ABSTRACT: Metabolism is fundamental to life, all life activities are sustained through metabolism. Metabolic engineering, while modifying host endogenous metabolic network or introducing heterologous pathway for biotechnological applications, often results in unfitness and suboptimal characteristics, because of the lack of full knowledge on host metabolism. Adaptive laboratory evolution (ALE), harnessing the nature of life -- evolution, has become a potent approach in phenotype optimization, environmental adaptation, and cell biology study1–3. Through ALE, alternative pathways of β-alanine biosynthesis4, pyridoxal 5’-phosphate (PLP) biosynthesis5, isoleucine biosynthesis6, as well as ATP generation7 have been uncovered; E. coli aerobic citrate utilization8,9 and various synthetic C1-trophy10–14 have been realized. In this study, we describe E. coli recruits the same workforce, Sad, in evolution through various strategies, i.e., catalytic efficiency improvement, gene dose increase, and gene expression regulations, to overcome metabolic damages (Fig. 1). Sad, i.e. NAD(P)+-dependent succinate semialdehyde dehydrogenase (SSADH), is an aldehyde dehydrogenase (ALDH) family protein. Sad oxidizes succinate semialdehyde to succinate, preventing accumulation of the toxic aldehyde intermediate in nitrogen metabolism, such as putrescine, arginine and γ-aminobutyrate (GABA) degradations15–17. Some bacteria, for example E. coli, possess also a NADP+-dependent SSADH, GabD15,18. SSADH is ubiquitous in both prokaryotes and eukaryotes. In human, it involves in catabolism of GABA, a major neurotransmitter, its malfunction leads to a disorder SSADH deficiency19. In plants, SSADH involves in leaf development and morphogenesis20 as well as defense of abiotic stress21. And in cyanobacteria, SSADH is an essential enzyme of its uncanonical tricarboxylic acid cycle. SSADH was also found to be able to oxidize glutarate semialdehyde, participating in lysine catabolism in bacteria.
Timo Glatter 
