Effect of the Gad system on Actinobacillus succinogenes during acid stress

Abstract

The glutamate decarboxylase (Gad) system is an important amino acid-dependent acid resistance system commonly found in microorganisms. Actinobacillus succinogenes is one of the best natural producers of succinic acid (SA) but lacks glutamate decarboxylase. This study assessed the effects of Gad system introduction into A. succinogenes. The recombinant strains gadB-SW and gadBC-SW were constructed by heterologous expression of gadB alone, or gadB together with gadC, respectively. After 1.0 and 1.5 h of acid stress at pH 4.6, cell survival of gadBC-SW was greater than gadB-SW. The growth of gadB-SW and gadBC-SW was both affected by the expression of heterologous proteins and by γ-aminobutyric acid, with gadBC-SW growth reduced at a neutral pH. SA production in acidic conditions was evaluated by a shake flask and by 3-L bioreactor fermentation. The results showed gadBC-SW to increase SA production by 8.4% in shake flask compared to the parent strain, SW. For a 3-L bioreactor batch fermentation under acidic environment, the highest conversion rate of sugar to SA was observed for gadBC-SW, reaching 96%. However, SA concentration by gadBC-SW was only 47 g/L and 31 g/L at pH 6.5 and pH 6.0, respectively. In summary, the introduction of heterologous gadB and gadC into A. succinogenes not only improved acid tolerance but also influenced the synthesis of SA and added a metabolic burden.

Development of highly efficient and specific base editors in Actinobacillus succinogenes for enhancing succinic acid production

“…2 a). The plasmid pLGZ922 was selected to drive the editing element, and the frd promoter was used to drive the gRNA element [ 37 – 39 ]. To facilitate rapid phenotype identification, lacZ gene ( Asuc_1398 ), which encodes a β-galactosidase in A. succinogenes , was selected as the editing target.…”

Recent advances in bio-based production of top platform chemical, succinic acid: an alternative to conventional chemistry

“…When two genes that encoded for two SA exporters, namely, Asuc_0716 and Asuc_0715, were individually knocked out, it had a prominent and deleterious effect on both cell homeostasis and SA biosynthesis [39]. In the same year Chen et al, developed an efficient, fast and precise gene manipulation toolkit for editing the genes of Actinobacillus by developing series of specific base editors (BE's) by fusing Cas nuclease and cytidine/adenine deaminase [40]. When they used BE's to delete the gene encoding of glucose transport (Asuc_0914), which shared homology with ptsG gene (encoding glucose permease) in E. coli, they found a 1.24 fold increase in titer and yield of SA compared to parent strain.…”

“…When they used BE's to delete the gene encoding of glucose transport (Asuc_0914), which shared homology with ptsG gene (encoding glucose permease) in E. coli, they found a 1.24 fold increase in titer and yield of SA compared to parent strain. In a 3L bioreactor, the ΔAsuc_0914 strain accumulated a maximum of 71.92 g/L SA with yield and productivity being 1.03 g/g and 1.18 g/L/h, respectively [40].…”

New Insights into the Biosynthesis of Succinic Acid by Actinobacillus succinogenes with the Help of Its Engineered Strains

“…A. succinogenes M 2012036 (ZK) was anaerobically cultured with TSB medium at 37 • C or on TSB agar plates with ampicillin (100 µg/mL) when necessary. The fermentation medium was described in reference [29]. The sgRNAs were designed online (https://chopchop.cbu.cib.no/ accessed on 1 September 2022), and the protospacer sequences are listed in Supplementary Materials: Table S3.…”

“…The method for the transformation refers to references [27,29]. After the gene-silenced plasmid was transformed into A. succinogenes, one colony was randomly picked and placed into TSB medium containing 100 µg/mL ampicillin and incubated anaerobically at 37 • C for 24 h. The cultures were spread on TSB agar plates until colonies were obtained.…”