An important aspect of any industrial scale bio-based production is the choice of biocatalyst used. Many commercially relevant microorganisms and industrial strains have been engineered to optimize the production of bio-based chemicals. One such chemical is succinate, listed as one of the top 12 building block chemicals from biomass by the US Department of Energy. Succinate is considered an important platform chemical as it has a number of applications and, most importantly, is a precursor to high-volume value-added commodity chemicals. Bio-based succinate is currently being produced at industrial scale levels using engineered microorganisms such as E. coli and S. cerevisiae. Actinobacillus succinogenes is one of the best natural succinate producers, which can grow on a wide variety of substrates, and, with the advance in genetic tools, can possibly be engineered for increased succinate production. Very few studies have focused on using succinate exporters as metabolic engineering targets for succinate production. Only a handful of studies have been carried out in E. coli and C. glutamicum with none whatsoever in A. succinogenes. With a combination of proteomics and transcriptomics we have identified candidate succinate transporters in A. succinogenes. Four of the top hits in our proteomics analysis were Asuc_1999, Asuc_0142, Asuc_2058 and Asuc_1990-91. To carefully tune the expression of these membrane proteins, we generated a library of promoters covering a large range of strengths below the strong, constitutive promoter (ppckA) we had been using. Some of the promoters were truncated versions of ppckA, and others were identified from our transcriptomics data. These promoters were tested using lacZ as the reporter gene in an A. succinogenes ∆lacZ background. Promoters ranged from ppckA as the highest down to pAsuc_0701 with a strength 209-fold lower than ppckA. The four succinate transporter candidates over-expressed under ppckA-92, a truncated version of ppckA, increased the succinate yield in glucose cultures compared to the control strain carrying the empty vector. Synthetic small RNAs (sRNAs) are another tool for metabolically engineering industrially relevant microorganisms. However, no sRNAs have been identified in A. succinogenes and only a few have been identified in other members of the Pasteurellaceae family. We identified sRNAs in A. succinogenes grown anaerobically on glucose and microaerobically on glycerol by RNA sequencing. We found 260 sRNAs in total, of which 39 were predicted by at least one of five computational programs. We validated 14 sRNAs identified from sequencing with RT-PCR. Additionally we identified probable Hfq-binding sRNAs through their Rho-independent terminators, a key feature of Hfq-binding sRNAs. Using additional characteristics of Hfqbinding sRNAs, we designed synthetic sRNAs targeting lacZ expression as a proof of concept. One plasmid-borne synthetic sRNA caused a 32% decrease in β-galactosidase activity in lactose- grown cultures. Using the same sRNAs scaffolds, we generated synthetic sRNAs that targeted the ackA and pta mRNAs to decrease the production of acetate, one of the major by-products of succinate production. One of the synthetic sRNAs targeting ackA caused a 14% decrease in the acetate yield of glucose-grown cultures. In summary, we have identified candidate succinate transporters and seen an increase in succinate production upon their overexpression. In the process, we have developed a promoter library for tunable expression of genes in A. succinogenes. We have also shown that sRNAs can be used as a tool for metabolic engineering in A. succinogenes, although additional studies are needed to make it more tunable and robust.
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