Acquisition of organic solvent tolerance using molecular chaperone in E.coli

在涉及有机溶剂-水相的全细胞生物过程中，微生物对有机溶剂耐受的分子机制变得越来越重要，这些过程涉及在水中溶解度相对较低的底物的生物转化。本研究通过引入分子伴侣构建了耐有机溶剂突变体。(1)来自超嗜热古菌的预折叠素增加了有机溶剂耐受性。极端微生物酶的分子稳定性一般反映了母体微生物的生长温度，并与变性酶（包括有机溶剂）存在下的稳定性正相关。来自嗜热古细菌堀堀焦球菌OT3 （PhPfd）的前折叠蛋白的表达增加了大肠杆菌细胞对有机溶剂的耐受性。在添加10%体积的辛烷和己烷有机溶剂混合物后，过表达PhPfd的细胞可以连续生长。过量表达PhPfd的细胞胞内水平低于对照细胞。古细菌前折叠蛋白由两种亚基组成，在水母样结构中以α2β4的六聚体存在。具有低伴侣活性的PhPFD截断突变体的过表达导致有机溶剂耐受性降低。由此可见，前折叠蛋白的分子伴侣活性导致大肠杆菌对有机溶剂的耐受性增强。(2)添加有机溶剂时，通过转录分析中上调基因的过表达构建耐有机溶剂突变体。从基因表达谱中选择的manXYZ过表达可提高大肠杆菌JA300菌株的有机溶剂耐受性。细胞可以在体积为10%的己烷和环己烷的有机溶剂混合物中生长。由于ManXYZ在细胞膜上的过表达改变了细胞表面特性，使其对碳氢化合物具有更高的亲和力，因此该菌株将在双相系统的全细胞生物过程中发挥作用。在葡萄糖分解代谢相关的全局调控基因分析中，发现环AMP受体蛋白（crp）和腺苷酸环化酶（cyaA）的敲除突变体增加了它们对有机溶剂的耐受性。在crp敲除突变体的转录分析中，与分子伴侣相关的基因被上调。少

The molecular mechanisms in organic solvent tolerance of microorganisms have become increasingly important in whole-cell bioprocesses involving an organic solvent-aqueous phase for the bioconversion of substrates that have relatively low solubility in water. In the present study, organic solvent tolerant mutants were constructed by introducing molecular chaperones.(1) Prefoldin from the hyperthermophilic archaea increased organic solvent tolerance.Molecular stability of extremophile enzymes generally reflects the growth temperatire of parent microorganisms and positively correlated with the stability in the presence of denaturing enzymes including organic solvents. Expression of prefoldin from hyperthermophilic archaeum, Pyrococcus horikoshii OT3 (PhPfd) resulted in increased organic solvent tolerance in Escherichia coli cells. The cells overexpressing PhPfd could grow continuously after addition of organic solvent mixture of octane and hexane at 10% volume. The intracellular level of … More cells overexpressing PhPfd was kept lower than the control cells. Archaeal prefoldin consists of two species of subunits and exists as a hexamer of α2β4 in jellyfish like structure. Overexpression of PhPFD truncation mutants that have low chaperone activities resulted in decreased organic solvent tolerance. Therefore, it was shown that molecular chaperone activity of prefoldin resulted in increased organic solvent tolerance in E.coli.(2) Construction of organic solvent tolerant mutants by overexpression of upregulated genes in transcriptional analysis when addition of organic solvents.Overexpression of manXYZ that was selected from gene expression profile was shown to increase organic solvent tolerance level of E.coli JA300 strain. The cells could grow in an organic solvent mixture of hexane and cyclohexane at 10% volume. Since overexpression of ManXYZ in cell membrane changed cell surface property to obtain higher affinity to hydrocarbons, this strain would be useful in whole-cell bioprocesses in biphasic systems. In the analysis of global regulatory genes related to glucose catabolism, knockout mutants of cyclic AMP receptor protein (crp) and adenylate cyclase (cyaA) was found to increase their tolerance to organic solvents. In the transcriptional analysis of a crp knockout mutant, genes related to molecular chaperones were upregulated. Less