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Biosynthetic insights into linaridin natural products from genome mining and precursor peptide mutagenesis Tianlu Mo, Wan-Qiu Liu, Wenjuan Ji, Junfeng Zhao, Tuo Chen, Wei Ding, Shaoning Yu, and Qi Zhang ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on April 30, 2017

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ACS Chemical Biology

Biosynthetic insights into linaridin natural products from genome mining and precursor peptide mutagenesis

Tianlu Mo1, Wan-Qiu Liu1, Wenjuan Ji1, Junfeng Zhao1, Tuo Chen2, Wei Ding1,2, Shaoning Yu1*, and Qi Zhang1*

1

Department of Chemistry, Fudan University, Shanghai 200433, China

2

Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Northwest

Institute of Eco-environment and Resource, Chinese Academy of Sciences, Lanzhou, Gansu, 730000, China

*

To whom correspondence should be addressed: 220 Handan Road, Fudan University,

Yuanchengying Building Room 415, Shanghai, 200433, China. Email: [email protected] (S.Y.) and [email protected] (Q.Z.)

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ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Linaridin is a small class of peptide natural products belonging to the ribosomally synthesized and posttranslationally modified peptides (RiPPs) superfamily. By an extensive genome-wide survey of linaridin biosynthetic genes, we show that this class of natural products are widespread in nature and possess vast structural diversity. The linaridin precursor peptides are relatively conserved in the N-termini but have diverse sequences in the core region, which appear to have coevolved with the biosynthetic enzymes. Using the prototypic linaridin cypemycin as a model, we have explored the structure-activity relationships involved in precursor peptide maturation and generated a diverse set of novel cypemycin variants, among which the T2S variant exhibits enhanced activity against Micrococcus luteus. Our results reveal valuable insights into linaridin biosynthesis and highlight the potential to explore this class of natural products by genome mining and by biosynthetic engineering studies.


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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products as revealed by the genome sequencing efforts of the past decade. These compounds are found in all three domains of life and possess vast structural and biological diversity, representing a promising area of chemical and genetic space for natural product exploration.1-5 Among these peptide natural products are linaridins (linear dehydrated peptides),6 which is a small but growing family of RiPPs thus far containing three characterized members. The prototypical member cypemycin (CYP), produced by Streptomyces sp. OH-4156, is an extensively modified linear peptide consisting of 21 amino acid (aa) residues, including a N,N-dimethylalanine, 2 allo-isoleucine residues, 4 dehydrobutyrine (Dhb) residues and an S-[(Z)-2-aminovinyl]-D-cysteine (AviCys) moiety (Figure 1A).7 This compound possesses potent in vitro activity against mouse leukemia cells, and also exhibits a narrow-spectrum antibiotic activity against Micrococcus luteus.8 Although CYP was initially believed to be a member of the lantibiotic family, Claesen and Bibb have later shown that the CYP biosynthesis does not involve any lanthionine synthase-like enzymes.6 Instead, an unusual set of enzymes, including CypH that consists of an N-terminal horizontally transferred transmembrane helix (HTTH) domain and a C terminal α/β hydrolase fold, participate in processing the precursor peptide CypA in CYP maturation (Figure 1B). A gene cluster highly similar to that for CYP biosynthesis was also found in the genome of Streptomyces griseus, which encodes a CYP structural analogue grisemycin (GRM) (Figure 1A).9 Despite possessing a similar linear scaffold decorated with multiple Dhb residues, the recently characterized linaridin member legonaridin (LEG) is structurally distinct from CYP and GRM.10 LEG has a much longer peptide scaffold consisting of 37 aa residues (in



contrast to 21 aa in CYP and 18 aa in GRM), contains an N,N-dimethylisoleucine (in contrast to the N,N-dimethylalanine in CYP and GRM) residue, and does not have an AviCys moiety (Figure 1A).10 Intriguingly, the LGE biosynthetic gene cluster does not have a full-length cypH homologue but instead, encodes two enzymes LegH and LegE that are homologous to the N- and C-terminal domains of CypH, respectively (Figure 1B), reflecting the distinct evolutionary pathways underlying linaridin biosynthesis.

To further explore the structural and biosynthetic diversity of linaridin natural products, we carried out a genome-wide examination of CypH homologues and their associated gene clusters. This analysis revealed more than 50 putative linaridin biosynthetic gene clusters that each encodes a CypH homologue (either a full-length CypH homologue or a pair of enzyme homologous to LegH and LegE, respectively), and one or more precursor peptides whose N-terminal part is homologous to that of CypA (hereafter a linaridin precursor peptide is generically termed LinA, a full-length CypH homologue is generically termed LinH, and the spilt LegH and LegE homologues are generically termed LinHS and LinE, respectively). A maximum-likelihood phylogenetic tree was then constructed using both the full-length LinH sequences and the catenated sequences of LinHS and LinE. This analysis showed that the CypH homologous enzymes fall into three major clades (Figure 2A). The clade A contains two subclades: A1 consists of full-length LinHs including CypH, whereas A2 consists of the split LinHSs and LinEs (Figure 2A). The clade B consists of split enzymes, including the LegH/LegE pair for LEG biosynthesis, whereas the clade C consists of full-length LinHs from gene clusters of as-yet uncharacterized linaridins (Figure 2A).


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One of the most notable findings in the bioinformatical analysis discussed above is that the group A LinAs contains two strictly conserved Cys residues in the C-termini (Figure 2B), which are converted to an AviCys moiety in the mature products, as has been observed in CYP and GRM (Figure 1A). The AviCys moiety is also found in some lanthipeptides such as epidermin and mutacin, which is formed by Michael addition of an enethiolate, resulting from oxidative decarboxylation of the C-terminal Cys residue, to a dehydroalanine (Dha) resulting from Ser dehydration.11,

12

It has been shown that a flavoprotein CypD catalyzes the oxidative

decarboxylation of the C-terminal Cys (Cys22, the first N-terminal Ala residue in the core peptide is designated as Ala1, and so forth, Figure 1C),6 in a way same to the LanD enzymes involved in lanthipeptide biosynthesis.11,

12

Hence formation of the linaridin AviCys moiety would entail

dethiolation of Cys residue at the C-terminal 4th position of LinAs, which is, to the best of our knowledge, unprecedented in biochemistry.

We asked whether the putative dethiolase, which is essential for producing the AviCys moiety in group A linaridins, could also catalyze Ser dehydration in a way similar to lanthipeptide biosynthesis. To this end, we used CYP as a model lindaridin and utilized a heterologous expression system for CYP production in Streptomyces coelicolor.6 Introduction of a cypA-expressing plasmid into the cypA-knockout mutant strain readily restored CYP production (Figure S1), establishing a working system to interrogate linaridin biosynthesis by CypA mutagenesis. We therefore replaced the CypA Cys19 by a Ser residue and introduced the resulting mutant-expressing plasmid into the cypA-knockout strain. Liquid chromatography with high-resolution mass spectrometry (LC-HRMS) analysis clearly showed that CYP was produced



in the resulting strain (Figure S2). The CYP yield is slightly decreased but comparable to that from the wild type CypA (Table 1), supporting that the linaridin dethiolase can aslo dehydrate Ser for AvisCys formation. However, it is possible that the newly introduced Ser in the C19S mutant is not dehydrated by the CYP dethiolase per se, but by the CYP dehydratase that is responsible for producing the Dhb residues. To exclude this possibility, we generated a mutant strain expressing the CypA C19T, which, however, did not produce the expected CYP variant (Table 1), as revealed by careful LC-HRMS analysis. This result suggests that the newly introduced Thr19 cannot be dehydrated by the CYP dehydratase, and the dethiolase activity in CYP biosynthesis is site-specific, which dehydrates Ser but not Thr.

Another notable finding in our bioinformatical analysis is that the core peptides of group A and B LinAs contain one or more strictly conserved Thr residues, which are all dehydrated in CYP and LEG (Figure 1A). Notably, group A LinAs have a strictly conserved Ser residue in the core region, which is not dehydrated in CYP and GRM (Figure 1A). Smilarly, the group B LinAs have a relatively conserved Ser, which also escapes dehydration in LEG (Figure 1A). These observations raise an intriguing possibility that the dehydratases in linaridin biosynthesis is Thr-specific and does not able to act on Ser residues. To test this hypothesis, we performed a Ser scanning by changing each Thr residue to Ser, generating 4 CypA mutants (T2S, T5S, T7S, and T17S). Introduction of each mutant-expression plasmid into the cypA-knockout strain showed that the expected CYP variants that carry a Dha residue (instead of Dhb) were all produced from these CypA Thr-to-Ser mutants (Table 1 and Figure S3). These results unequivocally show that the CYP dehydratase is able to dehydrate both Thr and Ser residues.


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It has been shown that for lanthipeptide dehydratases, Thr is a more preferred substrate than Ser because Ser escapes dehydration more often than Thr in lanthipeptide biosynthesis.13 The prevalence of Dhb and the absence of Dha in the known linaridins indicate that the dehydratases in lindaridin biosynthesis likely have a similar substrate preference. To interrogate the substrate preference of linaridin dehydratases, we developed a rapid fermentation procedure that produces CYP in 2 days (Supplementary Methods). LC-HRMS analysis of the cultures from this procedure showed that, for all the Thr-to-Ser mutants, the resulting products consist of both Dha and unmodified Ser (Table 1 and Figure S4 and S5). On the contrary, using the same fermentation procedure, the strain expressing the wild type CypA did not produce any detectable CYP variants with unmodified Thr. These results clearly show that the CYP dehydratase is more efficient with Thr, which is more prone to be dehydrated than Ser in both linaridin and lanthipeptide biosynthesis.

Group A LinAs contain a strictly conserved Ser (Ser16 in CYP), which escape dehydration in CYP (Figure 1A and Figure 2B). Group B LinAs also have a relatively conserved Ser residue (Ser21 in LEG), which also escapes dehydration in LEG (Figure 1A and Figure 2B). Based on the substrate preference of linaridin dehydratases discussed above, it is possible that the unmodified Ser is a result of the inefficient dehydratase activity with regard to Ser, raising an interesting possibility to replace Ser with a dehydroamino acid Dhb by a Ser-to-Thr mutation. To test this hypothesis, we constructed a mutant strain expressing the CypA S16T. Carefully analysis of the culture of the resulting strain clearly showed production of a CYP variant with an unmodified Thr, whereas the



expected Dhb16-containing product was not observed (Figure S6A). Detailed HR-MS/MS analysis revealed a b ion at m/z = 1557.84 and a y ion at m/z = 553.32 (Figure S6B), suggesting that Thr16 is not dehydrated in this S16T CYP variant. To test whether the adjacent Thr17 affects the dehydration of Ser16, we also generated a T17A mutant, and LC-HRMS analysis showed that Ser16 is still not dehydrated in the resulting CYP variant (Table 1 and Figure S7). Together with the failure in generation of a CYP variant from the CypA C19T, these results strongly support that, although linaridin dehydratase is able to carry out multiple round of dehydration reactions on both Thr and Ser, these reactions are site-specific. A similar scenario is also found for the nisin dehydratase NisB, which performs multiple rounds of dehydration but normally does not dehydrate the penultimate Ser in the C-terminus.14, 15 We also showed that the CypA S16A/T17A double mutant is also converted to the expected CYP variant (Table 1 and Figure S8), suggesting that the neither of the two adjacent dehydratable residues (i.e. Ser16 and Thr17) is essential for the maturation of the final compound.

The leader peptide of CypA contains a Cys residue at the -6 position (the first C-terminal Met in the CypA leader peptide is designated as Met-1, and so forth), which is a highly unusual observation, as in most cases, Cys is only present in the core peptides. However, the yield of CYP produced from the CypA C-6A mutant is roughly the same with that from the wild type CypA (Table 1), suggesting the Cys residue in the leader peptide does not have a specific role for CYP maturation, and this is consistent with the observation that such a Cys residue is not conserved in LinA leader peptides (Figure 2B). We also constructed a strain expressing the CypA P-3A mutant. LC-HRMS analysis of the culture of the resulting mutant showed that CYP was produced, albeit


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with decreased yield (Table 1), suggesting that although Pro-3 is conserved in all the identified LinAs (Figure 2B), it is likely not strictly essential for linaridin biosynthesis.

Because CYP is highly hydrophobic, we next attempted to introduce charge into to the CYP scaffold. To this end, we generated two Gln-to-Glu mutants, Q10E and Q14E, which carry the side chain carboxylate anion that could help improving the solubility. LC-HRMS analysis of the fermentation cultures showed that both mutants produced the expected CYP variants (Table 1 and Figure S9). We next interrogated whether the CYP biosynthetic machinery could tolerate a Lys residue, which is rarely found in LinA core peptides (Figure 2B) but could significantly improve the solubility because of the side chain cation. To this end, we generated 2 mutant strains that express the T5K and T7K mutants of CypA, respectively, which both produced the expected CYP variants containing a Lys residue (Table 1), as shown by LC-HRMS analysis (Figure S10). These results demonstrate the remarkable substrate promiscuity of linaridin biosynthetic enzymes, highlighting the potential to produce novel linaridins by bioengineering efforts. We also generated a mutant expressing the CypA T2K, but production of the expected Lys-containing CYP variant was not observed (Table 1). In addition, we showed the CypA T2L was also not converted to the corresponding CYP variant (Table 1). These results are consistent with the observation that Thr2 is strictly conserved in LinAs (Figure 2B), suggesting that the CYP biosynthetic enzymes do not tolerate large structural variation in the N-terminus of the core peptide. However, because the T2S mutant is converted to the expected CYP variant, minor structural modification in this region should be tolerable. Indeed, we showed that the CypA T2A mutant is converted to three CYP variants that carry 0, 1, and 2 methyl groups on the Ala1, respectively, among which the



non-methylated variant predominates (Table 1 and Figure S10). Notably, Ala1 in the wild type CYP and the T2S variant is exclusively di-methylated (Table 1), suggesting that, despite the remarkable substrate promiscuity of the α-N-methyltransferase CypM,16 this enzyme apparently prefers the dehydroamino acids substrate (Dha and Dhb) over Ala.

We finally investigated the antimicrobial activity of the CYP variants generated in this study. To this end, a subset of these compounds (T2S, T7K, Q14E, T17S) were produced from large scale of fermentation, purified by HPLC, and used for Micrococcus luteus bioassay in liquid medium. This analysis showed that CYP has a minimum inhibition concentration (MIC) of 96 nM, which is similar to that reported early.8 The CYP Q14E variant has a similar MIC value with that of CYP, whereas the T7K variant showed only a slightly decreased activity (Table 1, entries 14 and 16). These results suggested that introduction of charges into CYP did not significantly change the bioactivity, showcasing the potential to improve the chemophysical properties of CYP by structural modifications. Interestingly, although the T17S variant has a similar MIC value with CYP (Table 1, entry 7), the T2S variant demonstrated threefold higher activity against M. luteus (Table 1, entry 4). Although the mode of action of CYP and the detailed structure-activity relationship remains to be elucidated, these observations indicate that the N-terminus of CYP may possibly be an important structural element that interacts with the biomolecular target; future studies are awaited to test this hypothesis.

In summary, this study reveals the widespread pathways for linaridin biosynthesis and the rich genetic and chemical space to explore this class of natural products. We show that linaridins can


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be grouped into different types, and the precursor peptides and the biosynthetic machineries likely have co-evolved together. Like lanthipeptide dehydratases, the linaridin dehydratases are able to perform dehydration both on Ser and Thr, and Thr is the preferable substrate. Although the dehydratases perform multiple rounds of dehydration on LinAs, these reactions are site-specific. The unusual dethiolase responsible for the formation of the AviCys moiety in group A linaridins is also able to dehydrate a Ser residue, but not Thr. A series of site-directed mutagenesis study not only reveals valuable insights into linaridin biosynthesis, but also allows production a CYP variant with enhanced antimicrobial activity, highlighting the potential to obtain novel linaridin natural products with improved pharmacological properties by genome mining and by bioengineering efforts.

■ ASSOCIATED CONTENT Supporting Information Descriptions of all molecular biology procedures, fermentation and compound purification, bioinformatics analytical procedures, and supporting figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] ORCID



Qi Zhang: 0000-0002-8135-2221 Notes The authors declare no competing financial interest.

Acknowledgements We are grateful to M. Bibb and J. Claesen (John Innes Centre, UK) for providing Streptomyces coelicolor M1414 for CYP heterologous expression. This work is supported by grants from the National Key Research and Development Program (2016 Y F A0501302 to Q.Z.), from National Natural Science Foundation of China (1500028 and 31670060 to Q.Z., and 31470786 to S.Y.), and from State Key Laboratory of Microbial Technology Open Projects Fund (M2015-01 to W.D.). Q.Z. was also funded by the Thousand Talents Program and the Chemical Structure Association Trust.


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Figure 1. Biosynthesis of linaridin natural products. (A) Schematic representation of the three known linaridins, cypemycin (CYP), grisemycin (GRM), and legonaridin (LEG). Dhb residues are shown in red and the unmodified Ser residues are highlighted in yellow. (B) The biosynthetic gene clusters of CYP and LEG. (C) The sequences of the CypA and LegA. The red arrow indicates the proteolytic site from which the leader peptide is removed from the mature peptide. The blue residues in CypA are those that were investigated by mutagenesis in this study.

Figure 2. Widespread occurrence and structural diversity of linaridin natural products. (A) Coevolution of the linaridin precursor peptides and their modifying enzymes. Maximum likelihood tree was constructed using both the full-length LinHs and the catenated sequences of LinHS and LinE, and the nonparametric aLRT statistics were shown for the major braches. LinAs have relatively conserved N-terminal sequences whereas their C-termini can be grouped into three types, which correspond well with the three major clades of the phylogenetic tree of CypH-like enzymes. (B) The conserved motifs detected on the LinA sequences. Motif 1 is in the leader peptide region whereas motif 2 span both the leader and the core, and the red arrow indicates the proteolytic site from which the leader peptide was removed.



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Table 1. Summary of the CypA mutagenesis analysis and the CYP variants produced in this study. The antibiotic efficacies against M. luteus are shown for a subset of compounds (“-” means that assay was not performed). Entry

CypA

Product 2+ [a]

variant

([M + 2H] )

Yield (nmol)[b]

MIC (nM) 96

1

Wild type

1048.57

31 ± 5

2

C19S

1048.57

～20

96

3

C19T

N/A

NA

NA

4

T2S

21 ± 5

32

5

T5S

～25

-

6

T7S

～26

-

7

T17S

28 ± 5

96

8

S16T

1055.58

～30

-

9

T17A

1042.57

～18

-

10

S16A/T17A

1034.57

～15

-

11

C-6A

1048.57

～30

96

12

P-3A

1048.57

～10

96

13

Q10E

1049.06

～21

-

14

Q14E

1049.06

22 ± 4

128

15

T5K

1071.10

～8 ± 3

-

16

T7K

1071.10

10 ± 3

192

17

T2K

N/A

NA

NA

18

T2L

N/A

NA

NA

1028.56

～12

1035.56

～6

1042.57

～4

19

1041.56 (1050.57) 1041.56 (1050.57) 1041.56 (1050.57) 1041.56 (1050.57)

T2A

-

[a]

In most cases, the CYP variants were produced from solid culture medium. For T2S, T5S, T7S

and T17S mutants (entries 4-7), a rapid fermentation method using liquid culture medium was also utilized to investigate the substrate preference of the CYP dehydratase. By doing so the CYP variants with unmodified Ser were observed, and the protonated molecular ions of these CYP variants are shown in green font.

[b]

The yields (nmol) refer to the compounds produced from 1 L

fermentation culture. The T2S, T17S, Q14E, and T7K variants were quantified by HPLC from three parallel samples using CYP as a standard, whereas the yield other variants were only roughly estimated by comparing the MS intensities of the protonated molecular ions.


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Figure 1 174x184mm (300 x 300 DPI)



Figure 2 356x379mm (300 x 300 DPI)


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TOC 161x70mm (300 x 300 DPI)


Biosynthetic Insights into Linaridin Natural ... - ACS Publications

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