【作者】 朱亮；

【导师】 高海春； 吴雪昌；

【作者基本信息】 浙江大学 ， 微生物学， 2013， 博士

【摘要】 革兰氏阴性菌少动鞘氨醇单胞菌(Sphingomonas elodea ATCC31461)能发酵产结冷胶。结冷胶作为悬浮剂、凝胶剂、稳定剂、增稠剂、乳化剂、赋形剂等可应用于食品、医药、化工等领域,并且可以取代琼脂等同类食品添加剂,具有广阔的应用前景,市场需求量在逐年增加。然而,结冷胶产率相对较低,用于通氧搅拌的能耗高、下游提取纯化耗费大等问题限制了结冷胶的推广应用。大多数的鞘氨醇单胞菌能产生黄色类胡萝卜素—念珠藻黄素。但目前为止,类胡萝卜素合成相关基因还没有在鞘氨醇单胞菌中被鉴定。少动鞘氨醇单胞菌ATCC31461在发酵产结冷胶的同时也积累黄色类胡萝卜素,其中大部分为念珠藻黄素。此外,在整个发酵过程中,发酵液粘度不断增加,至发酵结束时,粘度变得很高,不仅影响发酵效率,还影响结冷胶的提取和纯化。在传统的结冷胶提取方法中,大量的异丙醇或乙醇被用于沉淀多糖、去除色素和其它杂质,从而增加了结冷胶的生产成本。本论文围绕结冷胶生产过程的优化(包括菌种选育和结冷胶提取方法的优化)以及克隆和鉴定少动鞘氨醇单胞菌ATCC31461念珠藻黄素合成相关基因等方面进行了系统研究,为提高结冷胶生产技术水平及降低生产成本、解析念珠藻黄素的代谢机理奠定了基础。主要研究结果如下：1、生产菌株的诱变选育对少动鞘氨醇单胞菌Sphingomonas elodea ATCC31461进行EMS、紫外复合诱变,筛选获得了一株黄色素生成缺陷的结冷胶高产突变株,命名为S. elodea βm007。突变株的结冷胶产量较亲株提高了13%,其所产结冷胶的甘油酰基和乙酰基含量比亲株所产结冷胶分别高了17%和65%。此外,pm007发酵液相比野生型菌株在室温下更稳定。以上结果表明,pm007相比野生型菌株更适用于高酰基结冷胶的大规模工业化生产,并且可以简化结冷胶提取方法中的脱色工艺,降低生产成本。2、克隆和敲除了八氢番茄红素脱氢酶基因克隆了少动鞘氨醇单胞菌ATCC31461的参与类胡萝卜素合成的八氢番茄红素脱氢酶基因(crtl)并对基因功能进行了分析。crtI基因全长为1479bp,编码492个氨基酸,其氨基酸序列与数据库中存在的其它八氢番茄红素脱氢酶序列有很高的同源性。敲除crtI后获得的重组菌株△crtI不产黄色的念珠藻黄素,而是积累无色的八氢番茄红素,从而确认了crtI为八氢番茄红素脱氢酶基因。进一步的发酵实验表明,△crtI重组菌株的结冷胶产量与野生型菌株相比没有明显的区别,因而可以选用△crtI重组菌株代替ATCC31461进行结冷胶的发酵生产,从而简化结冷胶提取方法中的脱色工艺。3、建立了一种高效经济的结冷胶提取方法基于突变株βm007和△crtI重组菌株在结冷胶发酵过程中不产黄色类胡萝卜素这一特点,设计了一种新的结冷胶提取方法并与传统的提取方法进行了比较。在新的提取方法中,200g/L的CaCl2溶液用于絮凝结冷胶,30%发酵液体积的乙醇用于沉淀多糖。新的方法对于△crtI重组菌株发酵液的提纯得率与传统方法对于野生型菌株发酵液的提纯得率相当。与传统的提取方法使用两倍发酵液体积的乙醇用于提取结冷胶相比,新方法所使用的乙醇量大大减少,从而可以降低结冷胶的生产成本。4、念珠藻黄素合成相关基因的克隆和功能分析克隆了少动鞘氨醇单胞菌ATCC31461的类胡萝卜素生物合成基因簇和位于基因簇外的p-胡萝卜素羟化酶基因(crtZ),基因簇中包含4个类胡萝卜素合成基因crtG.crtY、crtI和crtB. ATCC31461的类胡萝卜素合成基因与Brevundimonas(也合成念珠藻黄素)的相应类胡萝卜素合成基因具有中等程度的同源性(43%-58%)。对各个类胡萝卜素合成基因进行了敲除,用色谱和吸收光谱分析各基因缺失菌株的类胡萝卜素成分,鉴定了各个类胡萝卜素合成基因的功能。对两个羟基化p-胡萝卜素的羟化酶基因—crtZ和crtG(2,2’-β-羟化酶基因)在大肠杆菌中进行了表达,结果进一步验证了这两个基因的功能。crtG分别在积累玉米黄质和p-胡萝卜素的大肠杆菌中表达时,转化子分别积累念珠藻黄素和2,2’-二羟基-p-胡萝卜素。基于以上结果,解析了少动鞘氨醇单胞菌ATCC31461中类胡萝卜素的生物合成途径。更多还原

【Abstract】 Sphingomonas elodea ATCC31461was isolated as a Gram-negative bacterium capable of producing gellan gum. Gellan has been used as a suspending, gelling, stabilizing, thickening, emulsifying, texturing agent in the food, pharmaceutical, chemical and other industries. Moreover, its potential use as a replacement for gelatin and agar makes it the most important commercialized bacterial exopolysaccharide, and market demand for it has been increasing. However, its relatively low productivity, high stirring energy consumption and costly downstream purification process limit its economic viability. Most Sphingomonas synthesize a yellow carotenoid identified as nostoxanthin. However, the genes responsible for nostoxanthin synthesis have not been identified from any Sphingomonas species so far. During the synthesis of gellan, Sphingomonas elodea ATCC31461also accumulates yellow carotenoid pigments, mainly nostoxanthin. Furthermore, the viscosity of broth dramatically increases throughout the fermentation process and reaches a high value at the end of the process. These characteristics not only affect fermentation efficiency but also make it difficult to separate gellan from the fermentation broth. In the general procedure for recovering gellan, large amounts of isopropanol or ethanol have been used for precipitating the biopolymer, as well as removing pigments and other impurities, thereby increasing production costs. This study is aimed to optimize gellan production (including strain improvent and develop a low-cost method for gellan recovery) as well as cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC31461. The main results are as follows:1. Strain improvement by mutation. A carotenoid-free mutant strain of ATCC31461(βm007) was isolated by ethyl methanesulfonate mutagenesis combined with ultraviolet irradiation. The final gellan production of the mutant strain was13%higher than that of the parent strain. The glycerate and acetate substituents of gellan produced by the mutant strain were17%and65%higher than that produced from ATCC31461, respectively. Stability tests suggested that the broth produced by the mutant strain was more stable during storage at room temperature compared with that produced by the parent strain. The mutant strain seems to be an ideal strain for use in the commercial production of gellan.2. Cloning and knockout of phytoene desaturase gene. The phytoene desaturase gene(crtl) in the carotenoid bio synthetic pathway of S. elodea ATCC31461was cloned and identified. This gene contained1479bp and encoded a492-amino acid protein with significant homo logy to the phytoene desaturase of other carotenogenic organisms. Knockout of crtl gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production and cell growth by crtl gene knockout mutants (△crtl) is almost the same as that by the wild-type strain. Consequently, the△crtl mutant can be used as an industrial gellan gum-producing strain in place of ATCC31461.3. Develop a low-cost method for gellan recovery. Based on the non-pigment fermentation broths of βm007and the△crtl mutant, we designed and analyzed an economical recovery method. In this method, a200g/L CaCl2solution was used to flocculate gellan, followed by precipitation with30%ethanol (v/v). The recovery yield of gellan (including the unavoidable impurities) obtained from△crtl through this new method was quite close to that from ATCC31461recovered via the conventional method. Compared to the volume of alcohol for the parent strain (2volumes of broth), much less alcohol (30%) is required in the new recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.4. Cloning and characterization of genes involved in nostoxanthin biosynthesis. A carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtl and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster were cloned and characterized. The individual ATCC31461carotenogenic gene products showed moderate identity to the corresponding proteins derived from Brevundimonas (also synthesize nostoxanthin), ranging from43%to58%. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the2,2’-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of P-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and p-carotene carotene accumulating E. coli cells resulted in the formation of nostoxanthin and2,2’-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC31461is proposed.更多还原