Analysis Of Physiological Relationship Between Vitamin C Producing Stains Based On Biochemical Strategy And Omics Techniques

This dissertation chose Ketogulonicigenium vulgare and Bacillus megaterium, the industrial strains for production of vitamin C, as a model system to study the reciprocal physiological dependence in the mixed culture fermentation. The promotion role of B. megaterium on the growth of K. vulgare and 2-KLG production was firstly demonstrated in this dissertation, then the completed genome of K.vulgare and B.megaterium were sequenced and annotated, and the proteomic response in this co-culture system was investigated in detailed. The main results were described as follows:1. In order to increase K. vulgare cell growth and 2-KLG efficiency, B. megaterium growth status in the co-culture was manipulated by lysozyme, a enzyme catalyzes the degradation of bacterial peptidoglycan (PG) by hydrolyzing the glycosidic bond between the C-1 of N-acetylmuramic acid and the C-4 of N-acetylglucosame. Lysozyme is specific to damage B. megaterium cell wall structure and subsequently inhibits its cell growth. However, the growth of K. vulgare was not affected even 10,000 U/mL lysozyme was presented in the broth. The addition of 10,000 U/mL lysozyme at 12 h increased the biomass of K. vulgare, the L-sorbose consumption rate and 2-KLG productivity by 27.4%, 37.1%, and 28.2%, respectively, compared to the control (no lysozyme addition). As a result, the fermentation time decreased to 56 h, 20.6% shorter the control.2. The whole genome sequence of K. vulgare WSH-001 was released by a strategy combing Sanger shotgun approach with 454 single-end sequencing technology. The genomic feature was provided in this dissertation. The complete K. vulgare WSH-001 genome contains a single circular chromosome of 2,766,400 bp and two circular plasmids pKVU₁00 and pKVU₂00 267,986 bp and 242,715 bp, respectively. The overall G+C content of the chromosome is 61.69%, whereas the values of the plasmids are 61.33% and 62.58%, respectively. The chromosome contains 2604 protein-encoding genes, 51 tRNA-encoding genes, and 3 rRNA-encoding gene operons. Plasmids pKVU₁00 and pKVU₂00 contain 246 and 215 protein-encoding genes, respectively. According to the phylogenetic analysis of the house keeping genes, K. vulgare WSH-001 is most related to P. denitrificans PD1222 (5.24Mbp) and Rhodobacter sphaeroides ATCC 17025 (4.56 Mbp). However, the K. vulgare WSH-001 genome size is 1 Mbp smaller, this result implied that K. vulgare WSH-001 has undergone large-scale gene loss during the evolution process. A detailed inspection of the genome sequence revealed that many pathway are incomplete, such as glycolysis, amino acids and fatty acids metabolism, and biosynthesis of cofactors like folate, NAD, and biotin . The genome feature will facilitate the development of further strain engineering strategies and the better understanding of 2-KLG production machinery. Genes for 2-KLG production from D-sorbitol were identified, including genes encoding D-sorbitol dehydrogenase, L-sorbosone dehydrogenase (SNDH) and L-sorbose/L-sorbosone dehydrogenase (SSD). Among them, the sndh gene is located at pKVU₂00, the ssda1 gene at pKVU₁00. This result showed a great divergence from other four chromosomal ssd genes.3. K. vulgare cell growth and 2-KLG production were strongly dependent on B. megaterium in the co-culture fermentation. The complete B. megaterium WSH-002 genome contains a single circular chromosome of 4,047,912 bp and three circular plasmids pBME₁00, pBME₂00 and pBME₃00 of 74,613 bp, 9,699 bp and 7,006 bp, respectively. The total G+C content of the chromosome is 39.1%, whereas the values of the plasmids are 36.0% (pBME₁00), 32.2% (pBME₂00) and 33.2% (pBME₃00). The chromosome contains 5186 protein-encoding genes, 99 tRNA-encoding genes, and 10 rRNA-encoding gene operons. Plasmids pBME₁00, pBME₂00 and pBME₃00 contain 69, 11 and 14 protein-encoding genes, respectively. Except for the lack of the genes encodingθ,χ,ψsubunit gene of DNA polymerase III and the gene encoding Dam methyltransferase in DNA repair system, B. megaterium WSH-002 contains relatively completed metabolic pathways for amino acid transport and metabolism, carbohydrate transport and metabolism, transcription, translation, ribosomal structure and biogenesis, replication and recombination, signal transduction mechanisms and coenzyme transport and metabolism. Some gaps were found in the DNA repair system.4. Through using of the 2-DE electrophoresis and flight mass spectrometry technology, the proteome of K. vulgare under the conditions of single culture or co-culture with B. megaterium were carefully studied, and the effect of B. megaterium on K. vulgare physiological status were investigated in detailed. It was found that the expression level of K. vulgare intracellular protein can be promoted 30% with the presence of B. megaterium. Only 19 proteins existed in the single culture case, while 119 proteins were expressed in the co-culture case. Of the identified proteins on 2-D eletrophoresis gel pI 4-7 in K. vulgare in the co-culture, amino acid transport and metabolism was the highest proportion (about 20% of the total protein), followed by translation, ribosomal structure (about 10.5% of the total protein) and biogenesis, energy production and conversion(about 8.6% of the total protein). The 151 proteins were found to be up-regulated in K. vulgare when co-culture with B. megaterium, among of those up-regulated protein, about 41% are regarding of amino acid transport and metabolism, translation, ribosomal structure and biogenesis and energy production and conversion. Furthermore, B. megaterium also enhanced cofactor metabolism, cell wall and cell membrane synthesis in K. vulgare;5. With the purpose of developing a synthetic medium for 2-KLG production by K. vulgare and B. megaterium, to meet the demand of reconstruction and simulation of the genome-scale metabolic model of those two strains. The actual amounts of CSLP (corn steep liquor powder) components in 18 different batches CSLP were determined. It was found that amino acids were the crucial components in CSLP, account for about 30% of dry CSLP. Metal elements accounted for 10%. In addition, seven kinds of water-soluble vitamins were also detected in CSLP. The results suggested that glycine and threonine play key role on the growth of the two strains in the co-culture system. but serine, glycine, proline, nicotinic acid and biotin were the key components that affected 2-KLG production. The L-sorbose consumption rate was determined by proline. Based on the above quantitative and qualitative analysis, an synthetic medium with the combination of CSLP components was developed by the orthogonal method, and listed as follows: 0.28 g/L serine, 0.36 g/L glycine, 0.18 g/L threonine, 0.28 g/L proline, 0.19 g/L nicotinic acid and 0.62 m g/L biotin. With this chemical synthetic medium, a higher titer (58 g/L) and yield (0.76 g/g) of 2-KLG were achieved in a 7-L jar fermentor after 28 h culture. The chemical synthetic medium will facilitate the qualitative investigation of the metabolic functions and further developing of strain engineering strategies. Especial for the progress in the elucidation of physiological relationship between of K. vulgare and B. megaterium.