Increased glutarate production by blocking the glutaryl-CoA dehydrogenation pathway and a catabolic pathway involving l-2-hydroxyglutarate

Abstract

Glutarate is a five carbon platform chemical produced during the catabolism of l-lysine. It is known that it can be catabolized through the glutaryl-CoA dehydrogenation pathway. Here, we discover that Pseudomonas putida KT2440 has an additional glutarate catabolic pathway involving l-2-hydroxyglutarate (l-2-HG), an abnormal metabolite produced from 2-ketoglutarate (2-KG). In this pathway, CsiD, a Fe2+/2-KG-dependent glutarate hydroxylase, is capable of converting glutarate into l-2-HG, and LhgO, an l-2-HG oxidase, can catalyze l-2-HG into 2-KG. We construct a recombinant strain that lacks both glutarate catabolic pathways. It can produce glutarate from l-lysine with a yield of 0.85 mol glutarate/mol l-lysine. Thus, l-2-HG anabolism and catabolism is a metabolic alternative to the glutaryl-CoA dehydrogenation pathway in P. putida KT2440; l-lysine can be both ketogenic and glucogenic.

Glutarate metabolism in Pseudomonas putida is regulated by two distinct glutarate sensing transcription factors

“…coli, though only in E. coli has the CsiR homolog been characterized 14,18 . Our bioinformatic approach to predict the conserved binding site of CsiR largely agrees with the empirically determined binding site identified in E. coli (Figure 2), and our EMSA results also confirm the existence of four distinct CsiR binding sites (Figure 3).…”

“…In an effort to make production of these chemicals sustainable, many groups have developed engineered microbes to synthesize these precursors [9][10][11][12] . The L-lysine metabolism of Pseudomonas putida has been leveraged to produce valerolactam 13 , as well as the diacid glutarate both in the native host and heterologously 14,15 . Recently this utility has inspired much work to uncover missing steps in the lysine catabolism of P. putida.…”

“…Recently this utility has inspired much work to uncover missing steps in the lysine catabolism of P. putida. These missing steps included the discovery of an additional route of glutarate catabolism through a coA-independent route to succinate, in addition to the known coA-dependent route to acetyl-coA 14,16 (Figure 1). Recent work has also demonstrated that both glutarate catabolic pathways are highly upregulated in the presence of glutarate 16 .…”

Catabolism of biogenic amines in Pseudomonas species

“…Although some recent publications about amino acid catabolism in pseudomonads have indicated the role of these catabolic processes in the lifestyle of Pseudomonas (Zhang et al, 2018Thompson et al, 2019a,b), scarce information about the implications of BA catabolism in the behaviour and fitness of pseudomonads is actually available. However, putrescine catabolism has been indicated by Liu and colleagues (2018) as an important determinant of Pseudomonas sp.…”

“…Finally, GA is either converted into acetyl-CoA (via crotonyl-CoA) or into succinic acid through a reaction catalysed by a novel nonheme Fe(II) oxygenase (CsiD; Fig. 6; Zhang et al, 2018).…”