Green strategy from waste to value-added-chemical production: efficient biosynthesis of 6-hydroxy-3-succinoyl-pyridine by an engineered biocatalyst

Abstract

Value-added intermediates produced by microorganisms during the catabolism of N-heterocycles are potential building blocks for agrochemical synthesis and pharmaceutical production. 6-Hydroxy-3-succinoyl-pyridine (HSP), an intermediate in nicotine degradation, is an important precursor for the synthesis of drugs and compounds with biological activities. In the present study, we show that an engineered biocatalyst, Pseudomonas putida P-HSP, efficiently produced HSP from the renewable raw material of tobacco-waste that contains a high concentration of nicotine. The genetically constructed strain P-HSP realized a high accumulation of HSP, and HSP production was 3.7-fold higher than the non-engineered strain S16. Under optimal conditions, HSP was produced at high concentrations of 6.8 g l(-1) and 16.3 g l(-1) from tobacco-waste and nicotine, respectively. This work demonstrates a green strategy to block the catabolic pathway of N-heterocycles, which is a promising approach for the mutasynthesis of valuable compounds.

Figure 1. Transformation of N-heterocycles and potential use in chemical synthesis.

(1) Arthrobacter nicotinovorans can transform nicotine to 6-hydroxynicotine (precursor of insecticides). (2–6) Pseudomonas putida S16 transforms nicotine to 3-succinoylpyridine, 6-hydroxy-3-succinoylpyridine, and 2,5-dihydroxypyridine (precursor of 5-aminolevulinic acid) (red line). (7–11) Nicotinic acid can be transformed into 6-hydroxynicotinic acid (precursor of imidacloprid and nicotinoid insecticides) by Serratia marcescens IFO12648, 2,5-dihydroxypyridine by Pseudomonas fluorescens TN5, and 2-hydroxynicotinic acid by Proteobacteria sp.. (12, 13) 3-Cyanopyidine can be transformed into nicotinic acid and nicotinamide (a vitamin used as a food supplement) by Rhodococcus rhodochrous J1. (14, 15) Agrobacterium sp. DSM6336 has the capacity to transform 2-cyanopyrazine to 5-hydoxypyrazine-2-carboxylic acid, which can be used for the synthesis of 5-chloropyrazine-2-carboxylic acid esters. (16–20) Microbial degradation of quinoline and isoquinoline produces several intermediates, and these intermediates also have potential uses in pharmaceutical synthesis. Solid-lined arrows: biological processes. Blue-dashed arrows: chemical processes.

Figure 2. Characteristics of P. putida P-HSP.

(a) Metabolic pathway of nicotine in P. putida P-HSP was locally blocked (a, top). Enzymatic steps 1–5 are catalyzed by NicA2, nicotine oxido-reductase; Pnao, pseudooxynicotine amine oxidase; Sapd, DSP dehydrogenase; Spm, SP monoxygenase and HspB, respectively. The HSP monooxygenase activities of strain P. putida S16 and P-HSP are shown in (a, bottom panels). The photographs (a, bottom panels) were taken in the lab by the first author Yu. (b) Construction and verification of engineered strain P-HSP.

Figure 3. Degradation of nicotine and HSP by P. putida S16 and the hspB-complemented strain (P. putida S16 hspB::pK18mob (pME-hspB)).

(a) Both strains were grown in LB media with the addition of 1 g l⁻¹ nicotine for 24 h. (b, c) Resting cells of P. putida S16 (black line) and hspB-complemented strain (red line) were used for nicotine and HSP degradation at 30°C, 3.4 g DCW l⁻¹, in 0.05 mol l⁻¹ sodium phosphate buffer (pH 7.0). Each value is the mean of three parallel replicates ± SD. Symbols: HSP, ; nicotine, .

Figure 4. Feasibility of HSP production and optimization of biotransformation conditions.

(a) HPLC analysis of the reaction sample at 0 min (black line) and 5 h (red line) for the catalysis of HSP from nicotine by whole cells of P. putida P-HSP. The inset shows the spectrum of the HPLC signal at 12.93 min. (b) LC-MS analysis of the sample after a 5-h reaction. (c, d)¹H and ¹³C NMR profiles of the product. Effect of pH (e), temperature (f), and nicotine concentration (g) on HSP production. (h) Effect of biomass concentration on the specific biotranformation rate of HSP. Each value is the mean of three parallel replicates ± SD. t-test was used to calculate statistical significance. Symbols: ns, no significance (p > 0.05); **, p < = 0.01.

Figure 5. Time course of batch biotransformation by P. putida S16 and P. putida P-HSP at pH 9.0, 30°C, and 3.4 g DCW l⁻¹.

(a) P. putida S16 (black line) and P. putida P-HSP (red line) were used for HSP production under the same conditions. Cell-free system (blue line) and heat-killed cells (pink line) were used as controls. (b) For batch biotransformation, the catalysts were used four times after collection by centrifugation. (c) Total HSP production by four reactions. Each value is the mean of three parallel replicates ± SD. Symbols: HSP, ; nicotine, .

Figure 6. Time course of fed-batch biotransformation by P. putida P-HSP at pH 9.0, 30°C, and 3.4 g DCW l⁻¹.

Nicotine (a) and crude tobacco-waste extract (b) were used as substrates by P. putida P-HSP under optimal conditions. Each value is the mean of three parallel replicates ± SD. Symbols: HSP, ; nicotine, .