Yang V W, Marks J A, Davis B P, Jeffries T W
Forest Products Laboratory, U.S. Department of Agriculture, Madison, Wisconsin 53705.
Appl Environ Microbiol. 1994 Dec;60(12):4245-54. doi: 10.1128/aem.60.12.4245-4254.1994.
This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.
本文描述了用于木糖发酵酵母树干毕赤酵母的首个高效转化系统。该系统包括基于乳清苷-5'-磷酸脱羧酶（URA3）基因和从树干毕赤酵母CBS 6054分离的自主复制序列（ARS）元件（ARS2）构建的整合型和自主复制型质粒。通过选择对5-氟乳清酸的抗性获得尿嘧啶营养缺陷型菌株，并通过用树干毕赤酵母URA3转化鉴定为ura3突变体。树干毕赤酵母URA3通过与酿酒酵母URA3的同源性克隆，其编码区与酿酒酵母URA3的同源性为69%。树干毕赤酵母ARS元件通过质粒拯救进行功能克隆。当这些序列克隆到携带树干毕赤酵母URA3基因的载体中时，可赋予自主复制能力。树干毕赤酵母ARS2具有与酿酒酵母共有ARS及其他ARS元件相似的特征。携带树干毕赤酵母URA3基因及不同长度侧翼序列的环状质粒通过电穿孔法每微克DNA可产生600至8600个尿嘧啶原养型转化子。用环状载体获得的大多数转化子产生时未发生载体序列整合。一个载体在树干毕赤酵母URA3插入片段内的不同限制性酶切位点线性化后，每微克DNA可产生5200至12500个尿嘧啶原养型转化子。由线性化载体产生的转化子形成稳定的整合体，在所检测的20%的转化子中，整合事件对基因组ura3具有位点特异性。携带树干毕赤酵母URA3基因和ARS2元件的质粒每微克质粒DNA可产生超过30000个转化子。自主复制质粒在选择培养基中至少稳定50代，平均每个细胞核中有10个拷贝。
