Mining and engineering
natural-product biosynthetic pathways
LITERATURE SEMINAR#1 18 -12-25
触媒医療G Y.KAMIMURA

11. Introduction
・Secondary metabolites
2. Current progress of the field
・Genome mining approach

・Enzyme engineering approach
3. SummaryAppendix

2

Secondary metabolites
lCompounds created by bacteria, fungi, plants, etc.
lUsually, they are not directly involved in normal growth, development,or reproduction. (Vaishnav and Demain 2011)
lThey have multifarious structure, and bioactivity.

Vaishnav and Demain, Microb. Biotech., 2011, 4, 687-699

3Structural diversity and classification
Alkaloids

Nonribosomal peptides

Morphine

Bleomycin
Vinblastine
Reserpine

Polyketides

Vancomycin

Terpenoids
DigoxinAmphotericin B
Erythromycin
ResveratrolArtemisinin
Tetrahydrocannabinol
4Natural products as a druglNPs have been continuously exploited for drug discovery.

Percentage of NP origin drug in newly approved drug
(From : Newman and Cragg , J. Nat. Prod. 2016, 79, 629−661)
Feher and Schmidt, J. Chem. Inf. Comput. Sci., 2003, 43, 218-227

5Biologically relevant chemical spacelIn drug discovery, biologicallyrelevant chemical space is more
important than just a library size.
lNPs have wide range ofpharmacophores, and highdegree of stereochemistry.
lThe chemodiversity of NPs ismuch larger than those ofsynthetic compounds. (Feherand Schmidt, 2003)
Hervey et al., Nat. Rev. Drug Discov., 2015, 14, 111-129
Feher and Schmidt, J. Chem. Inf. Comput. Sci., 2003, 43, 218-227

6Number and diversity of new NPs
lHowever, number and diversity of new NPs seem to reached a plateau.
➡Method to discover structurally new NPs, or artificially diversify NPs
are needed.

Pie, et al. PNAS, 2017, 114, 5601-5606

7

1. Introduction
・Secondary metabolites
2. Current progress of the field
・Genome mining approach

・Enzyme engineering approach
3. SummaryAppendix

8

Genome mining approachlOver the past decade many biosynthetic gene clusters (BGCs) havebeen uncovered.
lHowever, most gene clusters are silent under laboratory conditions,presenting a bottleneck for natural product discovery.
lBio-informatics-based analysis predicted that several cryptic BGCs inmany species are likely to encode products with novel structures.

9Genome mining approachlThus, the activation of silent gene clusters will become a new
direction of natural product discovery.

Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.

10

Pleiotropic methods

Variation in
growth condition

Engineering thetranscription andtranslation machinery

Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.

Manipulating global
regulators

Epigenetic perturbation

11

Pathway-specific methods

Manipulating
pathway specificregulators

Reporter-guided mutant
selection

Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.

Refactoring

Heterologous expression

12

Example of epigenetic approach13

Pioneer work in epigenetic perturbation approachlBok and Keller found LeaA, which activate
the expression of transcription factor aflR.
lBy detailed analysis, LeaA was found to be・Global regulation factor conservedin filamentous fungi.
・Concerned to regulation of geneexpression.
・Bearing binding site of S-adenosylmethionine.

・Localizes in nuclei.
➡LeaA = regulating expression by PTM??
Bok and Keller et al., Eukaryotic Cell, 2004, 3, 527-535.
Asai and Oshima, 生化学, 2016, 88, 643-648.

14

Pioneer work in epigenetic perturbation approachlAlthough farther research elucidated that LeaA does not function as
methylation enzyme, Bok’s work connected the secondary metabolism
and epigenetic regulation.
lAnother basis : Many of the BGCs of filamentous fungi are located near
the telomere (Nierman et al., 2005)(Rehmeyer et al., 2006).
lTo confirm the relation, Keller’s group conducted further research.

Shwab et al., Eukaryotic Cell, 2007, 6, 1656-1664
Asai and Oshima, 生化学, 2016, 88, 643-648.

15

Pioneer work in epigenetic perturbation approachlShwab et al. (2007) examined the effectof HDAC loss on the 3 best characterized
BGCs in Aspergillus nidulans.
・ST : sterigmatocytin cluster
・PN : penicillin cluster

・TR: terrequinone A cluster
lST, PN is in subtelomeric region, but TR isnot.

Shwab et al., Eukaryotic Cell, 2007, 6, 1656-1664

16

Pioneer work in epigenetic perturbation approachlThey created isogenic lines losing one or more of hdaA, hosB, hstA. (HDACs)lIn ΔhdaA, production of ST and PN increased, but not TR.

STTR

Shwab et al., Eukaryotic Cell, 2007, 6, 1656-1664

17

Pioneer work in epigenetic perturbation approachlThey picked representative genes to see the change in mRNA production.
STPNTRFig

Shwab et al., Eukaryotic Cell, 2007, 6, 1656-1664

18

Pioneer work in epigenetic perturbation approachlTo see the generality of HDAC regulation, A.alternate and P.expansum wastreated with HDAC inhibitor TSA.

Fig

Genetic engineering / Chemical approach are possible.
Shwab et al., Eukaryotic Cell, 2007, 6, 1656-1664

19

Genetic engineering approachlBok et al., 2009
Induced loss-of-function CclA (involved in H3K methylation) in A. nidulans➡Activation of cryptic BGCs, and generation of several metabolites which
was not previously known to be produced by A. nidulans
cclA and hdaA are highly conserved in filamentous fungi➡These methods are applicable to various fungi.
e.g. : Calcarisporium arbuscula(Mao et al., 2015)
Pestalotiopsis fici(Wu et al., 2016)
Bok et al., Nat. Chem. Biol., 2009, 5, 462-464.
Mao et al., Angew. Chem. Int. Ed., 2015, 54, 7592-7596, Wu et al., Org. Lett., 2016, 18, 1832-1835

20

Genetic engineering approachlHenke et al., 2016
・PTM enzymes required for the survival is impossible to delete.

➡Replaced the promoter with xylose promoter

Henke et al., ACS Chem. Biol., 2016, 11, 2117-2123.

21

Chemical approach
lWilliams et al., 2008
Treated Cladosporium Cladosporioides with
DMTs or HDAC inhibitor➡Change in metabolic profile, and generationof new chladochrom.
DNMTi

Williams et al., Org. Biomol. Chem.,2008, 6, 1895-1897

HDACi

22

Chemical approachlHenrikson et al., 2008
Treated Aspergillus niger with the same HDAC inhibitor as Williams➡Discovered novel metabolite with unprecedented structure.
lAsai et al., 2015

Combined the chemical approach with semi-synthetic approach to produce novelcompound with bioactivity.

Henrikson et al., Org. Biomol. Chem.,2009, 7, 435-438
Asai et al., Nat. Chem.,2015, 7, 737-743

23

Limitations of epigenetic perturbation approachlApplication of genetic engineering approach is limited to few strains.

・Genome information, and establishment of transformation method isnecessary.
lPredictive change is difficult using pleiotropic approach, such as HDAC
inhibition by drug.
lBGCs that are not strongly regulated by epigenetic regulation are difficult toupregulate by this method.

24

1. Introduction
・Secondary metabolites
2. Current progress of the field
・Genome mining approach

・Enzyme engineering approach
3. SummaryAppendix

25

Nonribosomal peptides (NRPs)
lStructure of NRPs are made by amino acids, including nonproteinogenic
amino acids.

NRPs(From Kudo et al, J. Ind. Microbiol. Biotechnol., 2018, not yet assigned to an issue.)

26

Nonribosomal peptides (NRPs)
lNRPs are assembled by NRP synthetases, which consist of adenylation
domain and peptidyl carrier protein, and other domains for modification.

"The NRPS reactions catalyzed by the fundamental domains."©Xcrex(Licensed under CC BY 4.0)27

Nonribosomal peptides (NRPs)
lNRPs get various modification including reduction, oxidation, cyclization,epimerization, methylation, etc.
lEnzymes are modularized.
lSometimes NRPS and PKS are combined to form NRP-PKs.

NRP-PK(From : https://www.scripps.edu/shen/researchoverview/research2.html)
28

Engineering adenylation domain of NRPS
lMechanism of substrate recognition of A domainhave been extensively studied (Kudo et al., 2018).
・A1(D235) and A10(K517) recognize the aminoacids, including D-α-AA and β-AA
・A2～A9 recognize the sidechains.

lIt is now possible to predict the selectivity of theprotein by genome sequence.
lRecently, some study on rational redesign or
directed evolution of A-domain have been done.
Kudo et al, J. Ind. Microbiol. Biotechnol., 2018, not yet assigned to an issue.
Stachelhaus et al., Chem. Biol., 1999, 6, 493-505

29

Engineering adenylation domain of NRPS
lThirlway et al., 2012

・Rationally redesigned module 10 A-domain, CdaPS3, of calcium-dependent
antibiotic to change the selectivity.
・Lys278Gln at A4 : L-Glu (or 3-methyl-L-Glu) ➡ 3-methyl-L-Gln
・By mimicking the other A-domains which recognize Gln.

Thirlway et al, Angew. Chem. Int. Ed., 2012, 51. 7181-7184.

30

Engineering adenylation domain of NRPSlKries et al., 2014

・Modified TycA (Phe specific) to recognize OPropalgil Tyr
・Trp239Ser at A3 : L-Phe ➡ O-propalgyl-L-Tyr
・By altering A2～A9 by cassette mutagenesis
・This work is of importance since propalgil groupcan be a substrate of Huisgen Cycloaddition.

Kries et al, Angew. Chem. Int. Ed., 2014, 53. 10105-10108.

31

Engineering adenylation domain of NRPS
lNiquille et al., 2018 changed thespecificity from α-amino acid to β-aminoacid by high-throughput assay.
lTycAF was used as the target.

Niquille et al., Nat. Chem., 2018, 10, 282-287

32

Engineering adenylation domain of NRPSlIn the paper, TycApY was used to enableassay by fluorescence.
lThose strains were screened by FACS.

Niquille et al., Nat. Chem., 2018, 10, 282-287

33

Engineering adenylation domain of NRPS
lMutation was not fully random, but designed basedon structural analysis.
lReversion of W239S resulted in desired enzyme.

Superimposed structure of GrsA and TyrβF-AN
Niquille et al., Nat. Chem., 2018, 10, 282-287

34

Engineering adenylation domain of NRPS
lMutation was not fully random but designed basedon structural analysis.
lReversion of W239S resulted in desired enzyme.

Niquille et al., Nat. Chem., 2018, 10, 282-287

35

Engineering adenylation domain of NRPS
lAuthors confirmed that modified A domain correctly work in cooperation
with other NRPSs.

Niquille et al., Nat. Chem., 2018, 10, 282-287

36

Limitations
lScope is limited to structurally conserved changes.

lThe change in A domain specificity may result in unexpected effects on
overall NRPS function.(Uguru et al., 2004)

Uguru et al., J. Am. Chem. Soc., 2004, 126, 5032-5033

37

1. Introduction
・Secondary metabolites
2. Current progress of the field
・Genome mining approach

・Enzyme engineering approach
3. SummaryAppendix38

Summary
lDiversity of natural products is worth exploiting for drug discovery,biological tool, and so on.
lHowever, discovery of brand-new NP is becoming difficult.
lTo solve this problem, some approaches are possible, including
・Epigenetically activating silenced BGC.
・Engineering enzymes to alter the specificity.

39Current issues and futurelEpigenetically activating silenced BGC.
・Need Genome information, and establishment of transformation method.
・Or, analysis of the product may be complicated.
➡The method without genetic transformation to activate specific gene cluster
must be a breakthrough.
lEngineering enzymes to alter the specificity.
・Despite the great effort, scope is limited to structurally conserved changes.
➡Using artificial catalyst to activate desired amino acid may be a solution.
・The modification may result in unexpected overall effect.
40

41

1. Introduction
・Secondary metabolites
2. Current progress of the field
・Genome mining approach

・Enzyme engineering approach
3. Summary
Appendix42Appendix : The breakdown of all approved drug
B : Bio, V : Vaccine
N : NPs, NB : Botanical, ND : NP derivatives, S : synthetic, S* : Synthetic (NP pharmacophore)/NM : Mimic of NPsAll

Newman and Cragg , J. Nat. Prod. 2016, 79, 629−661

Small molecule43

Appendix : Morgan fingerprint
Fingerprint calculated using Morgan Algorithm(Extended-Connentivity Fingerprints)

For detail see the papers listed below.
Morgan, J. Chem. Doc., 1965, 5. 107-113.
Rogers and Hahn, J. Chem. Inf. Model., 2010, 50, 742-754.

From : http://chembioinfo.com/2011/10/30/revisiting-molecular-hashed-fingerprints/
44

Appendix : Tanimoto scoring
Similarity score of two bitmaps, definedby right formula.
Xi : i th bit of X
⋀ / ⋁ : bitwise operator And / Or.

45

Appendix : Pleiotropic methods
lVariation in growth conditions
Approach :Just change the growth condition(co-culture, temperature, pH, etc.)

Advantage :Easy to conduct.

Limitations :・Limited by the range of condition
under which organism will grow.

・Difficult to predict the effect of thechange.
e.g. : Aspoquinolons, Aspernidines, ClosthioamideA, etc.
Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.

Example of the approach.

46

Appendix : Pleiotropic methods
lEngineering the transcriptional and translational
machinery
Approach :Induce mutation in RNA polymerase
and ribosomal proteins to cause
upregulation of BGC expression.

Limitations :Only small number of antibiotics trigger
changes in a restricted group of bacteria.

e.g. : Piperidamycins, Coelimycins

Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.

47

Appendix : Pleiotropic methods
lManipulating global regulators
Approach :Change the expression levels of pleiotropic transcriptional regulatorof BGCs.

Advantage :Manipulation of single regulator may result in more than one pathway
and may lead to the discovery of multiple NPs.

Limitations :Only few such regulators are known.

e.g. : Terrequinone (A.nidulans), ClosthioamidesB-H, Pulvomycin (S.flavopersicus)
Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.

48

Appendix : Pleiotropic methods
lEpigenetic perturbation
Approach :Use mutagenesis or inhibitors totrigger global chromatin structure change.

Limitations :・Sites at which acetylation or methylation
may be altered are limited by accessibility.
・Not yet scope for predictive change.

e.g. : Emodin (A.nidulans), Cladochromes, NygeroneA

Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.

49

Appendix : Readily inducible promoter
・Tet-ON system

・Tet-OFF system(From : http://www.takara-bio.co.jp/goods/catalog/pdf/tet.pdf)
50Appendix : Pathway-specific methods
lManipulating pathway specific regulators
Approach :Identification and overexpression or deletion of pathway-specific regulators.

Advantage :Precise activation of specific BGC. Unambiguous identification of the products.

Limitations :Transcription factor should be identifiable, easy to manipulate and theperturbation should not complicate identification of the target metabolites.

e.g. : Aspyridones, Asperfuranone, Stambiomycins, Gaburedins, Burkholdacs, etc.

Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.

51

Appendix : Pathway-specific methods
lReporter-guided mutant selection
Approach :Couple target BGC to promoter-reporter system, and induce genome
scale random mutagenesis.

Advantage :Pleiotropic approach combined with pathway-specific detection ofvisualizing wonted mutant.

Limitations :Require cloning of promoter sequences. Only single example.

e.g. : Gaudimycin D, E

Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.
Guo, F et al. Metab. Eng., 2015, 28, 134-142

52

Appendix : Pathway-specific methodslRefactoring
Approach :Replace the promoter of the silent BGC withconstitutive, or readily inducible promoter .

Advantage :Afford precisely controlled activation of thepathway of interest.

Limitations :・Natural promoter must be identified.

・The BGC must be amenable to genetic
manipulation
e.g. :Burkholderic acid, 6-epialteramideA/B
Rutledge and Challis, Nat. Rev., 2015, 13, 509-523.
Franke et al., Angew. Chem. Int. Ed. Engl., 2012, 51, 11611-11615

53

Appendix : Pathway-specific methods
lHeterologous expression
Approach :・Expression of an entire BGC in aheterologous host・Often combined with refactoring.
Advantage :SImplifies the identification ofmetabolites.

Limitations :BGCs are usually difficult to handle
because of the size of the cluster.

e.g. : Terferol analogues, Epiisozizaene, Acermitilol,Haloduracin
Rutledge and Challis, Nat. Rev., 2015, 13, 509-523
Cobb and Zhao, Nat. Biotech., 2012, 30, 405–406

54Appendix : LeaA
NOR

Shwab et al., Eukaryotic Cell, 2007, 6, 1656-1664

PN55

Appendix : Asai et al. 2015

Asai et al., Nat. Chem.,2015, 7, 737-743

56

Appendix : Asai et al. 201557

Appendix : A domain structural analysis

58

Appendix : fluorescence activated cell sorting (FACS)
lCells are arranged in line, and analyzed bylaser.
lDrop containing target particle which satisfy
the criteria is charged.
lThe drops are deflected by charged plate,and collected.

59