School of Bioengineering, FH Campus Wien - University of Applied Sciences, Vienna, Austria.
FEMS Microbiol Lett. 2013 Jul;344(2):152-8. doi: 10.1111/1574-6968.12168. Epub 2013 May 23.
In this study, we developed a toolbox for genetic manipulation of Lactobacillus diolivorans, a promising production organism for 1,3-propanediol from glycerol. Two major findings play a key role for successful transformation of this organism: (1) the absence of a native plasmid, because a native plasmid is a major obstacle for transformation of L. diolivorans, and (2) the absence of DNA methylation. A suitable expression plasmid, pSHM, for homologous and heterologous protein expression in L. diolivorans was constructed. This plasmid is based on the replication origin repA of L. diolivorans. The native glyceraldehyde-3-phosphate dehydrogenase promoter is used for constitutive expression of the genes of interest. Functional expression of genes in L. diolivorans was shown with two examples: production of green fluorescent protein resulted in a 40- to 60-fold higher fluorescence of the obtained clones compared with the wild-type strain. Finally, the homologous overexpression of a putatively NADPH-dependent 1,3-propanediol oxidoreductase improved 1,3-propanediol production by 20% in batch cultures.
在这项研究中，我们开发了一个用于遗传操作乳酸乳球菌的工具箱，这是一种很有前途的从甘油生产 1,3-丙二醇的生产菌。两个主要发现对该生物的成功转化起着关键作用：(1) 缺乏天然质粒，因为天然质粒是乳酸乳球菌转化的主要障碍，(2) 缺乏 DNA 甲基化。构建了一个适合乳酸乳球菌同源和异源蛋白表达的合适表达质粒 pSHM。该质粒基于乳酸乳球菌的复制原点 repA。天然的甘油醛-3-磷酸脱氢酶启动子用于目的基因的组成型表达。通过两个例子证明了基因在乳酸乳球菌中的功能表达：绿色荧光蛋白的生产导致获得的克隆的荧光比野生型菌株高 40-60 倍。最后，推测 NADPH 依赖性 1,3-丙二醇氧化还原酶的同源过表达使分批培养中的 1,3-丙二醇产量提高了 20%。
