Development of a Markerless Knockout Method for Actinobacillus succinogenes

Abstract

cActinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, ␤-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

“…Despite delayed growth and onset of SA biosynthesis, effective reduction in acetate and formate was observed in all three mutants ( Fig. 3E and F), and nearly complete elimination of acetate and formate was observed in the ΔackA and ΔpflB single mutants, respectively, the latter of which is in agreement with previously reported pflB mutant generation (14). Interestingly, reduction of acetate was observed in ΔpflB mutants, and conversely, reduction in formate was observed in ΔackA mutants, indicative of interrelated and/or interdependent energetic and redox regulation of these biosynthetic pathways.…”

“…Genetic tool development. Prior metabolic engineering efforts in A. succinogenes have employed a positive selection strategy that leveraged the organism's glutamate auxotrophy to generate a series of genetic knockout mutants (14). Here, we report the development of an alternative set of positive selection tools via the conventional utilization of antibiotic resistance markers.…”

“…Prior studies have identified core metabolic and SA biosynthetic pathways and optimized fermentation engineering strategies in this organism, taking advantage of its unique incomplete TCA cycle, which natively terminates at SA ( Fig. 1) (13)(14)(15)(16)(17)(18)(19)(20)(21). A. succinogenes mutants with inactivated pyruvate-formate lyase (PFL) have been generated, effectively eliminating formate biosynthesis (14).…”

“…1) (13)(14)(15)(16)(17)(18)(19)(20)(21). A. succinogenes mutants with inactivated pyruvate-formate lyase (PFL) have been generated, effectively eliminating formate biosynthesis (14). Identification and manipulation of additional strain engineering targets offer a means of further enhancing SA flux and resultant productivity; however, to date, there have been few reports of successful metabolic engineering approaches in A. succinogenes, in part due to the limited tractability of the organism; thus, mechanisms governing flux to SA remain to be fully elucidated.…”

Bio‐based succinic acid: an overview of strain development, substrate utilization, and downstream purification

“…To elaborate the metabolic machinery of A. succinogenes clearly, 13 C metabolic flux analyses, flow calculation, several central carbon metabolism models, and the major biomass components models were used recently . Subsequently, several efficient expression methods like pLGZ920 plasmid and electroporation markerless knockout methods were proposed . The modified strain of A. succinogenes 130Z deleted with pyruvate formate lyase ( pfl ) and formate dehydrogenase ( fdh ) can only produce succinic acid in the presence of an external electron donor like reduced neutral red or H 2 with a final succinic acid concentration of 92.1 g L −1 .…”