Martin Judith A, Murphy Richard A, Power Ronan F G
Alltech Ireland, Sarney, Summerhill Rd., Dunboyne, Co. Meath, Ireland.
J Ind Microbiol Biotechnol. 2003 Sep;30(9):568-76. doi: 10.1007/s10295-003-0083-8. Epub 2003 Aug 28.
In an effort to produce phytases cost-effectively, and to determine the efficiency of a novel expression system, the genes for Aspergillus awamori ( phyA) phytase and Aspergillus fumigatus ( phyA) phytase (a putative thermostable enzyme) were cloned and overexpressed in A. awamori. Regulation of phytase expression was achieved by separately placing the genes under the transcriptional control of the glucoamylase A ( glaA) promoter of A. awamori. A gene fusion strategy was employed that involved the insertion of a hexapeptide Kex-2 protease cleavage site between the native glucoamylase and heterologous proteins and allowed for the efficient secretion and processing of the resultant chimeric proteins produced in this system by an endogenous Kex-2 protease. The genes for both of the above-mentioned phytases have already been cloned; however, this is the first report of either of the two phytases being fused with the glucoamylase gene, placed under the transcriptional control of the glaA promoter and overexpressed in A. awamori. Following transformation of A. awamori with separate expression vectors (one for each phytase), induction of phytase expression in submerged culture was effected by utilisation of a starch-containing medium. Optimisation of heterologous protein production in small shake-flask cultures involved changes in medium constituents. Maximum phytase expression levels of 200 phytase units (PU) ml(-1) and 62 PU ml(-1) for recombinantly expressed phytases from A. awamori and A. fumigatus, respectively, were obtained in crude fermentation extracts. Subsequent process scale-up to 4 l batch fermentation yielded phytase production levels comparable to those obtained on small scale. The enzyme yields herein reported demonstrate that the expression system developed and the host strain utilised were capable of expressing phytase at levels comparable to, or exceeding, previously reported data.
为了经济高效地生产植酸酶，并确定一种新型表达系统的效率，泡盛曲霉（phyA）植酸酶基因和烟曲霉（phyA）植酸酶基因（一种假定的耐热酶）被克隆并在泡盛曲霉中过表达。通过将基因分别置于泡盛曲霉糖化酶A（glaA）启动子的转录控制之下，实现了植酸酶表达的调控。采用了一种基因融合策略，即在天然糖化酶和异源蛋白之间插入一个六肽Kex-2蛋白酶切割位点，使得该系统中产生的嵌合蛋白能够通过内源性Kex-2蛋白酶进行有效分泌和加工。上述两种植酸酶的基因均已被克隆；然而，这是关于这两种植酸酶中的任何一种与糖化酶基因融合、置于glaA启动子的转录控制之下并在泡盛曲霉中过表达的首次报道。在用单独的表达载体（每种植酸酶一个）转化泡盛曲霉后，通过使用含淀粉的培养基在深层培养中诱导植酸酶表达。在小摇瓶培养中优化异源蛋白生产涉及培养基成分的改变。在粗发酵提取物中，泡盛曲霉和烟曲霉重组表达的植酸酶的最大植酸酶表达水平分别达到200植酸酶单位（PU）ml-1和62 PU ml-1。随后将工艺放大至4升分批发酵，得到的植酸酶产量与小规模实验相当。本文报道的酶产量表明，所开发的表达系统和所使用的宿主菌株能够以与先前报道的数据相当或更高的水平表达植酸酶。
