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

微生物耐有机溶剂的分子机制在全细胞生物过程中变得越来越重要，该全细胞生物过程涉及有机溶剂-水相，用于在水中具有相对较低溶解度的底物的生物转化。在本研究中，通过引入分子伴侣构建了耐有机溶剂突变体。(1)来自高温古生菌的前折叠蛋白提高了对有机溶剂的耐受性，极端嗜热酶的分子稳定性一般反映了亲本微生物的生长温度，并与包括有机溶剂在内的变性酶存在时的稳定性呈正相关。从超高温古生菌中表达的前折叠蛋白(PhPfd)能提高大肠杆菌细胞对有机溶剂的耐受性。过量表达PhPfd的细胞在加入体积为10%的辛烷和正己烷的有机溶剂混合物后仍能继续生长。…在细胞内的水平更多过表达PhPfd的细胞保持在较低水平。古细菌前折叠蛋白由两种亚基组成，以α-2、β-4的六角体形式存在于水母样结构中。过表达具有低伴侣活性的PhPFD截断突变体会导致有机溶剂耐受性降低。因此，预折叠蛋白的分子伴侣活性提高了大肠杆菌对有机溶剂的耐受性。(2)通过在转录分析中表达上调的基因构建耐有机溶剂突变体，从基因表达谱中选择的ManXYZ的过表达可以提高E.ColiJA300菌株的有机溶剂耐受性。该细胞可以在体积为10%的己烷和环己烷的有机溶剂中生长。由于ManXYZ在细胞膜上的过表达改变了细胞的表面性质，从而获得了对碳氢化合物更高的亲和力，因此该菌株将在两相体系的全细胞生物过程中发挥作用。在与葡萄糖分解代谢相关的全球调控基因的分析中，发现环AMP受体蛋白(CRP)和腺苷环化酶(CyaA)的敲除突变可以提高它们对有机溶剂的耐受性。在一个CRP基因敲除突变体的转录分析中，与分子伴侣相关的基因上调。较少

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