Construction and Characterization of Novel Shuttle Expression Vectors for Actinobacillus succinogenes

Abstract

Actinobacillus succinogenes is a ruminal microorganism of biotechnological importance due to its capacity to produce succinic acid at high yields. Despite the scientific interest in this organism, molecular vehicles for the transfer and expression of genetic material are limited compared to the existing demand. To facilitate gene cloning and expression in A. succinogenes, we report the generation and characterization of two novel shuttle expression vectors containing the chloramphenicol acetyltransferase gene (catA) as a selection marker and replication origins from A. succinogenes, other members of the Pasteurellaceae family, and Escherichia coli. Vector pAVP_trc includes the following features from the E. coli expression vector pTrc99A2: lacI^q gene, an IPTG-inducible trc promoter (P_trc), the lacZ ribosome-binding site, a multiple cloning site, and the rrnB transcription terminator. The second novel vector pAVP_mdh contains 200 bp of the promoter region (P_mdh) from the A. succinogenes malate dehydrogenase (MDH) gene Asuc_1612 (mdh). Gene mdh was cloned in the two novel vectors to generate pAVP_trcmdh and pAVP_mdhmdh. Promoter activity in these vectors was determined by measuring transcript levels with RT-qPCR analysis and MDH specific activity. In cultures with A. succinogenes/pAVP_trcmdh with 2 mM IPTG, a two-fold increase in MDH specific activity and a 22-fold increase in mdh transcript level were observed. In the case of pAVP_mdhmdh, a four-fold increase in MDH-specific activity and a 19-fold increase in mdh transcript level were observed. Analysis by qPCR showed plasmids pAVP_trc, pAVP_trcmdh, pAVP_mdh, and pAVP_mdhmdh to be present in a range of seven to nine copies per cell.