Lindahl Ann-Louise, Olsson Mikael E, Mercke Per, Tollbom Orjan, Schelin Jenny, Brodelius Maria, Brodelius Peter E
Department of Chemistry and Biomedical Science, University of Kalmar, SE-39182, Kalmar, Sweden.
Biotechnol Lett. 2006 Apr;28(8):571-80. doi: 10.1007/s10529-006-0015-6.
The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN.PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 microg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days' batch cultivation.
青蒿中紫穗槐-4,11-二烯合酶的编码基因通过两种根本不同的方式转化到酿酒酵母中。首先，将该基因亚克隆到半乳糖诱导型、高拷贝数的酵母表达载体pYeDP60中，并用于转化酿酒酵母菌株CEN.PK113-5D。其次，受相同启动子调控的紫穗槐-4,11-二烯合酶基因通过同源重组被导入酵母基因组。在半乳糖诱导的酵母细胞的蛋白质提取物中，观察到从质粒表达该酶的酵母具有更高的活性。基因组转化的酵母与野生型酵母生长速率相同，而携带质粒的酵母生长速度比野生型酵母稍慢。在16天的分批培养过程中，质粒转化和基因组转化的酵母分别产生了600和100微克/升的青蒿素前体紫穗槐-4,11-二烯。
