Biotransformation of nicotine by microorganism: the case ...

Appl Microbiol Biotechnol (2010) 86:11–17 DOI 10.1007/s00253-009-2427-4

MINI-REVIEW

Biotransformation of nicotine by microorganism: the case of Pseudomonas spp. Hongjuan Li & Xuemei Li & Yanqing Duan & Ke-Qin Zhang & Jinkui Yang

Received: 5 December 2009 / Revised: 23 December 2009 / Accepted: 24 December 2009 / Published online: 21 January 2010 # Springer-Verlag 2010

Abstract Several bacterial species are capable of using nicotine, the main alkaloid in tobacco plants, as a substrate for growth. The dominant species include members of two genera, Pseudomonas and Arthrobacter. The degradation pathway and genetic structure of nicotine catabolism in Arthrobacter nicotinovorans were recently reviewed (Brandsch Appl Microbiol Biotechnol 69:493–498, 2006). Here, we present up-to-date information on biodegradation of nicotine by Pseudomonas spp. Species in this genus capable of degrading nicotine are summarized and analyzed phylogenetically. Their metabolic intermediates and nicotine degradation-related genes were summarized, and the nicotine-biotransformation pathways were compared and discussed. Keywords Nicotine degradation . Pseudomonas spp. . Metabolic products . Nicotine degradation-related genes . Biotransformation pathway

H. Li : K.-Q. Zhang : J. Yang (*) Laboratory for Conservation and Utilization of Bio-Resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming 650091, People’s Republic of China e-mail: [email protected] X. Li Yunnan Academy of Tobacco Science, Kunming 650106, People’s Republic of China Y. Duan Technology Centre of Hongyun Honghe Tobacco (Group) Co, Ltd, Kunming 650202, People’s Republic of China

Introduction Nicotine is the principal alkaloid in the leaves of most Nicotiana species, and it contributes significantly to smoking properties (Doolittle et al. 1995). During the manufacturing and processing of tobacco products, powdery solid or liquid wastes are generated in high concentrations. Most often, nicotine is the main accumulated toxic compound and cannot be recycled. Because nicotine is soluble in water, it can easily contaminate ground water (Civilini et al. 1997). Nicotine is a well-known harmful substance to humans, and it can easily cross biological membranes and the blood–brain barrier (Schievelbein 1982). As a result, tobacco wastes have been classified as “toxic and hazardous wastes” by the European Union (Novotny and Zhao 1999). The bacterial community residing in the tobacco rhizosphere has presumably adapted to use nicotine as a growth substrate, including biochemical pathways to decompose this organic heterocyclic compound (Brandsch 2006). Several bacteria, such as Pseudomonas sp. no. 41 (Wada and Yamasaki 1954), Pseudomonas convexa (syn. Pseudomonas putida) PC1 (Thacker et al. 1978), Arthrobacter oxydans (Gherna et al. 1965; Sguros 1955), Arthrobacter nicotinovorans (Kodama et al. 1992), and Achromobacter nicotinophagum (Hylin 1959), have all shown capable of degrading nicotine. In the genus Arthrobacter, the pathway and related metabolic mechanism of nicotine degradation are thoroughly elucidated (e.g., Gherna et al. 1965; Schenk et al. 1998; Baitsch et al. 2001; Igloi and Brandsch 2003; Sachelaru et al. 2005; Brandsch 2006; Chiribau et al. 2006; Ganas et al. 2008). However, little is known about the biotransformation of nicotine by Pseudomonas spp., especially at the molecular level. Recently, Pseudomonas spp. began to receive increasing attention due to their potential roles in the processing of tobacco products and the treatment of tobacco wastes.

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Appl Microbiol Biotechnol (2010) 86:11–17

Significant advances have been achieved in recent years on nicotine biotransformation by Pseudomonas spp. (Wang et al. 2004, 2005, 2007, 2009; Ruan et al. 2005; Chen et al. 2008; Tang et al. 2008, 2009; Wei et al. 2008, 2009). In this paper, we present an up-to-date review on nicotine degradation by Pseudomonas spp. Our focuses are on the strains and nicotine degradation-related genes, pathways, and metabolic intermediates and products.

Nicotine-degrading bacteria of Pseudomonas spp. A wide range of Pseudomonas species and strains have shown capable of degrading pollutants. In 1954, Wada and Yamasaki (1954) isolated Pseudomonas sp. 41 from the soil and showed that it could degrade nicotine at pH6.4 and 30°C. It was not until over 20 years later that another nicotinedegrading bacterium P. convexa Pc1 was reported (Thacker et al. 1978). However, in recent years, an increasing number of nicotine-degrading strains of genus Pseudomonas were reported. These species are listed in Table 1. Many nicotine-degrading bacterium of Pseudomonas spp. can utilize nicotine as the sole carbon and nitrogen source for growth. The optimal condition for nicotine degradation in these species is typically at 30°C, with a pH range of 6.4–7.5 (Table 1). Most of these bacteria could grow at a concentration up to 4.0 g/L nicotine. The highest nicotine concentration for growth was 6 g/L by P. putida ZB-16A (Wan et al. 2009) and 5.5 g/L by Pseudomonas sp. SKD (Sun et al. 2008). They both degraded the supplied nicotine completely within about 48 h. The degradation rate of nicotine by Pseudomonas spp. corresponded to its growth rate, and a positive linear relationship between the growth of the isolate and the degradation of nicotine was observed (Ruan et al. 2005; Chen et al. 2008). Some Pseudomonas species could degrade nicotine in tobacco leaves and showed potential application in tobacco processing. For example, Pseudomonas sp. Nic22 was Table 1 List of nicotinedegrading strains of Pseudomonas spp.

– not reported

Strain

isolated from tobacco leaves, and it decomposed nicotine from different tobacco leaf samples including Burley, Oriental, and Zimbabwe LB4. Furthermore, the crude enzyme extracts of Pseudomonas sp. Nic22 also decomposed nicotine and significantly improved the quality of tobacco leaves (Chen et al. 2008). We performed a phylogenetic analysis of P. putida and its related species using their 16S rRNA gene sequences. Our analyses showed that these bacteria clustered into two clades (A, B) (Fig. 1). All nicotine-degrading Pseudomonas species were clustered in clade A. The phylogenetic results suggest that Pseudomonas sp. strain 41, HF-1, and Nic22 all likely belonged to the same species, P. putida. Therefore, all strains of genus Pseudomonas capable of degrading nicotine would be strains of P. putida. Moreover, Pseudomonas fluorescens was also clustered in clade A, and this species could decompose nicotinic acid into 2,5-dihydroxypyridine (DHP) that further degraded into maleamic acid and formic acid by a pathway similar to nicotine degradation in P. putida (Pinsky and Michaelis 1952; Thacker et al. 1978). Interestingly, none of the Pseudomonas species clustered in clade B are capable of degrading nicotine (Fig. 1).

Metabolic intermediates and nicotine degradation-associated genes in Pseudomonas spp. As early as 1942, oxidation of nicotine by microorganisms was observed and a purple crystalline substance was isolated from bacterial cultures growing on nicotine (Wenusch 1942; Bucherer 1942, 1943). In the 1950s, nicotine metabolic pathways were widely studied, and many metabolic intermediates were reported, such as oxynicotine, 3-pyridyl-methyl-ketone, nicotinic acid, and 3-succinoyl-pyridine N-oxide (Wada and Yamasaki 1954; Tabuchi 1955a, b, c; Wada 1957). The nicotine was degraded via intermediates pseudooxynicotine, 3-succinoyl pyridine, and 6-hydroxy 3-succinoyl pyridine to 2,5-dihydroxy pyridine in P. convexa

Optimal conditions for nicotine degradation

References

Pseudomonas sp. 41

2.0 g/L nicotine, pH6.4, 30°C

Wada and Yamasaki 1954

Pseudomonas convexa Pc1 (synonym: Pseudomonas putida) Pseudomonas putida Pseudomonas sp. HF-1 Pseudomonas putida S16 Pseudomonas sp. Nic22 Pseudomonas putida J5 Pseudomonas putida SKD Pseudomonas putida ZB-16A

–

Thacker et al. 1978

– 1.3 g/L nicotine, 3.0 g/L nicotine, 3.0 g/L nicotine, 2.0 g/L nicotine, 2.0 g/L nicotine, pH7.0, 30°C

pH6.5–7.5, 30°C pH7.0, 30°C pH6.5, 30–34°C pH7.0, 30°C pH7.5, 30°C

Thacker and Gunsalus 1979 Ruan et al. 2005 Wang et al. 2004, 2007 Chen et al. 2008 Wei et al. 2008, 2009 Sun et al. 2008 Wan et al. 2009


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Fig. 1 Phylogenetic analyses of P. putida and related species based on their 16S rRNA gene sequences. Accession numbers are provided. A. nicotianae (DQ015981) used as outgroup. Nucleotide sequences were aligned using Clustal X aversion 1.83 (Thompson et al. 1997) with default parameters. Phylogenetic analyses were performed using the UPGMA method in the MEGA 4.0 program (Kumar et al. 2008).

Confidence in the topology of phylogenetic trees was evaluated by performing 1,000 bootstrap replicates in the program. Bootstrap values (expressed as percentages of 1,000 replications) greater than 50% are indicated. Scale indicates percent of substitutions per nucleotide position

as well as other Pseudomonas strains (Thacker et al. 1978; Gauthier and Rittenberg 1971a, b). However, since the late 1970s, there has been little follow-up research at the molecular level to elucidate the bioconversion mechanism of nicotine by Pseudomonas spp. This area of research began to pick up steam after 2004. For example, Wang et al. (2004, 2005, 2007) isolated a nicotine-degrading strain and identified it as P. putida strain S16. Possible intermediates were identified based on the results of NMR, Fourier-transform IR and UV spectroscopy, GC-MS, and high-resolution MS analysis. The pathway of nicotine degradation in P. putida S16 was proposed (Fig. 2) to be from nicotine to DHP, and 3-hydroxybutyric acid, N-methylmyosmine, and succinic acid were detected as intermediate metabolites. In addition, an alcohol compound, 1-butanone-4-hydroxy-1-(3-pyridinyl), was found to be a novel product from nicotine degradation (Wang et al. 2007). At about the same time, Ruan et al. (2005) reported a highly efficient nicotine-degradation bacterium Pseudomonas sp. HF-1, and several previously unreported intermediates such as cotinine, myosmine, nicotyrine, and nornicotine were detected (Fig. 2). However, N-methylmyosmine was not detected in Pseudomonas sp. HF-1 (Ruan et al. 2005). Similar intermediates were also detected during nicotine degradation by Pseudomonas sp. SKD and Nic22 (Chen et al. 2008; Sun et al. 2008). Two compounds 6-hydroxy-3succinoylpyridine (HSP) and 3-succinoylpyridine (SP) similar to other intermediates of Pseudomonas species were also identified in Pseudomonas sp. HF-1 (Fig. 2) (Ruan et al. 2005). Interestingly, a viridescent pigment was observed during nicotine degradation in Pseudomonas sp. HF-1 (Ruan

et al. 2005). These findings provided new insights into the microbial metabolism of nicotine. Thacker et al. (1978) reported that nicotine degradation genes were located on the NIC plasmid in P. convexa Pc1. However, until recently, there has been little information about the molecular mechanism for nicotine degradation by Pseudomonas spp. In 2008, Tang et al. (2008) constructed a genomic DNA library of strain P. putida S16 and sequenced a 4,879-bp gene cluster involved in nicotine degradation. Intermediates N-methylmyosmine, pseudooxynicotine, SP, HSP, and DHP were identified from transformants containing this gene cluster, and these metabolites were shown identical to those of the pyrrolidine pathway reported in the wild-type strain P. putida S16 (Wang et al. 2007). These transformants contained the nic gene cluster with three genes, nicA (ORF1), an open reading frame with unknown function (ORF2), and hsp (ORF3) genes (Tang et al. 2008). The nicA gene encodes an oxido-reductase that converts nicotine to 3-succinoylpyridine through pseudooxynicotine. It showed 40% amino acid sequence identity to genes encoding NADH dehydrogenase subunit I and cytochrome c oxidase subunit I in eukaryotes. Based on enzymatic reactions and direct evidence obtained using H218O labeling, the degradation process likely consisted of enzyme-catalyzed dehydrogenation, followed by spontaneous hydrolysis, and then repetition of the dehydrogenation and hydrolysis steps (Tang et al. 2009). The hsp gene encodes a HSP hydroxylase which catalyzes HSP directly to DHP. Sequence analysis of hsp revealed that the deduced amino acid showed no significant similarity with any protein of known function (Tang et al. 2008).

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Fig. 2 Proposed pathway of nicotine degradation in Pseudomonas spp. Bracketed compounds were not detected. Metabolic compounds in pathway 1 (pyrrolidine pathway) were found in P. putida S16;

metabolic compounds in pathways 2 and 3 were found in Pseudomonas sp. Nic22 and HF-1, and metabolic compound in pathway 4 was found in Pseudomonas sp. HF-1

Many nicotine degradation genes have been found on plasmids in nicotine-degrading bacteria (Thacker et al. 1978; Baitsch et al. 2001; Sandu et al. 2005; Ganas et al. 2008). Recently, a nicotine catabolic plasmid pMH1 was identified in Pseudomonas sp. HF-1 using mitomycin C treatment. Concomitant with the loss of the pMH1 plasmid, the mutant lost the nicotine-degrading ability. When the pMH1 plasmid was transferred to the mutant and Escherichia coli Top10, a distant relative of Pseudomonas, they also gained the nicotine-degrading ability and showed a very high nicotine degradation efficiency. In addition, the hsp gene involved in nicotine degradation in P. putida, S16 was also present in pMH1 but not in pAO1, a well-known nicotine degradation plasmid in A. nicotinovorans (Igloi and Brandsch 2003). It was recently demonstrated that plasmid pMH1 is a novel nicotine-degrading plasmid (Wang et al. 2009). Recently, a nicotine-sensitive mutant was generated from the nicotine-degrading bacterium, P. putida strain J5, by mini-Tn5 transposon mutagenesis (Wei et al. 2009).

Sequence analysis showed that the Tn5 transposon inserted at the site of the ketopantoate hydroxymethyltransferase gene (panB), which had 54% identity to the PanB gene in E. coli K-12. In-frame deletion of the panB gene abolished the nicotine-degrading ability of strain J5, while complementation with panB from P. putida J5 and E. coli K-12 restored the degrading activity of the mutant to the wild-type level. These results suggest that panB is a crucial gene in nicotine metabolism in P. putida J5, but the detailed mechanism of panB involved in nicotine degradation by P. putida J5 is unknown (Wei et al. 2009).

Metabolic pathway for nicotine degradation in Pseudomonas spp. and comparison with other bacteria Previous studies demonstrated that nicotine catabolism in microorganisms occur via three main pathways: the pyridine pathway (in Arthrobacter), the pyrrolidine pathway (in


Pseudomonas), and the demethylation pathway (in some bacteria, fungi, and the tobacco plant) (Brandsch 2006). Some of the metabolic products and pathways were first reported in P. convexa and related Pseudomonas species (Thacker et al. 1978). Recently, the pathway of nicotine bioconversion was verified in P. putida S16 (Wang et al. 2007; Tang et al. 2008, 2009). Our comparisons of the nicotine metabolic pathway between A. nicotinovorans and P. putida S16 identified that they do share neither the same intermediates nor the final products. In P. putida S16, nicotine was catalyzed by the nicotine oxidoreductase (nicA) enzyme and was converted to N-methylmyosmine, followed by spontaneous hydrolysis of N-methylmyosmine to generate pseudooxynicotine. Pseudooxynicotine was further transformed with the nicA enzyme catalyzed dehydrogenation and then with spontaneous hydrolysis to generate SP. Subsequently, with the hydroxylation of the pyridine ring, SP was converted to HSP, and HSP was catalyzed directly to DHP by the HSP hydroxylase. After cleaving the pyridine ring, DHP was converted to maleamic acid and fumaric acid, with fumaric acid entering the tricarboxylic acid cycle for further metabolism (Wang et al. 2007; Tang et al. 2008, 2009). In contrast, in A. nicotinovorans, nicotine dehydrogenase catalyzes the first nicotine-degrading reaction, hydroxylating the pyridine ring to 6-hydroxynicotine (Brandsch 2006; Freudenberg et al. 1988). Subsequently, 6-hydroxynicotine is oxidized, hydroxylated, and hydrolyzed by a series of enzymatic reactions and converted finally into 2,6-dihydoxypyridine and γ-N-methylaminobutyrate (Schenk et al. 1998; Brandsch 2006; Sachelaru et al. 2006). Therefore, the metabolic intermediates during nicotine degradation between A. nicotinovorans and P. putida S16 were different. Similarly, during nicotine degradation by Pseudomonas sp. HF-1, SKD, and Nic22, several common metabolic intermediates (e.g., continine, nornicotine, and myosmine) were found, and these differed from the known metabolic products of P. putida S16, suggesting that these bacteria have different nicotine-degrading pathways (Chen et al. 2008; Sun et al. 2008). However, SP and HSP were also detected in Pseudomonas sp. HF-1 (Fig. 2), suggesting that the nicotine degradation pathway in Pseudomonas sp. HF-1 was partially analogous to that in P. putida S16. The different metabolites observed in different species may indicate that several nicotine-degradation pathways exist in Pseudomonas species. Figure 2 summarizes the putative nicotine-degradation pathways by Pseudomonas spp.

Conclusions and perspectives As a dominant group of bacteria involved in nicotine degradation, Pseudomonas spp. could tolerate high concen-

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trations of nicotine and utilize nicotine as a sole carbon and nitrogen source for growth. Based on our phylogenetic analysis (Fig. 1), all strains of the genus Pseudomonas capable of degrading nicotine likely belonged to the same species, P. putida. The nicotine metabolisms by Pseudomonas spp. were complex, and four putative catabolic pathways have been proposed (Fig. 2). While the nicotine degradation genes were reported to locate on the chromosome in P. putida S16 (Wang et al. 2007, 2009; Tang et al. 2008), in many other strains, they were located on plasmids (Thacker et al. 1978; Thacker and Gunsalus 1979; Wang et al. 2009). The evolutionary relationships among genes involved in nicotine degradation in Pseudomonas species will likely provide significant information for elucidating the molecular mechanism of nicotine biotransformation by Pseudomonas species. The biotransformation pathway of nicotine in P. putida S16 has been studied in detail, but whether it is the only pathway for nicotine degradation in P. putida S16 is presently unknown. Further, functional studies are still necessary. Moreover, several harmful metabolites, cotinine and myosmine, were detected in Pseudomonas sp. HF-1 and P. putida SKD (Ruan et al. 2005; Sun et al. 2008). Their mechanisms of degradation in Pseudomonas spp. might help us understand the nicotine degradation in human hosts and reduce nicotine damage to humans. To control and reduce the nicotine contents of tobacco and cigarettes and to eliminate the detrimental environmental effect caused by nicotine are two important areas that the tobacco industry is investing heavily at present to keep up with legislative demands. Pseudomonas species could break down nicotine in tobacco leaves without resulting in loss of desirable flavor, taste, and smoking properties (Chen et al. 2008). Moreover, nicotine could be converted to HSP and DHP by Pseudomonas sp. S16, and they are two important precursors for drug syntheses. Recently, a new technology for HSP production from (S)-nicotine in tobacco waste by Pseudomonas sp. S16 was reported (Wang et al. 2005). HSP could be easily purified from the reaction without complex separation steps. This biotransformation made it possible to convert nicotine in tobacco wastes with high nicotine content into valuable compounds. However, the majority of the research results on nicotine biotransformation and degradation are still not used by the tobacco industry or environmental protection agencies. Large-scale testing of the organisms and/ or the processes is needed before they can be used for bioremediation purposes. Acknowledgements We are grateful to Prof. Jianping Xu of the Dept. Biology, McMaster University for valuable comments and critical discussions. This work was supported by National Basic Research Program of China (approved no. 2007CB411600) by projects from the Department of Science and Technology of Yunnan Province (approved nos. 2006GG22, 2006C0004Q, 2007C170M, and 2009CI052).

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