Green route to synthesis of valuable chemical 6-hydroxynicotine from nicotine in tobacco wastes using genetically engineered Agrobacterium tumefaciens S33

Abstract

Background: Tobacco is widely planted as an important nonfood economic crop throughout the world, and large amounts of tobacco wastes are generated during the tobacco manufacturing process. Tobacco and its wastes contain high nicotine content. This issue has become a major concern for health and environments due to its toxicity and complex physiological effects. The microbial transformation of nicotine into valuable functionalized pyridine compounds is a promising way to utilize tobacco and its wastes as a potential biomass resource. Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways, in which several intermediates, such as 6-hydroxynicotine, can be used as renewable precursors to synthesize drugs and insecticides. This provides an opportunity to produce valuable chemical 6-hydroxynicotine from nicotine via biocatalysis using strain S33.

Results: To accumulate the intermediate 6-hydroxynicotine, we firstly identified the key enzyme decomposing 6-hydroxynicotine, named 6-hydroxynicotine oxidase, and then disrupted its encoding gene in A. tumefaciens S33. With the whole cells of the mutant as a biocatalyst, we tested the possibility to produce 6-hydroxynicotine from the nicotine of tobacco and its wastes and optimized the reaction conditions. At 30 °C and pH 7.0, nicotine could be efficiently transformed into 6-hydroxynicotine by the whole cells cultivated with glucose/ammonium/6-hydroxy-3-succinoylpyridine medium. The molar conversion and the specific catalytic rate reached approximately 98% and 1.01 g 6-hydroxynicotine h^-1 g^-1 dry cells, respectively. The product could be purified easily by dichloromethane extraction with a recovery of 76.8%, and was further confirmed by UV spectroscopy, mass spectroscopy, and NMR analysis.

Conclusions: We successfully developed a novel biocatalytic route to 6-hydroxynicotine from nicotine by blocking the nicotine catabolic pathway via gene disruption, which provides an alternative green strategy to utilize tobacco and its wastes as a biomass resource by converting nicotine into valuable hydroxylated-pyridine compounds.

Keywords: 6-Hydroxynicotine; 6-Hydroxynicotine oxidase; Agrobacterium tumefaciens; Biotransformation; Functionalized pyridine; Nicotine; Tobacco wastes.

Fig. 1

The hybrid pathway for nicotine degradation in A. tumefaciens S33 (a) and its nicotine-degrading gene cluster (b). NdhAB, nicotine dehydrogenase; Paz, pseudoazurin; Hno, 6-hydroxynicotine oxidase; Pno, 6-hydroxypseudooxynicotine oxidase; Ald, putative aldehyde dehydrogenase; Hsh, 6-hydroxy-3-succinoylpyridine hydroxylase; Hpo, 2,5-dihydroxypyridine dioxygenase; Nfo, N-formylmaleamate deformylase; Ami, maleamate amidohydrolase (amidase); Iso, maleate cis/trans-isomerase. The red cross in panel a indicates that the pathway has been blocked by disabling the enzyme Hno; the black cross in panel b shows our strategy of disrupting the hno gene

Fig. 2

Purification and properties of the Hno from A. tumefaciens S33. a The wild-type Hno purified from A. tumefaciens S33. M, markers; 1, DEAE Sepharose Fast Flow; lane 2, Q Sepharose. b The recombinant His-tagged Hno. Lane 1, cell extracts of the recombinant E. coli BL21 (DE3); lane 2, His Trap HP; lane 3, DEAE Sepharose Fast Flow; M, markers. c UV–visible absorption spectrum of 0.6 mg mL⁻¹ purified recombinant His-tagged Hno in 50 mM sodium phosphate buffer (pH 7.0). d Determination of the kinetic constants from the 6-hydroxynicotine oxidation. e UV–visible absorption spectra of the substrate 6-hydroxynicotine (solid line) and the products of 6-hydroxynicotine oxidation catalyzed by purified recombinant Hno (dashed line)

Fig. 3

LC–MS profiles of the reaction catalyzed by recombinant Hno from A. tumefaciens S33. The HPLC profile was obtained by monitoring with a PDA detector at 210 nm; a–c the mass spectra of the substrate 6-hydroxynicotine (m/z 179.1) and the products 6-hydroxy-N-methylmyosmine (m/z 177.1) and 6-hydroxypseudooxynicotine (m/z 195.1), respectively. Positively charged ions were detected

Fig. 4

Growth of wild-type A. tumefaciens S33, A. tumefaciens S33-∆hno, and A. tumefaciens S33-∆hno-C in LB (a), HSP (b), nicotine (c), and 6-hydroxynicotine (d) media. In panel d: 1, wild-type strain S33; 2, strain S33-∆hno; 3, strain S33-∆hno-C

Fig. 5

Biotransformation of nicotine into 6-hydroxynicotine catalyzed by the whole cells of A. tumefaciens S33-∆hno. a HPLC profile of the reaction mixtures sampled at different time after starting the reaction. The increase and decrease of the peaks during the reaction are indicated by upward or downward arrows, respectively. b Time course of the biotransformation reaction

Fig. 6

Optimization of biotransformation conditions to produce 6-hydroxynicotine. Effects of the amount of the catalyst added (dry cell weight, DCW) (a), the initial concentration of nicotine (b), pH (c), temperature (d), rotation speed (e), and the media used for preparation of the biocatalyst (f). In panel c: 50 mM sodium phosphate buffer (pH 6.0 and pH 7.0), 50 mM Tris–HCl buffer (pH 8.0), and 50 mM glycine–NaOH buffer (pH 9.0 and pH 10.0) were used. In panel f: HSP, HSP medium; GLU + HSP, glucose–ammonium–HSP medium; HSP + NIC, HSP-nicotine medium; GLU + NIC, glucose–ammonium–nicotine medium; GLU, glucose–ammonium medium; LB + NIC, LB-nicotine medium; LB, lysogeny broth. The reactions were carried out at 30 °C in 50 mM sodium phosphate buffer (pH 7.0) with 1.0 g L⁻¹ nicotine as the substrate and ~ 1.5 g L⁻¹ DCW whole cells of A. tumefaciens S33-∆hno, or as indicated

Fig. 7

Batch and fed-batch biotransformation reactions converting nicotine into 6-hydroxynicotine under the optimal conditions. a Time course of the batch biotransformation reactions. The whole cells were re-used three times after the initial reaction. b Time course of the fed-batch biotransformation reaction. Nicotine was supplemented three times

Fig. 8

Identification of the product 6-hydroxynicotine purified from the biotransformation reaction mixture. a UV–visible absorption spectrum of purified 6-hydroxynicotine in 0.1-M HCl solution. b HPLC profile of purified 6-hydroxynicotine. c MS profile of purified 6-hydroxynicotine