中文摘要：
维生素C是一种人体所必需的水溶性维生素,在体内不能合成,只能从外界摄取以维持机体正常的生命活动。工业维生素C的生产主要采用“二步发酵法”,其中第二步是由产酸菌和伴生菌共同组成的混菌发酵体系来完成,将L-山梨糖转化为维生素C的前体物质2-酮基-L-古龙酸(2-KGA)。产酸菌为普通生酮基古龙酸菌(Ketogulonicigenium vulgare,K.vulgare),具备糖酸转化的全部酶系,单独生长极其缓慢,产酸能力弱;伴生菌大多属于芽孢杆菌属,不具备糖酸转化的酶系,单独生长容易,不产酸,但能促进产酸菌生长和产酸。混菌发酵体系中两菌的关系为互利共生,但其具体互作机制并不完全明确。由活性氧引起的氧化胁迫一直制约着2-KGA产量的提高,本文通过对混菌发酵过程B.endophyticus ST-1解除K.vulgare 25B-1氧化胁迫机制进行解析,将有助于探究两菌互作的分子机制。同时为后续2-KGA产量的提高,奠定了理论和方法学基础。主要研究结果如下:(1)以普通生酮基古龙酸菌(K.vulgare 25B-1)和内生芽孢杆菌(B.endophyticus ST-1)为研究对象,建立单菌发酵体系和混菌发酵体系。K.vulgare 25B-1单独发酵时,2-KGA产量为7.64±1.4 mg/mL,ORP值在-28 mV上下波动;B.endophyticus ST-1单独发酵时,ORP值维持在-234 mV~-208 mV之间;混菌发酵体系中,2-KGA产量53.85±0.6 mg/mL,ORP值维持在-184 mV~-168 mV之间。对混菌发酵体系中B.endophyticus ST-1进行蛋白质组学分析,B.endophyticus ST-1共筛选到83个差异蛋白(40 h/18 h),其中上调蛋白45个,下调蛋白38个。GO富集和KEGG Pathway分析表明混菌发酵体系中B.endophyticus ST-1胞内糖代谢相关途径的差异蛋白表现为上调,胞内代谢能力加强,为细胞提供丰富的能量ATP和还原力NADPH,而糖代谢以外的代谢通路中差异蛋白表现为下调,可以为K.vulgare 25B-1提供充足的中间代谢产物。对B.endophyticus ST-1氧化应激相关蛋白进一步筛选,氧化应激相关的共有蛋白有:硫氧还蛋白还原酶、硫氧还蛋白过氧化物酶和硫氧还蛋白;氧化应激相关的差异蛋白有:铁硫蛋白、黄素腺嘌呤二核苷酸和二氢硫辛酰胺脱氢酶。氧化应激相关蛋白的存在,可以直接或间接清除体系中存在的活性氧,缓解氧化胁迫。此外,B.endophyticus ST-1可通过调控硒化合物代谢通路,积累硒代半胱氨酸,维持谷胱甘肽氧化还原系统和硫氧还蛋白系统中关键酶的活性,清除体系中的活性氧,促进K.vulgare 25B-1生长与产酸。(2)外源添加谷胱甘肽于混菌发酵体系中,GSH终浓度为1 mg/mL和GSH/GSSG浓度比为50:1时均能提高2-KGA产量,缩短发酵周期,其中添加GSH/GSSG的混菌发酵体系效果更佳,表现为2-KGA产量达到72.44±0.84 mg/mL,相比Co-culture体系中2-KGA产量提高了40.63%,发酵周期缩短24 h。GSH有利于维持B.endophyticus ST-1单个细胞的游离状态,为K.vulgare 25B-1提供更多的吸附面积;GSH/GSSG体系中K.vulgare 25B-1和B.endophyticus ST-1表现出大量生长聚集成团状的现象,保证了混菌发酵体系中菌数充足;混菌发酵体系中K.vulgare 25B-1存在膜壁分离现象;GSH/GSSG体系中K.vulgare 25B-1存在细胞膜面积增大现象,有利于细胞膜上山梨糖脱氢酶和山梨酮脱氢酶的表达。对于混菌发酵体系中B.endophyticus ST-1来说,胞内含有多个大小不一的“腔室”结构,GSH/GSSG体系中“腔室”结构数量更明显。进一步对混菌发酵体系中K.vulgare 25B-1和B.endophyticus ST-1相关抗氧化酶活和相关抗氧化基因表达量分析,谷胱甘肽主要通过调控胞内外T-AOC、T-SOD、CAT酶活力和基因sod、gst、gr、g6pd、gp表达,从而清除活性氧,缓解氧化胁迫,促进菌体生长和产酸。对T-AOC、T-SOD、CAT酶活力测定发现,GSH单独添加时主要清除胞内液中活性氧;GSH和GSSG共同添加时主要清除胞外液中活性氧。(3)对B.endophyticus ST-1中spx基因进行克隆,与B.endophyticus Hbe603相比,同源性可达94.14%。生物信息学分析显示Spx为核内蛋白质,蛋白分子量为15 kDa,具有亲水性,为水溶性蛋白;不含有跨膜结构域,但Spx蛋白的N端可能有一个强的跨膜螺旋;Spx蛋白二级结构主要以α-螺旋形式存在,三级结构中含有5个含硫氨基酸,包括3个甲硫氨酸和2个半胱氨酸。对单菌发酵和混菌发酵体系中B.endophyticus ST-1胞内spx基因表达分析发现,单菌发酵体系中spx基因表达呈现先下降后上升的趋势,且在24 h表达达到最低;混菌发酵体系中spx基因表达趋势为先上升后下降,且在24 h达到最高;40 h和48 h时,单菌发酵体系中和混菌发酵体系中spx基因相对表达量和增长趋势无明显差异。混菌发酵体系中B.endophyticus ST-1受到K.vulgare 25B-1以及发酵体系中活性氧的影响,触发胞内调控氧化应激的Spx调节因子,spx基因的表达上调有利于调控谷胱甘肽氧化还原系统和硫氧还蛋白系统关键酶的活性,缓解氧化胁迫,保证了伴生菌胞内代谢的平衡,从而更好地为K.vulgare 25B-1提供营养物质和维持良好的发酵微环境,促进其生长和产酸。
英文摘要：
Vitamin C is a water-soluble vitamin that is essential for the human body,but it cannot be synthesized and only can be taken in from food to maintain the normal activities of the body.The production of industrial vitamin C mainly uses “two-step fermentation”,in which the second step is completed by a co-culture fermentation system consisting of acid-producing strain and companion strain to convert L-sorbose into 2-Keto-L-gulonic acid(2-KGA,the precursor of vitamin C).The acid-producing strain is Ketogulonicigenium vulgare(K.vulgare),which includes all enzymes to convert L-sorbose to 2-KGA,it shows poor growth and low 2-KGA production ability in mono-culture fermentation system.Most of the companion strain belongs to Bacillus genus and lacks enzymes to convert L-sorbose to 2-KGA,it shows high growth in mono-culture fermentation system and can promote the growth of K.vulgare and 2-KGA production in co-culture fermentation system.The relationship between K.vulgare and companion strain is mutually symbiosis in co-culture fermentation system,but the interaction mechanisms between them are not very clear.Oxidative stress caused by reactive oxygen species has always restricted the increase of 2-KGA production.In this paper;the mechanism of oxidative stress can be studied in co-culture fermentation system composed of K.vulgare 25B-1 and B.endophyticus ST-1,which will help to explore the interaction of molecular mechanism between the two strains.At the same time,it can give the basis ideas for theoretical and methodological foundations to improve 2-KGA production.The main results are as follows:(1)Establishing mono-and co-culture fermentation system composed of K.vulgare 25B-1 and B.endophyticus ST-1.It was found that the 2-KGA production was 7.64 ± 1.4 mg/m L in mono-culture fermentation system by K.vulgare 25B-1,and the value of ORP was fluctuated around-28 m V.In mono-culture fermentation system by B.endophyticus ST-1,the value of ORP was maintained at around of-234 m V ~ 208 m V.In co-culture fermentation system composed of K.vulgare 25B-1 and B.endophyticus ST-1,2-KGA production was 53.85 ± 0.6 mg/m L,and the value of ORP was maintained at around of-184 m V ~-168 m V.Furthermore,proteomics of B.endophyticus ST-1 was carried out in co-culture fermentation system.Total of 83 differently expressed proteins(40 h/18 h)were screened from B.endophyticus ST-1 in co-culture fermentation system,among which 45 proteins were up-regulated and 38 proteins were down-regulated.GO enrichment and KEGG pathway showed that differently expressed proteins participated in relative glucose metabolism pathways of B.endophyticus ST-1 were up-regulated,which enhanced intracellular metabolic capacity and provides cells with more energy ATP and reducing power NADPH.The differently expressed proteins in the metabolic pathways other than glucose metabolism were down-regulated,which could provide sufficient intermediate metabolites for K.vulgare 25B-1.Oxidative stress-related of common proteins from B.endophyticus ST-1 included thioredoxin oxidoreductase,thioredoxin peroxidase and thioredoxin;oxidative stress-related of differently expressed proteins included iron-sulfur protein,flavin adenine dinucleotide and dihydrolipoamide dehydrogenase.The presence of oxidative stress-related proteins could directly or indirectly remove active oxygen and alleviate oxidative stress.In addition,B.endophyticus ST-1 could accumulate the selenocysteine through regulating the selenocompound metabolism process,which could maintain the activity of key enzymes in the glutathione redox system and the thioredoxin system to scavenge reactive oxygen species in the system,and enhance the growth of K.vulgare 25B-1 and 2-KGA production.(2)By adding glutathione into co-culture fermentation system,it was found that the final concentration of GSH was 1 mg/m L and the concentration ratio of GSH/GSSG was 50:1,which could increase 2-KGA production and shorten the fermentation cycle.The highest 2-KGA production was achieved at 72.44 ± 0.84 mg/m L while adding GSH/GSSG into co-culture fermentation system,which was 40.63% higher than that of co-culture fermentation system without addition GSH and GSSG,and the fermentation cycle was shortened by 24 h.The addition of GSH maintained the single cell state of B.endophyticus ST-1,and provided more adsorption areas for K.vulgare 25B-1.In GSH/GSSG fermentation system,it shown a large growth of K.vulgare 25B-1 and B.endophyticus ST-1 and aggregated phenomenon,which ensured the sufficient of cells in co-culture fermentation system;K.vulgare 25B-1 has membrane wall separation phenomenon.In addition,the increasing of cell membrane areas in GSH/GSSG fermentation system was significant,which could beneficial for the expression of sorbose dehydrogenase and sorbitol dehydrogenase.In co-culture fermentation system,B.endophyticus ST-1 had many "chambers" with different sizes,and the number of "chambers" was more in GSH/GSSG fermentation system.Further analysis of related enzyme activities and related antioxidant gene expression of K.vulgare 25B-1 and B.endophyticus ST-1,it revealed that glutathione mainly regulated through T-AOC,T-SOD,CAT activity and gene expression of sod,gst,gr,g6 pd,gp,thereby scavenged reactive oxygen species and alleviated oxidative stress to promote cell growth and 2-KGA production.For the determination of T-AOC,T-SOD and CAT activity,it was found that reactive oxygen species in the intracellular fluid was mainly removed by GSH with adding alone;reactive oxygen species in the extracellular fluid was mainly removed by adding GSH and GSSG together with ratio of 50:1.(3)The spx gene of B.endophyticus ST-1 was cloned.The homology of spx in B.endophyticus ST-1 is 94.14% compared with that of B.endophyticus Hbe603.Spx is a water-soluble nuclear protein with a molecular weight of 15 k Da through bioinformatics analysis;Spx does not contain a transmembrane domain,but its internal N-terminus may have a strong transmembrane helix;the secondary structure of Spx protein presents mainly with the alpha-helical forms,and its tertiary structure contains five sulfur-containing amino acids,which includes three methionine and two cysteine.The expression of spx gene of B.endophyticus ST-1 in mono-and co-culture fermentation system showed that: the expression trend of spx gene was decreased and then increased,and reached the lowest at 24 h in the mono-culture fermentation system;the expression trend of spx gene was increased and then decreased,and reached the highest at 24 h in co-culture fermentation system.At 40 h and 48 h,the expression and increase tendency of spx gene was no significant difference in mono-and co-culture fermentation system.In co-culture fermentation system,B.endophyticus ST-1 was stimulated by K.vulgare 25B-1 and reactive oxygen species,which triggered the Spx regulatory factor to regulate oxidative stress,and then caused the expression of spx gene to be up-regulated,which was beneficial for regulating the activity of key enzymes of glutathione redox system and thioredoxin system,and alleviated oxidative stress to maintain the balance of intracellular metabolism of B.endophyticus ST-1,which could provide more intermediate metabolites to K.vulgare 25B-1 and maintain a favorable micro-environment for the fermentation system,to promote its growth and 2-KGA production.
