Evolutionary Metabolic Engineering Using Transcription Regulator-based Synthetic Screening Devices

Abstract

For decades, biochemicals have been produced by regulating the cellular metabolism of microorganisms. Among the methods for regulating metabolism, evolutionary metabolic engineering uses an evolutionary approach to improve microbes, which involves two steps, generation of diversity in specific or non-specific genes and screening of the diversified pools. Although the approach has made great progress in improving metabolite-producing microbes, it is still important to develop an efficient screening method because the probability of positive mutants in a library is low. Therefore, in this study, synthetic screening devices using transcriptional regulators were constructed and optimized to effectively perform evolutionary metabolic engineering. The screening device needs to be redesigned to minimize disturbances, including growth inhibition caused by the metabolic burden, which may affect productivity in the host strain. After the optimization of the screening device, it can be applied to engineer metabolic pathways. As a target metabolic pathway, 3-hydroxypropionic acid (3-HP) production pathway was adopted. Despite the successful attempts of 3-HP production from glycerol, the biological process suffers from problems arising from low activity and inactivation of the enzymes. To apply the directed evolutionary approach to engineer the 3-HP production system, synthetic screening devices were constructed using 3-HP-responsive transcription factors. It was applied to an aldehyde dehydrogenase library, specifically the aldehyde-binding site library of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). Only two serial cultures resulted in enrichment of strains showing increased 3-HP production, and an isolated KGSADH mutant enzyme exhibited a 2.8-fold higher catalytic efficiency toward its aldehyde substrate than the wild-type one. This approach will provide a simple and efficient method to engineer pathway enzymes in metabolic engineering. Subsequently, adaptive laboratory evolution (ALE) was performed using the 3-HP screening device. As a result, enriched strains by acquiring mutations beneficial to productivity were identified. Interestingly, the mutations have occurred in cAMP receptor protein (CRP), a transcription factor involved in global transcription regulation, and adenylyl cyclase (AC), which supports CRP’s function. The additional genome-scale analyses were performed, and as a result, it was confirmed that the flux to the target metabolite increased as the central carbon flux was rewired. In addition, it was found that the yield is further improved by down-regulation of the genes involved in acetate production, which is a representative by-product formed by overflow metabolism. These results show that by performing ALE using a synthetic screening tool, the limitations of the rational approach can be overcome and productivity can be increased. Moreover, this strategy is worth in terms of the possibility of providing insights into non-intuitive targets. Collectively, these results indicate that the successful implementation of evolutionary metabolic engineering will be possible by using a high-throughput screening device. Therefore, the strategy used in this study could expedite strain improvement in the field of metabolic engineering.