An Artificial Pathway for N-Hydroxy-Pipecolic Acid Production From L-Lysine in Escherichia coli

Abstract

N-hydroxy-pipecolic acid (NHP) is a hydroxylated product of pipecolic acid and an important systemic acquired resistance signal molecule. However, the biosynthesis of NHP does not have a natural metabolic pathway in microorganisms. Here, we designed and constructed a promising artificial pathway in Escherichia coli for the first time to produce NHP from biomass-derived lysine. This biosynthesis route expands the lysine catabolism pathway and employs six enzymes to sequentially convert lysine into NHP. This artificial route involves six functional enzyme coexpression: lysine α-oxidase from Scomber japonicus (RaiP), glucose dehydrogenase from Bacillus subtilis (GDH), Δ1-piperideine-2-carboxylase reductase from Pseudomonas putida (DpkA), lysine permease from E. coli (LysP), flavin-dependent monooxygenase (FMO1), and catalase from E. coli (KatE). Moreover, different FMO1s are used to evaluate the performance of the produce NHP. A titer of 111.06 mg/L of NHP was yielded in shake flasks with minimal medium containing 4 g/L of lysine. By this approach, NHP has so far been produced at final titers reaching 326.42 mg/L by 48 h in a 5-L bioreactor. To the best of our knowledge, this is the first NHP process using E. coli and the first process to directly synthesize NHP by microorganisms. This study lays the foundation for the development and utilization of renewable resources to produce NHP in microorganisms.

Engineering an Artificial Pathway to Improve the Bioconversion of Lysine into Chiral Amino Alcohol 2-Hydroxycadaverine Using a Semi-Rational Design

“…L-lysine, 3-hydroxylysine, and 2-hydroxycadaverine were analyzed and quantitated using HPLC (Agilent Technologies 1200 series, Hewlett-Packard, Palo Alto, CA, USA). The sample was derived with phenyl isothiocyanate (PITC) for the detection of lysine [18]. Samples were analyzed using HPLC with a C18 column (4.6 × 250 mm) and a Chirex ® 3126 (D)-penicillamine LC column (4.6 × 250 mm, Phenomenex, Torrance, CA, USA) as described by Cheng et al [3].…”

“…For example, 5-hydroxytryptophan is the functional precursor for the synthesis of serotonin, exhibiting important pharmaceutical activity [17]. Moreover, N-hydroxy-pipecolic acid is an important systemically acquired resistance signal molecule [18]. Trans-4-hydroxy-L-pipecolic acid is a direct intermediate for the synthesis of bioactive cyclopeptide MBJ-0110 and bromopyrrole alkaloid damipipecolin [19].…”