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

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.