Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for l-2-hydroxy-carboxylate production

Abstract

As an important method for building blocks synthesis, whole cell biocatalysis is hindered by some shortcomings such as unpredictability of reactions, utilization of opportunistic pathogen, and side reactions. Due to its biological and extensively studied genetic background, Pseudomonas putida KT2440 is viewed as a promising host for construction of efficient biocatalysts. After analysis and reconstruction of the lactate utilization system in the P. putida strain, a novel biocatalyst that only exhibited NAD-independent d-lactate dehydrogenase activity was prepared and used in l-2-hydroxy-carboxylates production. Since the side reaction catalyzed by the NAD-independent l-lactate dehydrogenase was eliminated in whole cells of recombinant P. putida KT2440, two important l-2-hydroxy-carboxylates (l-lactate and l-2-hydroxybutyrate) were produced in high yield and high optical purity by kinetic resolution of racemic 2-hydroxy carboxylic acids. The results highlight the promise in biocatalysis by the biotechnologically important organism P. putida KT2440 through genomic analysis and recombination.

Two NAD‐independent l‐lactate dehydrogenases drive l‐lactate utilization in Pseudomonas aeruginosa PAO1

“…After inspecting the P. aeruginosa PAO1 genome sequence, putative enzymes involved in lactate catabolism were identified (Table ). Three adjoining genes, lldP (PA4770, encoding a lactate permease), lldD (PA4771, encoding an l ‐iLDH) and dldD (PA4772, encoding a d ‐iLDH), constitute a putative lactate utilization operon that has been annotated in many different Pseudomonas strains (Wang et al, ). Unexpectedly, gene lldA (PA2382), which encodes another putative l ‐lactate dehydrogenase, was found in the P. aeruginosa PAO1 genome (Table ).…”

“…LldD of P. aeruginosa PAO1 showed strikingly high amino acid sequence identity to l ‐iLDHs from E. coli K12 (83.1%) and most Pseudomonas strains, including Pseudomonas putida KT2440 (88.5%) and Pseudomonas stutzeri SDM (85.7%.) (Gao et al, ; Wang et al, ). By contrast, LldA of P. aeruginosa PAO1 showed < 50% amino acid sequence identity to l ‐iLDHs from the strains mentioned previously, but exhibited 65.4% homology to the l ‐iLDH from Neisseria meningitidis (Erwin and Gostchlich, ).…”

Coexistence of two d‐lactate‐utilizing systems in Pseudomonas putida KT2440

“…P. putida, which is an important rhizobacterium, has long been known to be capable of utilizing lactate (O'Brien, 1977). While the L-lactate oxidizing enzyme of its typical strain, KT2440, has already been identified (Wang et al, 2014a), it is still unclear which enzyme(s) enable the strain to utilize D-lactate. Both nLDHs and iLDHs can participate in lactate oxidization in bacteria (Jiang et al 2014).…”

“…A comparison of the genes in the vicinity of the L-iLDHencoding gene in P. putida KT2440 with those in P. stutzeri SDM revealed a high degree of synteny (Nelson et al, 2002;Wang et al, 2014a). The L-iLDH belongs to a three-gene operon pp4735-37 in the P. putida KT2440 genome (Supporting Information Fig.…”

2,3‐Butanediol catabolism in Pseudomonas aeruginosa PAO1

“…The plasmids and primers used for the gene knockout and complementation procedures are listed in Supporting Information Tables S4 and S5 respectively. P. aeruginosa PAO1 knockout mutants were generated via allelic exchange using the suicide plasmid pK18mobsacB-tet (Schäfer et al, 1994;Wang et al, 2014b). pBBR1MCS-5, a broad-host-range plasmid (Biovector Science Lab, Inc.), was used to complement the gene knockouts.…”