,16,17,17a-decahydro-2H-benzo[k][1,8]dioxacyclopentadecine-4,9,15(11H)-trione and 4-methylhexanoic acid.After literature retrieval,compounds T01,T05,T12,T13 and T16 were 5 new compounds,and the structures of other compounds have been reported.Then,the crude extracts of the wild-type S.albulus M-Z18 strain and the?-poly-L-lysine synthase gene deletion mutant strain under the same fermentation and extraction conditions were compared by high performance liquid chromatography.Compared with the wild-type strain,the yields of compounds T05 and T13 in the mutant strain were significantly increased,at the same time,compounds T12 and T16 were decreased,and compound T12 were significantly decreased,and compounds T01,T04,T09,T10 and T18 didn't change significantly.Aspartate kinase is a key enzyme in the biosynthetic pathway of ?-poly-L-lysine,and its enzymatic activity is regulated by feedback inhibition by the metabolic terminal L-lysine.In the mutant strain of S.albulus M-Z18 ?-poly-L-lysine synthase gene deletion,?-poly-L-lysine couldn't be synthesized due to the lack of ?-poly-L-lysine synthase,and L-lysine accumulated in large amount.The activity of aspartate kinase is inhibited by the feedback of lysine,resulting in the blocking of the metabolic pathway for the synthesis of ?-poly-L-lysine,and eventually the intermediate acetyl-Co A is diverted to other metabolic pathways,preferentially to T05 and T13.Finally,the inhibitory activities of ?-glucosidase,acetylcholinesterase,antioxidant activity and antibacterial activity of the isolated compounds were tested.Compounds T04,T05,T09,T10,T12,T13,T16 and T18 have weak inhibitory activity on ?-glucosidase at a concentration of 1 mg/mL,and the inhibitory rate of compound T12 is 10.05%.Compound T18 has weak inhibitory activity on acetylcholinesterase at a concentration of1 mg/mL,and the inhibitory rate is 6.13%.The detection of thin layer chromatographybioautography showed that compound T11 has antioxidant activity at a concentration of 4mg/mL.Compounds T01,T04,T05,T09,T10,T12,T13,T16 and T18 have no antibacterial activity at a concentration of 1 mg/mL.The results of this research showed that when the ?-poly-L-lysine synthetase gene of S.albulus M-Z18 is knocked out,the production of compounds T05 and T13 was significantly increased,while compounds T12 and T16 was reduced.The results would provide regulation method for high yield ?-poly-L-lysine from the level of metabolic flow.At the same time,the discovery of new compounds adds new chemical resources to natural products.