Metabolic Engineering of Acinetobacter baylyi ADP1 for L-Leucine Production.

Acinetobacter baylyi ADP1 has garnered attention as a promising synthetic biology chassis due to its compact genome, rapid growth, innate competence for horizontal gene transfer, and ease of genetic manipulation. To assess its potential for natural product biosynthesis, we engineered ADP1 for the production of l-leucine. First, feedback inhibition was relieved by overexpressing the endogenous leuA and ilvBN genes, alongside the replacement of transcriptional attenuation regions within the leuBCD operon. These interventions derepressed the native biosynthetic pathway, resulting in a substantial increase in l-leucine titers from 0.10 to 0.82 g/L. Next, we augmented the eda gene in the Entner-Doudoroff pathway, while disrupting poxB, which diverts carbon toward acetate, further promoting l-leucine biosynthesis. To resolve carbon competition between the tricarboxylic acid (TCA) cycle and l-leucine synthesis, an inducible sRNA-based system was developed to dynamically repress TCA cycle-associated genes. This balanced the cell growth with l-leucine anabolism, ultimately achieving a titer of 1.16 g/L with a yield of 0.08 g/g glucose. Interestingly, the l-leucine feedback regulation diverges markedly from classical prokaryotic chassis like Escherichia coli and Corynebacterium glutamicum, in which feedback-resistant variants of leuA and ilvBN are typically required to overcome repression. In contrast, in ADP1, overexpression of the native, wild-type genes was sufficient to drive efficient product synthesis. Moreover, the unique glucose catabolism network in ADP1 limits its pyruvate availability, supplementing pyruvate and minimizing carbon loss proved critical for optimizing l-leucine production. Collectively, our findings offer mechanistic insights into chassis-specific metabolic regulation and optimizing precursor supply in nonmodel organisms.