1-Butanol is a C4 aliphatic alcohol that is widely used as a gasoline blendstock. In this study, we report an underexplored metabolic route for the de novo biosynthesis of 1-butanol in Escherichia coli. Specifically, the 1-butanol biosynthetic pathway comprises a redesigned Clostridium-derived pathway for butyryl-CoA synthesis, followed by a CoA-acylating aldehyde dehydrogenase (PduP from Salmonella enterica) and an endogenous alcohol dehydrogenase (YqhD from E. coli). We found that abolishing thioesterases to block the formation of butyrate byproduct would lead to deleterious effects on the cell growth. Upon introducing a metabolic buffering pathway towards polyhydroxyalkanoate biosynthesis, the toxicity caused by metabolic imbalance was alleviated, accompanied by enhanced cell growth and 1-butanol productivity. A similar strategy was also validated to be effective for improving the production of hexane-2,3-diol, a potential cosmetic component with high value. In summary, this work successfully demonstrates that the metabolic buffering strategy exerts a synergistic effect on enhancing biochemical production with improved carbon recovery efficiency.

2-Hydroxyisovalerate (2-HIV) is a value-added chemical that is widely applied in the synthesis of bioactive compounds and polymers. Here, we report an underexplored metabolic route for the de novo production of S-type 2-HIV (S-HIV) in Escherichia coli. In particular, we identified promiscuous activity of 4-hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis towards the conversion of 2-keto-4-methyl-pentanoate (2-KMP, an immediate precursor for L-leucine) to S-HIV. Next, we designed a variant HmaS (S201F) with abolished activity for mandelate and 4-hydroxymandelate synthesis, thereby minimizing byproduct formation. Coupled with systematic optimization of the L-leucine biosynthetic pathway, we achieved de novo production of S-HIV at 8.1 ​mM (0.95 ​g/L) in shake flasks and 33.9 ​mM (4.0 ​g/L) in 2-L fed-batch fermentation. In summary, this work represents the first time to realize the efficient synthesis of S-configuration 2-HIV in metabolically engineered E. coli.

A great number of multifactorial diseases, including neoplastic, metabolic, and autoimmune diseases, have been associated with microbiota dysbiosis. Recently, there has been an increasing understanding of the importance of microbiome and their impact on human health. Advances in synthetic biology have led to the development of probiotics as diagnostic tools and disease treatment approaches. In this review, we briefly summarize recent examples of engineered probiotic-based therapeutics in human diseases, including cancers, gastrointestinal disorders, infectious diseases, and metabolic disorders. Finally, we discuss the challenges and opportunities in developing engineered probiotics for disease treatments.