An Actinobacillus succinogenes plasmid vector which provides a means to overexpress proteins in A. succinogenes . The plasmid can be transformed efficiently by electroporation, and replicates in a stable manner in A. succinogenes . The plasmid comprises at least one marker gene, operably linked to a first promoter functional in Actinobacillus succinogenes , an origin of replication functional in Actinobacillus succinogenes , a second promoter isolated from Actinobacillus succinogenes , and a cloning site downstream from the second promoter. Plasmids pLGZ901, pLGZ920, pLGZ921, and pLGZ922 are disclosed. The pckA gene polypeptide sequence and nucleic acid sequence of Actinobacillus succinogenes , including the promoter and ribosome binding site, is disclosed. Furthermore, a method for producing a recombinant Actinobacillus succinogenes is described, including a method of transformation. Additionally, a recombinant Actinobacillus succinogenes is disclosed and a method for producing succinate utilizing this recombinant Actinobacillus succinogenes is described.
1. A method for producing succinate comprising:
(a) providing Actinobacillus succinogenes transformed with a plasmid comprising at least a 1.5 kB PstI-HaeII fragment of a pGZRS-1 replicon so as to be capable of autonomous replication in Actinobacillus succinogenes , at least one selectable marker gene which is expressed in Actinobacillus succinogenes so as to confer resistance to an antibiotic selected from the group of ampicillin, tetracycline, and chloramphenicol, and a recombinant gene expressed under the control of an Actinobacillus succino genes pckA promoter;
(b) providing a growth medium for culturing the transformed Actinobacillus succinogenes ; and
(c) culturing the transformed Actinobacillus succinogenes in the growth medium to produce the succinate.
2. The method of claim 1 wherein the promoter comprises substantially the nucleic acid sequence set forth in SEQ ID NO: 21 from between about nucleotide 25 and nucleotide 255.
3. The method of claim 1 wherein the plasmid is pLGZ902.
4. The method of claim 2 wherein the growth medium comprises an antibiotic selected from the group of ampicillin, tetracycline, and chloramphenicol.
5. The method of claim 1 wherein the plasmid further comprises a ColE1 origin of replication.
6. The method of claim 5 wherein the plasmid is pLGZ920 comprising the recombinant gene under control of the Actinobacillus succinogenes pckA promoter of pLGZ920.