Open AccessGenetic engineering of Lactobacillus diolivorans
Author(s) -
Pflügl Stefan,
Marx Hans,
Mattanovich Diethard,
Sauer Michael
Publication year - 2013
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/1574-6968.12168
Subject(s) - plasmid , biology , transformation (genetics) , gene , heterologous , heterologous expression , expression vector , homologous chromosome , microbiology and biotechnology , genetics , recombinant dna
In this study, we developed a toolbox for genetic manipulation of L actobacillus 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.