Studies On The Alteration Of Metabolic Function Of Streptomyces Fimicarius HLF-43 And The Antitumor Metabolites Newly Produced By Bioactive Mutants Of HLF-43

Actinomycetes had played an important role as an abundant source of new drugs for many years. However, the expected activity can't be found for most of the actinomycete stains isolated from nature under a given bioassay in general. How to make good use of the microbial strains without the ability to produce bioactive metabolites will be a meaningful research. Ribosome engineering is a rational approach to gain high yielding mutants by using the antibiotic- resistance screening method. Classical drug-resistance screening methods were still effective even without specific genetic information, but these methods always caused a serious waste of time and resource because they were based on the spontaneous mutation of microorganisms. Therefore, under the dual-requirement of obtaining bioactive mutants and increasing mutation rate of actinomycete strains, three new methods, which were used in obtaining bioactive mutants from inactive actinomycete strains, were established in succession: 1. Microwave mutagenesis coupled with neomycin-resistant mutant screening; 2. Magnetic nanoparticle (MNPs)-aided microwave mutagenesis coupled with neomycin resistant mutants screening. 3. Multi-antibiotic resistant mutants screening coupled with MNPs-aided microwave mutagenesis. Several mutants were obtained by applying above three methods to HLF-43 (a marine-derived S. fimicarius, the inhibition rate of 3.9% at the 1000μg/mL sample concentration on K562 cells) and the antitumor activity was tested by the MTT method using K562 cells for the samples from the fermentation of these mutants, through which several antitumor mutants were obtained eventually. Three antitumor mutants were chosen, each of which was obtained by one of the above three new methods respectively, to investigate their main antitumor metabolites for the exploration of relationship between the main antitumor metabolite production and the metabolic pathway(s) probably activated in the mutants. Then, the newly produced bioactive metabolites from the mutant N73 was investigated systematically in the following research.1.Alteration of metabolic function of S. fimicarius HLF-43 by microwave mutagenesis coupled with neomycin-resistant mutant SelectionTotal 128 neomycin-resistant (neo) mutants including the 117 mutants from microwave mutagenesis and the 11 mutants from auto-mutagenesis were obtained by neo mutant selection on neomycin-containing plates coupled with microwave-induced mutation technique. Among the neo mutants obtained, the 8 neo mutants from microwave mutagenesis appeared to be bioactive with the inhibition rates over 20% at the 100μg/mL of sample concentration on K562 cells. By combination of the different time of microwave irradiation and the different concentration of neomycin, it has been appeared that the 120 s of microwave irradiation is likely to be favorable for obtaining more neo mutants.2.Alteration of metabolic function of S. fimicarius HLF-43 by magnetic nanoparticle (MNPs)-aided microwave mutagenesisA new method was investigated for altering metabolic function of inactive actinomycete strain by MNPs-aided microwave mutagenesis to obtain antitumor mutants. HLF-43 was used as the initial strain in the present study, and the spore suspension of HLF-43 was exposed to the microwave radiation under the presence of Fe₃O₄ MNPs then spread on neomycin-containing plates and the neomycin-resistant (neo) mutants were selected. The antitumor neo mutants were obtained by the test of antitumor activity for those samples. By the method coupled with the neo mutant selection, total 80 of neo mutants were obtained from the inactive-initial strain HLF-43, and 28 of them are antitumor mutants with the inhibition rates over 20% at the 100μg/mL sample concentration on the K562 cells. The antitumor mutants could be obtained at quite high frequency of 35.0% (28/80), and showed stable genetic ability. By comparison with the microwave mutagenesis without the aid of Fe₃O₄ MNPs, the Fe₃O₄ MNPs-aided microwave mutagenesis is more favorable to obtain the neo mutants resistant to higher concentration of neomycin and to obtain the antitumor neo mutants at higher frequency.3. Alteration of metabolic function of S. fimicarius HLF-43 by microwave mutagenesis coupled with multi-antibiotic resistances mutant selectionIn order to further improve the screening effect, a new antibiotic resistance screening method by using multi-antibiotic was explored in this chapter. On the basis of the method of the MNPs-aided microwave mutagenesis, the multi-antibiotic resistance screening method with four kinds of antibiotics, such as neomycin, gentamycin, kanamycin and Rifampicin, was used to treat the inactive initial strain HLF-43. By the method above, the total of 16 multi-antibiotic resistance mutants were obtained, and 7 of them were antitumor mutants with the inhibition rates over 20% at the 100μg/mL sample concentration on the K562 cells. The antitumor mutants could be obtained at quite high frequency of 43.7% (7/16). Compared with the previous methods, the method in this chapter can obtain the antitumor mutants with higher frequency.4. Isolation and comparison of the main newly produced metabolites with antitumor activity of the mutants (N27,36,15G) derived from three different methods.The three different active mutants ( N27,36,15G), derived from inactive strain HLF-43 by three different selecting methods respectively, were used as the target strains for researching the product. The best fermentation time was determined by time course experiment of each strain. Meanwhile the mutants were fermented with the same condition of the strains. The three bioactive metabolites (compound 1-3), different from initial mutants, were isolated from the metabolites of mutants by a bioassay-guided separation procedure. Compound 1 (14-methylpentadecanoic acid) was gained from all three mutant strains, and it was also the main metabolite with antitumor activity. Compound 2 (Glycerol 1-(14-methylpentadecanoate)), separated from mutant strain 36, was the derivant of compound 1. Compound 3 (β-sitosterol)), separated from mutant strain 15G, was common phytosterols. The compound 1, 2 and 3 were bioactive on K562 cells with an inhibition rate of 58.1, 74.5 and 25% at 100μg/mL, respectively. IC₅₀ of compound 1 and 2 were 95μg/mLå’Œ52.5μg/mL, respectively The inhibition activity of compound 1 and 2 on K562 cells was reported for the first time. The structures of compounds above were shown in Table.1. The documents and experiement results showed that metabolic function of the initial strains HLF-43 could be altered by each of the three methods above, which resulted in Malonyl-coenzyme A(fatty acid synthetase, Fab)expressed highly and long-chain saturated fatty acids over-produced(More than 350 milligrame of compound 1 can be obtained from strain N27). It was probable that all the changes were originated from the correlative mutation on the ribosome when the resistance was gained by the mutant strains.5. Studies on the newly produced bioactive metabolites from the mutant N73The antitumor activity mutant strain N73 was chosen for investigating the newly produced activity products. The best fermentation time was determined as 13d by time course experiment. 7 compounds different from initial strain HLF-43 were isolated and identified from the mutants strains N73 by bioassay-guiding metabolizes of the mutant and comparing the metabolize of mutants to the initial strains, and they were three compounds of fatty glycerides: Glycerol 1-(14-methylpentadecanoate) (2), monolaurin (4), glycerol monooleates (5); two compounds of diketopiperazines: cyclo-(4-hydroxyl-Pro-Leu) (6), and cyclo-(4-hydroxyl-Pro-Phe) (7); one compound of sterol: daucosterol (8) ;and one compound of flavonoid: Genistein (9). Literature showed that, apart from compound 6 and 9 being reported by our research group, the other compounds hadn't been reported on the metabolites of S. fimicarius. Furthermore, these compounds were compared with fermentation sample from HLF-43 by HPLC. Results proved that all the compounds were newly produced bioactive compounds, and didn't exist in fermentation of the initial strains HLF-43. All the results above further indicated that the combining methods, based on MNPs-aided microwave mutagenesis and neomycin resistance screening, could alter the metabolic function of inactive actinomycetes. Antitumor activity testing showed that all the compounds (4-9)mentioned above showed antitumor activity on K562 cells. The compounds 4 and 5 were bioactive on K562 cells with an inhibition rate of 84.4 and 55.5% at 100μg·mL^-1, their IC₅₀ was 57.7μg/mL and 82.9μg/mL. The compounds 6, 7, 8, 9 were bioactive on K562 cells with an inhibition rate of 33.2%,21.0%,33.3%,41.3% at 100μg·mL^-1 respectively. The inhibiting activities of compounds 4, 5, 7, 8 on K562 cells were not reported so far. All the results above further indicated that the combining methods, based on MNPs-aided microwave mutagenesis and neomycin resistance screening, could alter the metabolic function of inactive actinomycetesIn conclusion, S. fimicarius strains HLF-43, isolated from the sea mud samples collected from the intertidal zone, were used as initial strains. Three new different screening methods (Neomycin-resistant mutant selection coupled with microwave mutagenesis; MNPs-aided microwave mutagenesis ; Screening multi-antibiotic resistances actinomycetes coupled with microwave mutagenesis) were developed, by which 117, 80 and 16 resistant mutants were gained respectively. The rate of antitumor activity mutants on K562 cells over 20% at the 100μg·mL^-1 concentration of sample was 6.8% (8/117), 35.0% (28/80) 43.7% (7/18), respectively, which indicated that all the three new methods can effectively change the metabolic function of HLF-43. Compared with the microwave mutagenesis without the aid of Fe₃O₄ MNPs, the latter two methods were more favorable to obtain the antitumor mutants. The main metabolites with antitumor activity from the three different-derived mutants (N27, 36, 15G) was isolated, characterized and tested. Results identified that the main activity metabolites were long-chain saturated fatty acids. It was deduced that the mutation of ribosome made Malonyl-coenzyme A expressed highly, and finally caused long-chain saturated fatty acids over-produced. Moreover, the mutant strains N73 with best antitumor activity were chosen by the preliminary screening to investigate the newly produced activity products. 7 compounds, different from the initial strains, were isolated from the metabolism of mutants. Furthermore, the effectivity of the methods was provedby activity test and comparation of HPLC between compounds and fermentation of initial strains.The research in this paper was guided by a new idea on secondary development of inactive actinomycete. Results showed that it is feasible to change the inactive actinomycete into bioactive strain sources by utilizing the three new methods mentioned above.