Effect of a functional recombinant cytochrome P450 enzyme of Helicoverpa armigera on gossypol metabolism co-expressed with NADPH-cytochrome P450 reductase in Pichia pastoris

https://doi.org/10.1016/j.pestbp.2019.01.003Get rights and content

Highlights

  • Helicoverpa armigera CPR and CYP6AE14 or CYP9A12 were functionally co-expressed in Pichia Pastoris.

  • CYP9A12 is able to metabolize free gossypol intermediate metabolites in vitro.

  • The free gossypol would be spontaneously degraded to compound G1 (m/z 265) and G2 (m/z 293).

Abstract

Gossypol is a polyphonic toxic compound that is present in cotton plants. The P450 cytochromes CYP6AE14 and CYP9A12 of Helicoverpa armigera are highly induced by gossypol and have been reported to be possibly involved in gossypol degradation. To determine whether the candidate H. armigera CYP6AE14 and CYP9A12 enzymes could metabolize gossypol in vitro, functional recombinant H. armigera CYP6AE14 and CPR (CYP9A12 and CPR) enzymes were successfully expressed in Pichia pastoris (P. pastoris). UPLC-QTOF/MS demonstrated the following results: (1) Free gossypol was spontaneously degraded to the gossypol metabolites G1 (m/z 265) and G2 (m/z 293) without the addition of any enzyme. (2) Free gossypol was observed following the addition of the endogenous or recombinant H. armigera P450 cytochrome CYP6AE14/CYP9A12 enzyme: in the first pathway, free gossypol was dehydroxylated and decarboxylated to G3 (m/z 453), and in the second pathway, the aldehyde group of gossypol and its metabolite were covalently bound with the amine products to form G4 (m/z 437) and G5 (m/z 783). (3) In addition to the gossypol binding pathways, the recombinant H. armigera CPR and CYP9A12 enzymes was found that could further decarboxylate the gossypol intermediate demethylated reduction of gossypolonic acid (m/z 294) and demethylated gossic acid (m/z 265) to G0 (m/z 209) and G0′ (m/z 249) respectively.

Introduction

Gossypol, is a toxic polyphonic dialdehyde present in cotton plants in two forms (free gossypol and bound gossypol) (Firestone, 2003). Free gossypol exists in (+)- and (−)-isomers, with the (−)-gossypol considered more active than the (+)-gossypol as an antifertility agent (Matlin and Zhou, 1984; Matlin et al., 1988). Studies of gossypol have focused on a range of biological activities, including antiviral, antiparasitic and anticancer activities (Dodou, 2005). In the feed industry, a large amount of cottonseed meal can be used as a protein source for monogastric animals. However, excess intake of free gossypol can depress animal growth and feed efficiency (Yildirimaksoy et al., 2004), reduce of fertility of bulls (Chenoweth et al., 2000), and compromise gamete viability in cattle (Brocas et al., 1997). Gossypol consumed by swine is primarily excreted in faeces (Abou-donia and Dieckert, 1974, Abou-Donia and Dieckert, 1975).

H. armigera is tolerant to gossypol. A low level of gossypol may stimulate the growth and development of cotton bollworms, probably due to the insects' Cytochrome P450 enzymes (Kong et al., 2010; Krempl et al., 2016a, Krempl et al., 2016b). Insect P450 enzymes are heme-thiolate proteins, which are involved with diverse endogenous substrates in the biosynthesis, biotransformation and detoxification of xenobiotics (Feyereisen et al., 1990). The H. armigera P450 monooxygenases CYP6AE14 and CYP9A12 are gossypol-inducible (Celorio-Mancera et al., 2011; Zhou et al., 2010) and are thought to be involved in pyrethroid resistance and gossypol detoxification(Jia et al., 2008; Krempl et al., 2016a, Krempl et al., 2016b). Functional H. armigera CYP9A12 and CYP9A14 obtained from Saccharomyces cerevisiae have xenobiotic-metabolizing activities (Yang et al., 2008), but no further gossypol metabolism research has been reported. When H. armigera larvae are fed double-stranded RNA (dsRNA) CYP6AE14 plant material, the growth and development of the cotton bollworm is retarded (Mao et al., 2007; Mao et al., 2011). H. armigera CYP6AE14 heterogeneously expressed in Sf9 cells could not directly metabolize gossypol (Krempl et al., 2016a). However, CYP6AE14 microsomes (Krempl et al., 2016a) and house flies co-expressing the NADPH P450 reductase with H. armigera CYP6AE14 (Tao et al., 2012) could metabolize aldrin into dieldrin due to the sufficient redox environment, which was necessary for Cytochrome P450 enzyme activity. The NADPH-cytochrome P450 reductase (CPR) is a membrane-bound protein that donates electrons to support the involvement of the Cytochrome P450 monooxygenase in the metabolism and detoxification of substrates and xenobiotics (Guengerich et al., 2009).

To verify whether H. armigera CYP6AE14 or CYP9A12 are capable of degrading gossypol, a reconstituted system consisting of the H. armigera monooxygenase and reductase linked with foot-and-mouth disease virus (FMDV) 2A peptide was constructed in pPICZαA by Gibson assembly (GA). The advantage of the 2A peptide is its ability to combine two genes and “self-cleave” when translated, which generates equal amounts of the co-expressed proteins (Donnelly et al., 2001; Sharma et al., 2011). Using this system, we obtained a functional microsomal membrane complex of H. armigera CPR and CYP6AE14 (H. armigera CPR and CYP9A12) and assayed the complex with selective substrates and gossypol in vitro.

Section snippets

RNA isolation, cDNA synthesis and cloning of H. armigera CPR, CYP6AE14 and CYP9A12

Ten H. armigera larvae with marginal resistance to free gossypol were originally collected from Shihezi in the Xinjiang Autonomous Region of China in 2015. The larvae were selected when newly moulted, and caterpillars of the 5th instar were fed a semi-artificial diet containing gossypol (25 mg/kg). Then, the midgut of the 5th instar was dissected for RNA extraction. Total RNA was extracted using TRIzol (Invitrogen, CA, USA) reagent according to the manufacturer's instructions. The quality and

Construction and expression of CPR-2A-signal-CYP6AE14 /CPR-2A-signal-CYP9A12 in P. pastoris GS115

The purified overlapping fragments (2A-α-factor signal, CPR, CYP6AE14 and CYP9A12) were assembled in one single-step reaction using GA, which could overcome the limiting multiple cloning site (MCS) in the pPICZαA vector. The constructed plasmids pPICZαA-CPR-2A-α-factor signal-CYP6AE14, pPICZαA-CPR-2A-α-factor signal-CYP9A12, and the empty pPICZαA vector were linearized and subsequently transformed into P. pastoris GS115 by electroporation. The genome of the corresponding transformed P. pastoris

Discussion

Gossypol is a natural insecticide that protects cotton plants against pathogens and herbivorous insects and is toxic to most organisms (Brocas et al., 1997; Chenoweth et al., 2000; Eisele, 1986) except rumen microorganisms (Nagalakshmi et al., 2003). Helicoverpa zea and H. armigera are tolerant to gossypol (Stipanovic Jr et al., 2006; Yang et al., 1999), this tolerance is attributed to the insect P450 monooxygenase. The potential candidate enzymes, H. armigera CYP6AE14 and CYP9A12, which are

Conclusion

The recombinant H. armigera CYP6AE14 and CYP9A12 microsomal proteins were successfully expressed and characterized in our study. The candidate reconstituted H. armigera CYP6AE14 microsomal protein was proved that no gossypol metabolism activity but the CYP9A12 was able to further decarboxylate the gossypol intermediate demethylated reduction of gossypolonic acid (m/z 294) and demethylated gossic acid (m/z 265) to G0′ and G0 respectively. This study is the first report of the co-expression of H.

Acknowledgments

This work was supported by National Natural Science Foundation of China (grant no. 31860660). The authors would like to thank professor Lee J. Johnston from University of Minnesota for reviewing an early draft of this article.

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