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

Abstract

The production of bio-succinic acid (SA) from renewable feedstocks is a promising and sustainable approach to mitigating the high carbon emissions associated with the current energy crisis. Actinobacillus succinogenes was recognized as one of the most promising SA producers; however, lack of genetic background and the scarcity of genetic manipulation tools hinder the improvement in A. succinogenes by metabolic engineering. Here, for the first time, we successfully developed a series of A. succinogenes base editors (BEs) mediated by the fusion of Cas9 nickase and deaminase, including CBE, ABE, Td-GABE, and Td-CBE. Among these, ABE and Td-CBE based on a fusion of Cas9 nickase and TadA-8e variant (Escherichia coli TadA) can efficiently convert A to G and C to T, respectively, with editing efficiencies of up to 100%. We also investigated the multiplex base editing of ABE and Td-CBE, and the results showed that the editing efficiency of ABE reached 100% for six sites and 10% editing efficiency of Td-CBE for two sites. In addition, cytosine base editors were applied to inactivate hypothetical sugar and SA transporters of A. succinogenes. We found that the inactivation of Asuc_0914 encoding sucrose-specific IIBC subunit enhanced SA production, while the inactivation of hypothetical SA transporters Asuc_0715 and Asuc_0716 significantly reduced SA production. Therefore, the tools have great application potential in the metabolic engineering of A. succinogenes.

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 ].…”