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 a recombinant Actinobacillus succinogenes comprising:
(a) providing a plasmid comprising at least one marker gene which confers resistance to an antibiotic comprising a Tn3 Amp R gene, or an ampicillin, tetracycline or chloramphenicol resistance gene operably linked to a first promoter comprising one or more pairs of −35 and −10 promoter elements of an Actinobacillus succinogenes pckA gene; an origin of replication comprising at least a 1.5 kB PstI-HaeII fragment of a pGZRS-1 replicon so as to be functional in Actinobacillus succinogenes ; a second promoter comprising one or more pairs of −35 and −10 promoter elements of the Actinobacillus succinogenes pckA gene; and a cloning site for a nucleic acid downstream of the second promoter;
(b) transforming Actinobacillus succinogenes with the plasmid; and
(c) selecting in the presence of the antibiotic the recombinant Actinobacillus succinogenes from non-transformed Actinobacillus succinogenes.
2. The method of claim 1 , wherein the transformation is electroporation.
3. The method of claim 1 wherein the recombinant Actinobacillus succinogenes is selected from non-transformed Actinobacillus succinogenes by culturing in the presence of the antibiotic.
4. The method of claim 1 wherein the second promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 21 from between about nucleotide 25 and nucleotide 255.
5. The method of claim 1 wherein the second promoter provides a ribosome binding site comprising the nucleotide sequence AGGTG.
6. The method of claim 1 wherein the plasmid further includes a ColE1 origin of replication.
7. The method of claim 1 wherein the plasmid is pLGZ901 or pLGZ920.