【作者】 朱雷；

【导师】 唐欣昀；

【作者基本信息】 安徽农业大学 ， 微生物学， 2006， 硕士

【摘要】 本文围绕红曲色素的生物合成机理和混合培养对色素生物合成的影响进行研究。首先,采用刺激实验和静息细胞培养实验法来确定红曲色素的合成前体。考察了不同氨基酸和脂肪酸等对抗生素合成的影响,结果表明甘氨酸(5mmol·L-1)、缬氨酸(2mmol·L-1)、异亮氨酸(2mmol·L-1)、蛋氨酸(1mmol·L-1)、谷氨酸(2mmol·L-1)、精氨酸(1mmol·L-1)以及乙酸(2mmol·L-1)、丙酸(3mmol·L-1)等能够最大的刺激色素的生产,具有前体的作用。而且这些前体物质都具有转化为聚酮合成前体的共同特征。所以聚酮途径被认为是红曲色素合成的主要途径。为了证明红曲色素由聚酮合成,考察不同代谢途径的抑制剂对红曲色素合成的影响,结果表明莽草酸途径的抑制剂三甲胺(15mmol·L-1)和甲羟戊酸途径的抑制剂邻氨基苯甲酸(15mmol·L-1)及3,4-二羟苯甲酸(21mmol·L-1),对红曲色素的生物合成以及菌体生长没有影响;而许多聚酮合成关键酶的抑制剂对红曲色素的生物合成具有强烈的抑制作用,如β-酮酯酰-ACP合成酶专一性抑制剂碘乙酰胺、咪唑、硫酯酶抑制剂2,4-二硝基氟苯、甲基丙二酰异构酶的抑制剂Cu2+和Zn2+。其中碘乙酰胺的浓度为1mmol·L-1、咪唑浓度为1mmol·L-1、2,4-二硝基氟苯浓度为2mmol·L-1、Cu2+浓度为4mmol·L-1、Zn2+浓度为10mmol·L-1时就已经完全抑制色素合成。从而从正反两面都证明了红曲色素由聚酮途径合成。甲基转移酶抑制剂磺胺(200mg·L-1)对色素合成以及菌体生长没有影响,表明红曲色素中的甲基并非由甲基转移酶合成。研究了红曲霉与酵母菌和乳酸菌混合培养,由于酵母菌和乳酸菌生长迅速消耗了大量的营养物质,不利于红曲霉的生长和色素的分泌。但酵母菌和乳酸菌的代谢产物能促进红曲色素的合成。更多还原

【Abstract】 The biosynthesis mechanism of monascus pigment and effect of mix-culture on pigment biosynthesis were studied in this dissertation.First, feeding experiment and resting cell system were used to confirm precursors of pigments. The effects of different amino acids and branched fatty acids on pigments production were investigated, and the results showed those amino acids and branched fatty acids, such glycine ( 5mmol·L-1 )、valine(2mmol·L-1)、isoleucine(2mmol·L-1)、methionine(1mmol·L-1)、glutamine(2mmol·L-1)、arginin(e1mmol·L-1)、acetat(e2mmol·L-1)、propionat(e3mmol·L-1)could act as precursors and mostly stimulate pigments biosynthesis. Besides, all of these compounds could be transformed to the precursors of polyketide biosynthesis. So the ployketide pathway was presumed to be the main biosynthetic pathway of monascus pigments.In order to testify the hypothesis above, influences of different metabolic pathways’ inhibitors on monascus pigment biosynthesis were examined and the following results were obtained. Inhibitor of shikimic acid pathways -trimethylamine or inhibitors of mevalonic acid pathways-anthranilic acid and 3,4-dihydroxybenzoic acid had no effects on pigment biosynthesis. Whereas, pigment biosynthesis was severely inhibited by inhibitors of the key enzymes in the polyketide pathways, such as the specific inhibitors ofβ-ketoacyl-acylcarrier protein synthase-iodiacetamide or imidazole, the thioesterase inhibitor- 2,4-dinitrofluorobenzene, methyl-malonylCoA isomerase Cu2+, Zn2+. Besides,pigment biosynthesis was totally inhibited when added 1mmol·L-1 iodiacetamide, 1mmol·L-1 imidazole, 2mmol·L-1 2,4-dinitrofluorobenzene, 4mmol·L-1 Cu2+, 10mmol·L-1Zn2+. Polyketide pathway was therefore confirmed as the main biosynthetic pathways of monascus pigment. It had no effects on pigment biosynthesis when added 200mg·L-1 sulphanilamide .Because the growth of lactic acid bacteria and saccharomyces were faster than monascus, they quickly consumed lots of nutrient substance. Mixed culture is adverse to monascus growth and pigment excretion. But the metabolic products of them are good for pigment excretion.更多还原