Targeted Genome Mining Reveals the Biosynthetic Gene Clusters of Natural Product CYP51 Inhibitors

Natural Product CYP51 Inhibitors

Communication

Targeted Genome Mining Reveals the Biosynthetic Gene Clusters of

Nicholas Liu, Elizabeth D. Abramyan, Wei Cheng, Bruno Perlatti, Colin J.B. Harvey, Gerald F. Bills,

and Yi Tang *

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sı Supporting Information

ABSTRACT: Lanosterol 14α-demethylase (CYP51) is an important target in the development of antifungal drugs. The fungal-

derived restricticin 1 and related molecules are the only examples of natural products that inhibit CYP51. Here, using colocalizations

of genes encoding self-resistant CYP51 as the query, we identiﬁed and validated the biosynthetic gene cluster (BGC) of 1. Additional

genome mining of related BGCs with CYP51 led to production of the related lanomycin 2. The pathways for both 1 and 2 were

identiﬁed from fungi not known to produce these compounds, highlighting the promise of the self-resistance enzyme (SRE) guided

approach to bioactive natural product discovery.

he sterol pathway has been successfully targeted for drug

structurally related, glycinated tetrahydropyran polyketides, are

proposed to inhibit CYP51 through coordination of the free

amine to the heme iron, in a similar mechanism as that of the

development, including the cholesterol lowering statins

and the antifungal azoles. Azoles such as ﬂuconazole and

ketoconazole (Figure 1 ) inhibit lanosterol 14 α -demethylase

imidazole and triazole in azoles. Restricticin 1, while relatively

unstable, has an antifungal spectrum close to that of

ketoconazole. Due to its attractive bioactivity, several total

−11

syntheses of 1 have been reported.

However, the

biosynthetic routes to 1 and 2, as well as related compounds

such as chaunopyrone A, have remained elusive. Identifying

the enzymatic basis for constructing 1 may lead to the

discovery of new CYP51 inhibitors to counter the threat of

drug resistance.

Because the genomes of the reported producers of 1,

Penicillium restrictum, and 2, Pycnidophora dispersea, have not

been sequenced, we used genome mining to identify possible

restricticin or lanomycin biosynthetic gene clusters (BGCs)

12,13

from the public database.

Given that any producer of 1

must circumvent the toxicity of 1 toward the housekeeping

CYP51, we hypothesized that a self-resistance gene encoding

an additional copy of CYP51 that either increases expression

levels, or is insensitive to 1, may be colocalized with the

restricticin BGC. Colocalizations of a gene encoding SRE are

particularly well-documented for natural products that inhibit

the sterol pathways, due to the necessity of the producing

organism to self-protect. For example, lovastatin and

squalestatin BGCs each encode SREs that represent additional

copies of hydroxymethyl glutaryl coenzyme A reductase

16,17

Figure 1. CYP51 inhibitors: (A) restricticin family and (B) synthetic

azoles.

(

HMGR) and squalene synthase, respectively.

ing the presence of an SRE encoding gene to predict

Leverag-

(

CYP51), a cytochrome P450 that catalyzes the ﬁrst step in

Received: February 7, 2021

Published: April 15, 2021

ergosterol biosynthesis. Considering the dearth of eﬀective

fungal targets, azole and other CYP51 inhibitors will remain

indispensable in the antifungal arsenal. Given the eﬀectiveness

of inhibiting CYP51 to limit fungi growth, it is surprising that

only a small group of natural products target CYP51. The

fungal restricticin 1 and lanomycin 2 (Figure 1), which are

2021 American Chemical Society

Journal of the American Chemical Society

J. Am. Chem. Soc. 2021, 143, 6043−6047

043

Communication

Figure 2. Compounds produced by rstn cluster in A. nidulans. (A) BGC of restricticin in A. nomius and a related cluster in P. dematioidea (TTI-

096). Abbreviations: (i) for PKS, KS, ketosynthase; MAT, malonyl-CoA:ACP transacylase; DH, dehydratase; MT, methyltransferase; ER,

enoylreductase; KR, ketoreductase; ACP, acyl-carrier protein; (ii) for NRPS, A, adenylation; T, thiolation; C, condensation; R, reductase (*R

domain is fused to the P. dematioidea NRPS, but not in rstn8); (iii) FMO, ﬂavin-dependent monooxygenase; SDR, short-chain dehydrogenase/

reductase; HP, hypothetical protein. (B) HPLC analysis of A. nidulans extracts. (C) Proposed mechanisms of modiﬁcation of 1 in A. nidulans.

Molecules indicated with “characterized” were isolated and NMR conﬁrmed.

bioactivity of the product of a BGC has emerged as a useful

bioinformatics tool in genome mining.

(Figure S15). To verify that Rstn2 has CYP51 function, we

overexpressed Rstn2 in S. cerevisiae treated with ﬂuconazole

and monitored growth recovery. Mild recovery of yeast growth

was observed when either yeast CYP51 or Rstn2 was

overexpressed (Figure S16 ). In contrast, Rstn2 was unable to

rescue growth when yeast was treated with amphotericin which

has a diﬀerent mode of action. Therefore, we are conﬁdent that

rstn2 likely encodes a CYP51.

18−22

To locate BGCs that encode CYP51 as an SRE, we

developed an algorithm that can identify BGCs based on user-

input gene colocalization criteria. The algorithm scores the

BGCs based on conﬁdence scores for predictions of (i) core

enzymes (PKS, NRPS, etc.), (ii) SRE, and (iii) tailoring

enzymes (Figure S1). Utilizing available fungal genome

databases, our algorithm identiﬁed various BGCs in which

core enzymes are colocalized within 20 kb with a gene

encoding a copy of CYP51 that is in addition to the

housekeeping one. To ensure we were searching for the

CYP51 subfamily and not biosynthetic P450 enzymes, we

reﬁned our SRE search by setting stringent restrictions with

minimum thresholds of 60% identity to CYP51 and an expect

value of 1 × 10⁻. This resulted in ﬁ ve cluster candidates

containing potential CYP51 SREs (Figure S2), none of which

have been studied or linked to any natural products.

To investigate the product of the rstn cluster, we

reconstituted the entire eight gene pathway in A. nidulans

A1145ΔEM. New metabolites were detected in both

minimal (CD) and rich (CDST) media (Figure 2B). In CD

media, a set of three nonpolar compounds 4−6 were observed.

In CDST media, a compound with the same molecular weight

of 1 (Figure S26) was produced, while 3 and 7 were present at

higher titers. All compounds except 1 were puriﬁed from

scaled-up cultures and structurally characterized. Surprisingly,

4 (11.4 mg/L, Figures S39 −S43, Table S7) is a molecule fused

from 1 and the known A. nidulans metabolite aspernidine

One candidate BGC (rstn) from Aspergillus nomius encoded

eight putative biosynthetic genes (Figure 2A), including two

core enzymes: a highly reducing PKS (rstn3) and a single-

module NRPS (rstn8). We reasoned the NRPS can catalyze

the esteriﬁcation of glycine to the PKS-derived pyran in 1.

While most ﬁlamentous fungi encode two conserved paralogs

(Figure 2C). The restricticin portion of the molecule fully

matches that of 1, including the four contiguous stereocenters

on the tetrahydropyran ring. The connectivity of the glycyl

amine in the isoindolone ring of aspernidine was unambigu-

ously supported by HMBC correlations (Figure S42, Table

S7 ). 5 (7.6 mg/L, Figure S29 −S33, Table S7) and 6 (7.1 mg/

L, Figure S34 −S38, Table S7) diﬀer in the methylation

patterns from 4. We veriﬁed that glycine is indeed incorporated

of CYP51, CYP51A and CYP51B, the A. nomius genome

encodes a third copy in the rstn cluster (rstn2). Phylogenetic

analysis showed Rstn2 clades with the CYP51C paralog found

in some azole-resistant strains, such as A. flavus (Figure S14).

in 4−6 through the feeding of 2,2-d -glycine into the CD

Sequence alignment revealed Rstn2 contains mutations found

in CYP51s isolated from azole resistance strains, such as S240A

media, which led to increases in mass of 2 mu of the products

(Figure S7). 3 (5.5 mg/L, Figures S44 −S48, Table S8) and 7

(4.3 mg/L, Figures S49 −S54, Table S9) were structurally

characterized to be N-acetyl-restricticin and desmethyl-

(

A. flavus) and T315A (C. albicans), as well as unique

mutations in the active site that may interact with inhibitors

044

Journal of the American Chemical Society

J. Am. Chem. Soc. 2021, 143, 6043−6047

Communication

Figure 3. Biosynthesis of 1 and 2. (A) Proposed biosynthetic pathway to 1. Molecules indicated with “characterized” are isolated and NMR

conﬁrmed. (B) (i−iii) Production of intermediates of 1 through diﬀerent combinations of rstn genes. (iv−vi). Veriﬁcation of the last two steps

using puriﬁed enzymatic assays. (C) Bioactivity guided discovery and isolation of 2. (D) Extracted ion chromatograms for lanomycin (m/z: 310).

restrictinol, respectively, both of which were isolated from the

of any of the enzymes led to the appearance of diﬀerent

compounds all with extended π-conjugation as indicated by the

λ_m_a_x(Figure S4 ). Notably, expression of the rstn3 (HRPKS)

and rstn7 (ER) led to the emergence of a new peak, 8, with an

MW of 246 that matches a polyene aldehyde that could be the

product of the HRPKS (Figure 3A and Figure S4). However, 8

and other polyene intermediates were too unstable to be

isolated for characterization. On the basis of the structure of 7

and the requirement of Rstn3−7 for biosynthesis of 7, we can

propose a putative pathway as shown in Figure 3A. Rstn3 and

Rstn7 biosynthesize 8, of which the reductive oﬄoading can be

original producer of 1. Therefore, although 1 produced in the

heterologous host was not structurally veriﬁed due to low titer

and noted instability, the isolation of 3 and 4−7 strongly

indicate the transplanted rstn genes are involved in the

biosynthesis of 1.

The derivation of 1 to 3 or 4−6 represents mechanisms by

which the host organism can sequester amine nucleophiles. A

proposed route for incorporation of 1 into 4−6 is shown in

Figure 2C. Aspernidine is biosynthesized from orsellinalde-

hyde, which is a common fungal metabolite. Orsellinalde-

hyde can be farnesylated and modiﬁed into a 2-hydroxyme-

catalyzed by Rstn4 or by endogenous reductase. Further

thylbenzoic acid intermediate. Dehydration into the quinone

methide sets up the conjugated addition by diﬀerent primary

amines, which is followed by lactamization to the isoindolone.

Aspernidine derivatives in which the isoindolone is substituted

reduction by Rstn4 aﬀords the alcohol 9, which can be

epoxidized by the FMO Rstn6 to 10. The 6-endo cyclization via

epoxide opening to the diol 7 could be catalyzed by the HP

protein Rstn5, which does not display homology to any

characterized enzymes.

From 7, the remaining two modiﬁcations, C4 O-methylation

and C3 esteriﬁcation with glycine, are likely performed by

Rstn1 (MT) and Rstn8 (NRPS), respectively. Coexpression of

rstn1 with rstn2−7 indeed led to the appearance of a new

compound, 11 (Figure 3B(iii)). NMR analysis con ﬁ rmed the

structure of 11 (3.2 mg/L, Figures S55 −S59, Table S8 ) to be

the known restrictinol. The activity of Rstn1 (Figures S12 and

S17) was conﬁrmed using recombinantly expressed enzyme,

which readily converted 7 to 11 in the presence of S-

with diﬀerent amino acids have been isolated, likely formed

through the same mechanism as 4−6 (Figures S9 and S13).

The overproduction of 1 in the minimal CD media could lead

to activation of this amine-scavenging pathway.

We next tested the roles of rstn genes in the biosynthesis of

. First, exclusion of the predicted SRE-encoding rstn2 did not

aﬀect the production of 1-related compounds, con ﬁrming

Rstn2 does not catalyze biosynthetic steps (Figure S6 ). We

found that coexpression of rstn3 (HRPKS), rstn4 (SDR), rstn5

(

HP), rstn6 (FMO), and rstn7 (ER) is required to generate the

ﬁrst stable intermediate 7 (Figure 3B and Figure S5 ). Exclusion

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J. Am. Chem. Soc. 2021, 143, 6043−6047

Journal of the American Chemical Society

adenosylmethionine (SAM) (Figure 3B(iv) (k_c_a_t= 112 s , K

https://pubs.acs.org/doi/10.1021/jacs.1c01516.

Communication

−

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at

■

1.14 mM).

From the A. nidulans hosts that coexpressed rstn2−7 and

rstn1−7, we also observed the related metabolites 12 and 13

Figure 3B(i,ii)). While we were unable to isolate 12,

sı

(

Experimental details and spectroscopic and computa-

tional data (PDF)

puriﬁcation and NMR characterization revealed that 13 (0.9

mg/L, Figures S60 −S64, Table S9) contained a terminal C14-

hydroxyl group, compared to 11. The structure of 12 is then

proposed on the basis of those of 13 and 7. We attribute the

hydroxylation modiﬁcation to the activities of the yet

unidentiﬁed P450s in the A. nidulans host.

The function of NRPS Rstn8 was con ﬁ rmed using the

puriﬁed enzyme from E. coli (Figures S12 and S18). When

Rstn8 (5 μM) was added to 11 (20 μM) in the presence of

ATP and glycine, a product with the same retention time and

mass as 1 (Figure 3B(v)) was observed. To corroborate that

this product is 1, we treated the reaction mixture with acetic

anhydride which led to the formation of N-acetyl-restricticin 3

AUTHOR INFORMATION

Corresponding Author

■

Yi Tang − Department of Chemical and Biomolecular

Engineering and Department of Chemistry and Biochemistry,

University of California, Los Angeles, California 90095,

United States; orcid.org/0000-0003-1597-0141;

Email: yitang@ucla.edu

Authors

Nicholas Liu − Department of Chemical and Biomolecular

Engineering, University of California, Los Angeles, California

90095, United States

(Figure S11). Hence, Rstn8 represents an example of the

Elizabeth D. Abramyan − Department of Chemical and

Biomolecular Engineering, University of California, Los

Angeles, California 90095, United States

emerging class of single-module NRPS-like enzymes that

27,34,35

performs esteriﬁcation reactions.

Rstn8 displays strict

substrate speciﬁcity toward glycine as no other natural amino

acid was accepted (Figure S10). Rstn8 does not recognize 7 as

a substrate, demonstrating that Rstn1-catalyzed methylation,

possibly protecting the C4-OH, must precede the ﬁnal

esteriﬁcation step.

Having demonstrated that SRE guided mining led to the

discovery of the rstn pathway from a host not reported to

produce 1, we next explored whether other hosts/pathways

identiﬁed from our algorithm and homology searches of the

rstn cluster (Figures S2 and S3) can also produce CYP51

inhibitors. A conserved cluster found in both Curvularia lunata

and Pyrenophora dematioidea (TTI-1096) was targeted (Figure

Wei Cheng − Department of Chemical and Biomolecular

Engineering, University of California, Los Angeles, California

90095, United States; orcid.org/0000-0002-5297-7046

Bruno Perlatti − Department of Chemistry and Biochemistry,

University of California, Los Angeles, California 90095,

United States; Texas Therapeutics Institute, The Brown

Foundation Institute of Molecular Medicine, The University

of Texas Health Science Center at Houston, Houston, Texas

7054, United States

Colin J.B. Harvey − Hexagon Bio, Menlo Park, California

4025, United States

Gerald F. Bills − Texas Therapeutics Institute, The Brown

Foundation Institute of Molecular Medicine, The University

of Texas Health Science Center at Houston, Houston, Texas

A), because the cluster contains both HRPKS and NRPS

homologues to Rstn3 and Rstn8, respectively. This cluster does

not contain a homologue to the Rstn7 (ER) and Rstn4 (SDR).

However, an SDR-like reductase is fused to the end of the

NRPS homologue, giving an unusual domain architecture of

A−T−C−R. When the strains are cultured on PDA, no

compounds related to 1 can be detected. We then cultured P.

dematioidea on diﬀerent media and used a bioactivity guided

approach to determine if any antifungal products could be

produced. These extracts were assayed against Staphylococcus

aureus, Candida albicans, and Cryptococcus neoformans, and the

inhibition zones were visualized (Figure 3C). When grown on

Wheat1, the P. dematioidea extract led to signiﬁcant inhibition

of both C. albicans and C. neoformans but not against S. aureus.

LC−MS analysis showed that, only in the Wheat1 extract, a

major metabolite with the same UV absorption proﬁle as 1,

and the same MW of 309 as lanomycin 2, was present (Figure

7054, United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/jacs.1c01516

Notes

The authors declare the following competing ﬁnancial

interest(s): N.L., C.J.B.H., and Y.T. are shareholders of

Hexagon Bio, Inc.

ACKNOWLEDGMENTS

■

This work was supported by the NIH R35GM118056 to Y.T.

and R01GM121458 to G.F.B. We thank the Stanford Genome

Technology Center for providing the BY4743ΔYHR007C

background yeast strain.

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