Noncatalytic Chalcone Isomerase-Fold Proteins in Humulus Lupulus
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Noncatalytic chalcone isomerase-fold proteins in PNAS PLUS Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis Zhaonan Bana,b, Hao Qina, Andrew J. Mitchellc, Baoxiu Liua, Fengxia Zhanga, Jing-Ke Wengc,d, Richard A. Dixone,f,1, and Guodong Wanga,1 aState Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101 Beijing, China; bUniversity of Chinese Academy of Sciences, 100049 Beijing, China; cWhitehead Institute for Biomedical Research, Cambridge, MA 02142; dDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; eBioDiscovery Institute, University of North Texas, Denton, TX 76203; and fDepartment of Biological Sciences, University of North Texas, Denton, TX 76203 Contributed by Richard A. Dixon, April 25, 2018 (sent for review February 6, 2018; reviewed by Joerg Bohlmann and Mattheos A. G. Koffas) Xanthohumol (XN) and demethylxanthohumol (DMX) are special- braries have been deposited in the TrichOME database [www. ized prenylated chalconoids with multiple pharmaceutical appli- planttrichome.org (18)], and numerous large RNAseq datasets from cations that accumulate to high levels in the glandular trichomes different hop tissues or cultivars have also been made publically of hops (Humulus lupulus L.). Although all structural enzymes in available. By mining the hops transcriptome data, we and others have the XN pathway have been functionally identified, biochemical functionally identified several key terpenophenolic biosynthetic en- mechanisms underlying highly efficient production of XN have zymes from hop glandular trichomes (1, 18–23); these include car- not been fully resolved. In this study, we characterized two non- boxyl CoA ligase (CCL) genes and two aromatic prenyltransferase catalytic chalcone isomerase (CHI)-like proteins (designated as (PT) genes (HlPT1L and HlPT2) (22, 23). We have shown that HlCHIL1 and HlCHIL2) using engineered yeast harboring all genes HlPT2 physically interacts with HlPT1L to form an active metabolon required for DMX production. HlCHIL2 increased DMX production that catalyzes the major prenylations in the β-bitter acid pathway with by 2.3-fold, whereas HlCHIL1 significantly decreased DMX produc- high efficiency: PT1L catalyzes the first prenylation step and tion by 30%. We show that CHIL2 is part of an active DMX bio- PT2 catalyzes the subsequent two prenylation steps. We then suc- synthetic metabolon in hop glandular trichomes that encompasses cessfully reconstructed the whole β-bitter acid pathway by coex- a chalcone synthase (CHS) and a membrane-bound prenyltransfer- pressing two CoA ligases (HlCCL2 and HlCCL4), the polyketide ase, and that type IV CHI-fold proteins of representative land synthase valerophenone synthase (HlVPS), and the dimethylallyl di- plants contain conserved function to bind with CHS and enhance phosphate (DMAPP)-consuming PT complex in an optimized yeast its activity. Binding assays and structural docking uncover a func- system (DD104 strain, in which the endogenous farnesyl pyrophos- tion of HlCHIL1 to bind DMX and naringenin chalcone to stabilize phate synthase activity was down-regulated by site-mutation of the ring-open configuration of these chalconoids. This study re- K197G) (23). veals the role of two HlCHILs in DMX biosynthesis in hops, and In XN biosynthesis, p-coumaroyl-CoA is produced by the se- PLANT BIOLOGY provides insight into their evolutionary development from the quential actions of L-Phe ammonia-lyase (PAL), cinnamate 4- ancestral fatty acid-binding CHI-fold proteins to specialized auxil- hydroxylase (C4H), and p-coumaroyl-CoA ligase (HlCCL1 from iary proteins supporting flavonoid biosynthesis in plants. hops) (Fig. 1). Chalcone synthase (CHS; EC 2.3.1.74) then cat- alyzes the condensation of p-coumaroyl-CoA with malonyl-CoA chalcone isomerase-like | chalcone synthase | flavonoid | to form naringenin chalcone (NC). A trichome-specific CHS Humulus lupulus | trichome Significance ops (Humulus lupulus L., Cannabaceae) is a dioecious Hperennial vine, whose female cones are a key ingredient that Here, we identify two noncatalytic chalcone isomerase-fold pro- provide unique flavor and aroma for brewing beer. Essential oils, teins, which are critical for high-efficiency prenylchalcone pro- bitter acids, and prenylchalcones account for the major three duction in Humulus lupulus. Our results provide insights into their categories of specialized metabolites that are highly accumulated evolutionary development from the ancestral noncatalytic fatty in the glandular trichomes (lupulin glands) of female cones, acid-binding chalcone isomerase-fold proteins to specialized aux- while different combinations of these compounds dictate the iliary proteins supporting flavonoid biosynthesis in plants, and bittering and finishing of beer (1–3). Trace amounts of preny- open up the possibility of producing high-value plant prenyl- lated flavanones have also been detected in hops (4). Recent chalcones using heterologous systems. studies have demonstrated that hop terpenophenolics (a term for both bitter acids and prenylchalcones) exhibit diverse bio- Author contributions: R.A.D. and G.W. designed research; Z.B., A.J.M., and B.L. performed activities with a high potential for pharmaceutical applications research; H.Q. and F.Z. contributed new reagents/analytic tools; Z.B., J.-K.W., and G.W. (5–8) (Fig. 1), with the prenylchalcones exhibiting higher bio- analyzed data; and Z.B. and G.W. wrote the paper. activity than the prenylflavanones, mainly due to the α,β- un- Reviewers: J.B., University of British Columbia; and M.A.G.K., Rensselaer Polytechnic Institute. saturated ketone functional group in chalcones (9–11). Among The authors declare no conflict of interest. these prenylchalcones, xanthohumol (XN, 3′-prenyl-6′-O- meth- This open access article is distributed under Creative Commons Attribution-NonCommercial- ylchalconaringenin) (Fig. 1) has received much attention due to NoDerivatives License 4.0 (CC BY-NC-ND). its cancer-preventive, antiinflammatory, and antioxidant prop- – Data deposition: The sequences reported in this paper have been deposited in the Gen- erties (3, 12 16). XN exhibits more powerful antioxidant activity Bank database (accession nos. HlCHIL1, HlCHIL2, MG324004, and MG324005). than resveratrol, the well-known antioxidant found naturally in 1To whom correspondence may be addressed. Email: [email protected] or red wine (17). [email protected]. To understand the molecular basis for the biosynthesis of terpe- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nophenolics in hop trichomes, a total of more than 22,000 expressed 1073/pnas.1802223115/-/DCSupplemental. sequence tags (ESTs) from several hop trichome-specific cDNA li- Published online May 14, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1802223115 PNAS | vol. 115 | no. 22 | E5223–E5232 Downloaded by guest on September 30, 2021 Fig. 1. The proposed biosynthetic pathway for terpenophenolics in hop glandular trichomes. The XN pathway, starting from phenylalanine, is boxed in blue. The possible R-groups in bitter acids are isobutyryl, isopropyl, and butan-2-yl groups. DD-acylphloroglucinols, di-dimethylallylated acylphloroglucinol; Iso-XN, isoxanthohumol; MEP pathway, plastid-localized methylerythritol phosphate pathway; OMT, O-methyltransferase; VPS, valerophenone synthase. gene, CHS_H1, has been identified from hops (24). NC is then CHIs do not possess bona fide CHI activity, which led to the prenylated by HlPT1L, and further methylated by an O-meth- renaming of both types of CHIs as CHI-like proteins (CHIL). yltransferase (HlOMT1) to form XN (20, 23, 25). Paradoxically, Recently, type III CHI folds from Arabidopsis were shown to previous transcriptome data had indicated that several chalcone bind fatty acids in vitro, and play a role in fatty acid metabolism isomerase (CHI; EC 5.5.1.6) genes represent the most abundant in planta (25). Type III CHIs are thus divided into three fatty- ESTs from hop glandular trichomes that accumulate massive acid binding protein subfamilies (FAP1, FAP2, and FAP3). A amounts of chalcone (1, 21). While bona fide CHI enzymes were loss-of-function mutation in type IV CHI from Japanese morn- the first to be identified in the context of plant flavonoid me- ing glory (Ipomoea nil) led to low amounts of anthocyanin, al- tabolism, CHI-fold family proteins are more widespread in other though the underlying mechanism remains unknown (31). domains of life, such as fungi and bacteria (26). The plant CHI Previous phylogeny and sequence analyses suggest that bona fide family can be classified into four subfamilies (type I to type IV) CHIs are diverged from type IV CHIs, which evolved from the according to their phylogenetic relationships. Types I and II common ancestor FAP3 (type III CHIs). However, the role of CHIs are bona fide catalysts having CHI enzymatic activity. Type type IV CHIs in flavonoid biosynthesis during land plant evo- I CHIs widely exist in vascular plants and are responsible for the lution remains to be determined (25, 32, 33). production of plant flavonoids (27–29). Type II CHI proteins Here, we used DD104 yeast strain to characterize two hop CHIL appear to be legume-specific and are involved in isoflavonoid genes (HlCHIL1 and HlCHIL2). We demonstrate that although production (27, 30). Type III CHIs are widely present in land lacking CHI activity, both CHILs play