【作者】 张静；

【导师】 陈坚；

【作者基本信息】 江南大学 ， 发酵工程， 2010， 博士

【摘要】 本文以能在胞外大量积累2-酮基-L-古龙酸(2-KLG)的工业生产菌株酮古龙酸菌(Ketogulonicigenium vulgare)和巨大芽胞杆菌(Bacillus megaterium)组成的混菌体系为研究对象,在充分理解B. megaterium在维生素C混菌发酵体系中重要生理作用的基础上,借助两菌全基因组测序和蛋白质组学解析方法,就混菌发酵培养中B. megaterium如何促进K. vulgare生长和2-KLG高效合成的生理机制开展研究。主要研究结果如下:1.采用可定向破坏细胞壁中N-乙酰胞壁酸和N-乙酰氨基葡糖之间β-1,4糖苷键的溶菌酶以研究混菌发酵体系中调控B. megaterium生长状态对K. vulgare生长和2-KLG合成的影响。添加溶菌酶显著降低了B. megaterium的细胞存活率,但不影响K. vulgare细胞的正常生长。在B. megaterium对数末期(12 h)添加10,000 U/mL溶菌酶导致B. megaterium迅速裂解快速释放胞内活性物质,显著促进了K. vulgare生长。与对照组相比,K. vulgare最大细胞浓度和2-KLG生产强度分别提高了27.4%和28.2%,发酵周期缩短12 h;2.利用Sanger shotgun法与454高通量基因组测序(GS FLX Titanium System)相结合的测序策略,对K. vulgare WSH-001进行全基因组测序。K. vulgare WSH-001基因组全长为3.28 Mb (染色体+质粒),包括一条长度为2,766,400 bp的染色体和长度分别为267,986 bp和242,715 bp的两个质粒。进化关系分析表明,K. vulgare WSH-001比其亲缘关系最相近的P. denitrificans PD1222和R. sphaeroides KD131均少了一个1 Mb以上的小染色体,推测K. vulgare单菌生长较弱的原因在于其与伴生菌B. megaterium长期共存过程中进行了基因组缩减,丢掉了其单独生长所必需的部分基因,导致其DNA复制系统、氨基酸代谢系统、能量代谢系统、辅因子代谢系统等大量相关基因在基因组中未找到。基因组信息显示,K. vulgare WSH-001基因组中含有从D-山梨醇到2-KLG转化所需要的全部蛋白所需的基因;3.混菌培养过程中,K. vulgare细胞生长和2-KLG合成强烈依赖于B. megaterium。对B. megaterium WSH-002进行全基因组测序,得到由一条长度为4,047,912 bp的染色体和长度分别为74,613 bp、9,699 bp和7,006 bp的三个质粒组成的全长4.14 Mb基因组。对基因组进行注释发现,除了DNA修复系统中未发现编码DNA聚合酶III中θ、χ、ψ亚基的基因和Dam甲基化酶编码基因外,B. megaterium WSH-002的其他代谢途径基本完整;B. megaterium WSH-002与已经测序的B. megaterium QM B1551的亲缘关系最近,两者的16S rDNA序列的相似性为100%,核酸序列的相似性为95-97%。从进化树来看,B. megaterium WSH-002与B.cereus组群亲缘关系较近,有40%的保守基因;4.为了阐明B. megaterium对K. vulgare的伴生机制,研究了产酸稳定期K. vulgare在B. megaterium伴生与否时胞内蛋白表达差异情况。对质谱鉴定得到的419种蛋白点进行COG注释发现:(1)约有20%蛋白点与氨基酸转运与代谢相关;(2)与蛋白质翻译、核糖体结构、生物合成、能量产生与转化相关的蛋白占总鉴定蛋白的10.5%和8.6%。当B. megaterium和K. vulgare共同培养时, K. vulgare胞内总蛋白点(已表达)比K. vulgare单独培养时增加了约30%。进一步进行差异表达谱分析发现,151种蛋白在B. megaterium伴生时表达量显著升高,其中氨基酸代谢、蛋白质翻译、能量代谢等相关蛋白约占41%。此外,B. megaterum还明显促进了K. vulgare的辅因子代谢、细胞壁和细胞膜合成等;5.为了获得一种K. vulgare和B. megaterium生长和高效产酸的全合成培养基,以满足两菌基因组规模代谢网络构建与验证和定量模拟的需求。作者详尽分析了18批次玉米浆组分(约为40种,其中17种为氨基酸、9种为维生素和14种为金属元素)的含量,其中氨基酸约占30%,金属元素占10%左右,此外还含有8种水溶性维生素。玉米浆组分浓度变化高度线性关联2-KLG发酵参数的变化:丝氨酸、甘氨酸、苏氨酸、脯氨酸、烟酸和生物素这6种组分对2-KLG生产效率产生显著影响。基于上述定量测定与定性分析,结合正交实验得到影响2-KLG生产效率的关键玉米浆组分的最佳组合为:丝氨酸0.28 g/L、甘氨酸0.36 g/L、苏氨酸0.18 g/L、脯氨酸0.56 g/L、烟酸0.19 g/L、生物素0.62 mg/L。采用这一最佳营养组合,经28 h培养,K. vulgare最大细胞浓度、2-KLG产量、生产强度和转化率分别达到4.14×109 cfu/mL、58 g/L、2.07 g/(L·h)和0.76 g/g。实现了K. vulgare和B. megaterium混菌体系在全合成培养基上的快速生长和2-KLG的高效生产。更多还原

【Abstract】 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.更多还原