| 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. |
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