Institute of Pharmacy and Biomedical Science

9 month report for the PhD project

A new synthetic biology platform for expanding the DNA

binding repertoire of minor groove binding natural products
By Emilio Corts Snchez
(Registration Number: 201254764)
First supervisor:
Dr. Paul A. Hoskisson,
Strathclyde Institute of Pharmacy and Biomedical Sciences,University of Strathclyde.

Second supervisor:

Dr. Glenn Burley,

Department of Chemistry,University of Strathclyde.

Third Supervisor:

Dr Daniel Walker,

Institute of
Institute of Infection, Immunity and Inflammation, University of Glasgow.

Infection, Immunity and Inflammation

1
Appendix:Material suppliers 3
Appendix:Figure and Table index 5
Appendix:Acronyms list 6

1.Abstract 7
2.Introduction 8

2.1 Motivation 8

2.2 Streptomyces 10

2.3 NRPS 14

2.4 Congodicine 23

2.5 Distamycin 25

3.Project 28

4.Materials and methods 31

4.1 Bioinformatics 31

4.2 Media 31

4.3 Cultivation of Streptomyces strains 34

4.4 MGB extraction and bioassay 35

4.5 Molecular Biology 38

5.Results 44

5.1 Transformation of host strains 44

5.2 Growth in different media 44

5.3 Protein expression of the cgc19 peptidyl carrier protein 46

5.4 Antibiotic production and isolation 55

6.Discussion and future work 64

6.1 Discussion 64

6.2 Future work 65

7.Bibliography 69

2
Appendix: Materials suppliers
Compound Supplier
ZnCl2 Sigma Aldrich

FeCl3 Sigma Aldrich

CuCl2 Sigma Aldrich

MnCl2 Sigma Aldrich

Na2B4O7 Sigma Aldrich

(NH4)6Mo7O27 Sigma Aldrich

Agar Meldford

Mannitol Fischer

Soya Flour Neils Yard Wholefood

NaCl Sigma Aldrich

Glucose Fischer

Yeast extract Sigma Aldrich

Peptone Fischer

Malt extract Oxoid

Sucrose Fischer

MgCl2 Fischer

Glycerol Fischer

Tryptone Fischer

Ethanol Sigma Aldrich

Chloroform Fischer

3
Appendix: Figure and table index

Figure 1. Discovery of important antibiotics and other natural products with antimicrobial activity over
time (Pag 9 )

Figure 2.Life cycle of Streptomyces46(Pag 14 )

Figure 315 A) Detail of how acyl groups are joined together(Pag 18 )

B)C domain with conserved His motiv in black, N terminal in red and C terminal in blue joined with a
linker region in purple (Pag 18 )

Figure 4.NRPS PCP protein domain 15, in blue, red ,orange and purple can be seen the helix I,II,III IV
that aquires different folding conformations depending of the state of the enzyme (Pag 20 )

A-apo form

B-apo/holo form

C-holo form

Figure 5a. Adenilation reaction carried out by the adenylation domain in an NRPS (Pag 21 )

Figure 5b.Reaction between a PCP and its cognate activated aminoacid (Pag 21 )

Figure 6. Examples of non ribosomal peptides69 ( A: destruxin. B: amanitin.C: cyclosporin. D:

ergotamine. E: HC-toxin. F: AM-toxin. G: ferrichrome. H: enniatin. I: victorin. J: ACV. K: peptaibol.)

(Pag 22 )

Figure 7. Kirromycin , a MGB antibiotic produced by Streptomyces collinus70(Pag 22 )

Figure 8. Linear NRPS and example metabolite vancomycin (Pag 23 )

Figure 9.Iterative NRPS and example metabolite enniatin15 (Pag 23 )

Figure 10. NonLinear NRPS and example metabolite vibriobactin15 (Pag 23 )

Figure 11.Chemical structure of congocidine22 (Pag 25 )

Figure 12.Genetic organisation of the congocidine gene cluster18 (Pag 26 )

Figure 13.Chemical structure of Distamycin22 (Pag 28 )

Figure 1487.a)Congocidine gene cluster.b)Proposed pathway to congocidine (Pag 30 )

Figure 15.a) pyrrolamide b)Pyrrolamide derivates to feed to Streptomyces Congodicine producer to

produce novel compounds (Pag 31 )

Figure 16. Streptomyces M1146 plated in MS medium (left) vs MS medium+R2 solution (right) (Pag 46 )

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Figure 17. Map of pEX(. T7 promoter,CMV promoter Ampicillin resistant eurofins mwg operon)
containing the cgc19 gene(Pag 48 )

Figure 18. PCR amplification of a region of the cgc19 gene, 166 bp size were expected (Pag 49 )

Figure 19. a)pET15b cut with NdeI/EcoRI vector expected band of 5375bp,b)pEX cut with NdeI/EcoRI
insert expected band of 419 bp,c) final assembly of an overnight ligation 1:2 . (Pag 51 )

Figure 20. Gel purification of vector:insert before cloning. Lanes 1,2 containing the digestion of pET15b
with EcoRI/NdeI, upper band corresponding to desired vector. Lanes 3,4 containing the digestion of pEX
cgc19 with EcoRI/NdeI, lower band corresponding to desired insert. (Pag 52 )

Figure 21. Digestion of potential clones containing pET15b-cgc19. Left part of the gel shows the
digestion with NcoI/PstI cutting the inside of cgc19. Colonies 1,2,4 shown expected bands. Digestion
with EcoRI/NdeI shows correct size of vector but no insert. (Pag 53 )

Figure 22. Overexpresion of cgc19 protein before purification revealed in an acrylamide gel (Pag 54 )

Figure 23. Cgc19 protein post purification in AKTA, different alicuotes. From left to right, first eluted
portion to last one coming out of the column. It can be appreciated a small dimer band at 24 kDa when
looking to the right. (Pag 55 )

Figure 24. Growth curve of Streptomyces heterologous host and Streptomyces netropsis in a 72 hours
run. Only one sample per timepoint. (Pag 57 )

Figure 25. Growth curve of Streptomyces heterologous host and Streptomyces netropsis in a 108 hours
run. Only one sample per timepoint. (Pag 58)

Figure 26. Congodicine elution in H2O/CH3CN (pH 7.00), congodicine peak at 9.872 min (Pag 62 )

Figure 27. Congodicine elution in CF3CO2H 0.1 %/CH3CN (pH 2.00 ) Congodicine peak at 10.922 min (Pag
62)

Figure 28. Congodicine elution in HCO2H 0.1 % /CH3CN (pH 3.00 ) Congodicine peak at 9.663 min (Pag
63)

Figure 29. Gibson cloning strategy overview, adapted from https://www.neb.com/ (Pag 67)

Table 1 .R2 salt solution composition (Pag 33)

Table 2. Buffer composition (Pag 40)

Table 3. Acrylamide gel composition per given concentration (Pag 43)

Tables 5,6,7. Showing the inhibition percentage of Bacillus subtilis growth with different dosage of
culture extract. Meaning 100% for complete inhibition (Pag 60)

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Appendix: Abbreviation list
WHO-World Health Organization

MGB-Minor Groove Binder

NRPS-Non Ribosomal Peptide Synthetase

NRP- Non Ribosomal Peptide

PCP-Peptidyl Carrier Protein

PPTase- phosphopantotheinyl transferase

A-domain- Adenylation domain

DMSO-Dimethyl sulfoxide

UV-Vis-Ultra Violet and Visible

CFU-Colony Forming Units

6
1.Abstract
In 2011 it was announced by the WHO that antibiotic-resistant infections have
reached unprecedented levels and outstrip the ability to fight them with existing
drugs1. With a passive pharmaceutical industry over last decade regarding the
discovery of new antimicrobial compounds2, a united push from both academia and
industry is urgently needed in order to create a pool of new drugs big enough to
address this raising problem.

Minor groove binding metabolites (MGBs) are chemical compounds able to

bind at the minor groove of the DNA double helix. This property makes them suitable
as antimicrobial compounds as they can interfere with transcription and potentially
other cellular processes. This makes MGBs promising therapeutic agents and have
potential as novel gene silencing tools. MGBs have been extensively synthesized
chemically nevertheless poor yield are achieved3. Streptomycetes are the major source
of known antibiotics of microbial origin4 and are also producers of natural MGBs.
Natural MGBs have better yields than synthetic but the binding regions are fixed by
the nature of the compound; which are usually very short in length, around 4-6 base
pairs. The natural synthesis of these compounds is carried out by proteins called non-
ribosomal peptidase synthases (NRPS), that are large multi-modular enzymes capable
of incorporating a huge variety of chemicals beyond the classic L-amino acid based
synthesis of proteins.

To achieve the aims of the project, the NRPSs of MGBs biosynthetic clusters
will be engineered to allow the synthesis of new MGBs based on the structure of
existing ones.

In addition to synthesizing novel MGBs using our combinatorial/synthetic

biology approach to discover new metabolites, we will try to activate the so called
silent or non-expressed secondary metabolites clusters using a library of random
small peptide aptamers. These may activate the unexpressed antibiotic clusters
through interaction with regulators or directly with DNA.

7
2.Introduction
2.1 Motivation

After the golden era of antibiotics discovery, which was during the 1950-70s,
the discovery of natural antibiotics was successful until a sharp decrease began in the
1970s 5 (Fig. 1). Due to the continued increase of antimicrobial resistance in pathogens
for existing antibiotics all over the world there is an urgent need of new antibiotics67

This is not an easy task as it has been proven that modification of natural
molecules does not always makes them more effective than the metabolites from
which they are derived 8, but recent advances in DNA sequencing may allow the search
for novel secondary metabolite clusters with hitherto unknown activity which could be
useful as clinical antibiotics 9, whether this is through the use of synthetic biology to
create novel biosynthetic clusters or through the activation the non-expressed
antibiotic clusters that are found within the genomes of organisms that have been fully
sequenced.

Actinomycetes are by far the most prolific producers of antimicrobials, with

about two thirds of compounds coming from this source. As a major source of
antibiotics, the mining of their genome is one logical option in the search for new
compounds5. There exist two different approaches to reveal the compounds encrypted
by those secondary metabolite clusters either through induction of the cluster in the
native strain or by heterologous expression of the cluster in a surrogate strain. The
induction of gene clusters could be made by using random peptide aptamers,
containing about 16 random amino acids expressed under the control of an inducible
promoter 10. Those aptamers then could be controlled by the promoter and used for
interfering with the secondary metabolic capacity of the strain. The strains can then be
screened for changes in activity.

Moreover there exists other novel approaches to address this problem, and
one of the most promising ones is to use synthetic biology for manipulating existing
strains, to produce new antibiotics.

8
Figure 1. Discovery of important antibiotics and other natural products with antimicrobial activity over time .44

9
 Mutasyntesis can produce artificial derivatives from existing natural antibiotics ,
introducing mutasynthons in to the structure of them. These muthasynthons are
synthetic analogues of the intermediates found in the original biosynthetic pathway
which are fed to the strain, and the strain can use them and incorporate them to the
final antibiotic, creating a new metabolite11,12. Usually the chemical precursors fed
compete with the natural substrates resulting in a low yield of the new compound. To
avoid that, mutation of the genes encoding the presursors can be carried in order to
force the strain to only use the muthasynthons as substrates. The second technique in
synthetic biotechnology is the combinatorial biosynthesis, which consists in
engineering a natural pathway or to redesign a de novo pathway which could make
new unnatural compounds 13-14.

Applying these techniques in Streptomyces strains, the key to designing new

drugs may lie in the Non Ribosomal Peptides (NRPs) , which are a huge class of natural
compounds comprising of not only the natural amino acids present in the common
peptides but also for non-proteinogenic amino acids, and other biomolecules such as
fatty acids, sugars or more complex compounds like heterocyclic rings 15.

Those compounds are arranged by enzymes called Non-ribosomal Peptide

Synthetases (NRPS)16-17. Due to their modular configuration, with key conserved
residues, and their iterative nature of molecular assembly they are suitable for
engineering in order to make them accept non natural compounds that finally could be
incorporated in the final NRP. 18-19

Two such antibiotics made via the NRPS route are congocidine and distamycin
and belong to a class of molecule called Minor Groove Binders (MGBs), they inhibits
the action of the different enzymes implicated in the reading and transcription of the
DNA such gyrases, helicases or topoisomerases, RNA polymerases 20. MGBs typically
have aromatic rings connected by single bonds that can rotate and relieve the torsional
rotary tension when those rings interact with the DNA groove. The most interesting
part of derivatives from congocidine or distamycin is that they bind strongly but in a
reversible fashion, which modulates the DNA expression without damaging it 21 since
so many other minor groove binders bind covalently to DNA causing permanent
damage 22.

2.2 Streptomyces

Actinomycetes were first described as a separate group from bacteria or fungi,

based on knowledge about their morphology23, later the genus Streptomyces was

10
proposed by Waksman and Henrici24. Despite of this, for a long time they were
believed to be part of fungi or the missing link between fungi and bacteria. The
Streptomyces genus is the largest among the actinomycetales, comprising about 600
validly described species25 and is increasing each year26. Streptomyces species are
aerobic, Gram-positive soil saprophytic bacteria that have the ability to form an
extensive branched substrate mycelium and also to produce aerial hyphae that
typically differentiate into chains of spores (Fig. 2). It has been found among
decomposing plants and soil, and are famous for the earthy smell that it has due to
production of geosmin27, one of the multiple secondary metabolites made by
Streptomycetes. Soil is a complex and highly variable environment due to its physical,
nutritional and biological characteristics. Streptomyces is one of the most numerous
and widespread bacteria in a range of soils28.

Streptomyces coelicolor A3(2) is the model species for the genus since is the
best known strain . It has an 8,667,507 bp genome arranged in one single linear
chromosome with an origin of replication situated in the center at 4,269,853 bp29. The
largest chromosome among Streptomyces is 10.1 Mb belonging to Streptomyces
scabies 30. The chromosome terminal ends (telomeres) are formed by inverted repeats
of diverse length that also have binding proteins in their 5 end. Spontaneous deletions
into the arms of the chromosome occur at a rate 0.1% per spores and can target these
telomeres leading to circularization of the chromosome.31

The genus is characterized by a high GC percentage (69-78%) in its DNA 32. One
enigmatic genetic feature of Streptomyces, is that any of the ends of the chromosome
can suffer deletions or amplifications up to 2 Mbp without showing any change in its
metabolism in laboratory cultivation31. However most streptomycetes shows synteny
(conservation in the gene order) in the conserved central core region of the genome 33.
Coding density is uniform over the whole chromosome, with all the indispensable
genes located in this conserved core. The non-essential loci coding for secondary
metabolites are largely, in contrast, in the arms of the chromosome.29.

2.2.1 Streptomyces habitat and life cycle

Most of the known species have been isolated from soil, where they bear part
as saprophytes in the ecological system. They are able to breakdown complex
molecules such as cellulose or lignine through enzyme secretion to the medium 34. This
is a adapation to soil as it is a rich carbohydrate environment but low abundance of
nitrogen or phosphate 28. Considering that soil is an oligotrophic environment ,
Streptomyces has proven that is able to growth in both poor and rich nutrient
conditions making them facultative oligotrophs.28.

11
 The life cycle is summarized in Fig. 2; a single spore germinates, the resulting
hyphae grows by tip branching forming a dense vegetative mycelium. Once nutrients
are exhausted, aerial hyphae emerge from the substrate mycelium and grow upwards,
curl and then septate to form uniform, individual spores that can act as dispersal
agents.35 It is at the onset of aerial hyphae formation that antibiotics are often
biosynthesized, suggesting that the processes have common regulatory features.

2.2.2 Streptomyces liquid cultivation

Streptomycetes have a proneness to form aggregates or pellets when cultivated

in liquid media. This creates a myriad of different physiological states among cells in
culture, since the cells inside the pellet will behave differently as the ones in the
outside28. To address this problem various methods have been tested, such the
inclusion of springs, cultivation in vessels with baffles and addition of polymers to
inhibit cell adhesion, however this does not remove the problem yet minimize it. It is a
particular problem during the industrial fermentation of streptomycetes.

2.2.3 Streptomyces metabolism

Primary metabolism stands for both catabolic and anabolic reactions

that are indispensable to promote the growth and life of the microorganism. The study
of Streptomyces revealed an unusual feature: the lack of a phosphotransferase system
for glucose36. It also has been observed the presence of other transport systems
involved in anaplerotic metabolism such the one for PEP 37, and the ones reflected in
the KEGG database (http://www.genome.jp/kegg/) Maltose, N-Acetyl glucosamine , -
Glucosides, L-Ascorbate. Streptomyces species have a complex secondary
metabolism32 producing a plethora of secondary metabolites, among which are many
well-known antibiotics, representing more than two thirds of known antibiotics38.

Streptomyces coelicolor A3(2) synthesise at least four compounds with

antibiotic activity39, actinorhodin or ACT, a blue pigmented polyketide compound 40, a
calcium dependent antibiotic41, the NRPS/PKS derived prodigenines and the type I
polyketide CPK 42. However genome sequencing revealed the presence of biosynthetic
clusters for up to 20 secondary metabolites within the genome.

Antibiotic production is dependant on the state of cell differentiation,

influenced by the growth rate, signalling molecules such as ppGpp or -butyrolactone
trigger production 43 and is also associated with physiological stress44. Recent studies
on transcriptional levels shows that the strongest switch happens during phosphate

12
limiting conditions.45 . No general conclusion can be drawn,however what is clear, is
that multiple levels of regulation occur for each metabolite.

13
Figure 2.Life cycle of Streptomyces46

14
 2.3 Nonribosomal Peptide Synthetases (NRPS) and
non-ribosomal peptides (NRP)
NRPS are large enzymes consisting of multiple independent modules with
different catalytic centers, within the same transcription unit, that are responsible for
the assembly of non-ribosomal peptides 47. They are natural molecules from bacteria
or fungi that have tremendous importance in terms of their pharmacological
applications 17. Research regarding their biosynthesis could help to understand how to
produce new drugs 47-48. The importance of these kind of peptides lies in their
diversity; while ribosomal encoded peptides use only the proteinogenic amino acids,
NRPS are capable of using non-proteinogenic amino acids, lactones, heterocyclic rings
and multiple other precursors11-48, in fact, more than 300 different precursors have
been identified in the past century49, that lead to the synthesis of a myriad of different
natural products (fig. 6 & 7)

2.3.1 Modular biosynthesis

Even though NRPs are structurally very diverse, their synthesis follows a
conserved pattern. Depending on which of the three kind of different NRPS the
synthesis of the NRPs can occur in a linear, iterative or non-linear manner. This three
different NRPS are classified due to structural differences:

1. Type I: the different constituent modules are arranged in one single

polypeptide (characteristically in fungal species50;51 ; fig. 8). The number
and sequence of the amino acids in the final peptide matches the
modules in the NRPS 15
2. Type II: modules are single proteins which interact between each other
more than once, creating repeated sequences of amino acids
(characteristically in bacteria52-53). (Fig. 9)
3. Type III: As type II that works together in an iterative or non-linear
fashion generating peptides that do not match the order in the NRPS
template .15 (Fig.10)

2.3.1 Core NRPS domains

NRPS regardless of the type contain catalytic centers in each single module
which work coordinately to direct the synthesis of the final peptide product 54.
Furthermore, beyond the variety of catalytic centers there are core motifs which
always are present15:

15
 First the adenylation domain (A) which is responsible for the recognition and
activation of the amino acid building block by forming an amino acid adenylate
through ATP consumption.

Second the Peptidyl carrier protein (PCP) which is a small protein (80-100 aas)
acting as a tRNA, transporting the activated amino acids between catalytic centers
through a thioester bond and third the condensation Domain (C) which catalyzes the
peptide bond formation with the downstream amino acyl unit that is tethered to the
PCP of the adjacent module.

2.3.1.1 Adenylation domain

The so called A domain is the first domain of the NRPS production line, the
gatekeeper. It is responsible for the recognition and activation of its cognate amino
acid in a two step reaction (Fig. 5a).

This adenylation costs one molecule of ATP, as AMP is incorporated to the

amino acid. Once the amino acid is adenylated it submits a nucleophilic attack from a
thiol domain in the phosphopantetheine moiety of the PCP domain, creating an
aminoacyl-S-PCP with a thioesther bound (fig. 5b).

According to previous studies 55 the main structure of an adenylation domain

consists of a small C-terminal domain and a larger N-terminal domain, with the
catalytic centre at the junction of these two regions 15. This adenylation binding motif
is conserved among the family of adenylate forming enzymes such the 4-coumarate-
CoA ligases, acyl-CoA ligases and the oxidoreductases. This module catalyses the same
reaction as the aminoacil-tRNA-synthetases even no structural homology has been
observed 56.

Among NRPS, the A domain shares an identity ranging from 30 to 60 % which

results in a core of conserved residues. Acting as the codons in the NRP synthesis, the
eight to ten catalytic residues are degenerated and were exploited for synthesis of new
antibiotics 57, using directed mutagenesis to modify the selectivity in order to accept
non cognate amino acids.

Recently, a prediction algorithm has been used for computational prediction of

point mutations resulting in alteration of substrate specificity58 based in solved
structures of existing A domains.

More studies were carried out in this family of enzymes, showing that at least
two conformational changes are needed in order to recognise and activate NRPs
substrates 15. This discovery lead to create bioinformatics tools that are based on the

16
structure of solved adenylation domains and can predict the susbtrate specificity of
novel NRPSs clusters such the NRPSs predictor2 59 integrated into the online webtool
ANTISMASH 60.

2.3.1.2 Peptidyl carrier protein domain

The peptidyl carrier protein is a small domain which compromises an average of
80 to 100 residues 15. It carries the growing product chain across the NRP catalytic
steps. This enzyme is first synthetized in an inactive apo form. The PCP is then post-
translationally modified by a phosphopantetheinyl transferase (PPTase) that add
covalently the 4 arm of a CoA molecule onto a serine residue that is conserved among
the catalytic center in the PCP. After that the tiol moeity is added and the PCP is
considered in its holo or active form, and is able to create a thioester bond among the
phosphopantetheine and its cognate aminoacyl substrate (Fig. 4) After the bond is
formed, the peptide is transferred from one PCP to the next one located in the next
module throughout the NRP assembly. In the NRPS modification the PPTase involved is
a type II thioesterase which is more promiscuous, leading to some mis-priming events
that leads to an incapacity to produce the peptide.15

2.3.1.3 Condensation domain

The adenylated precursors in the NRP synthesis are condensated by mediation

of the C domain which is the largest of the catalytic modules with an average size of
450 amino acids. It is usually fused to a N-terminal end accepting acyl groups from the
previous module (Fig.3a)61. The catalytic centre is situated in the junction of two
subdomains (C and N terminals) arranged in a V-shape (Fig. 3b). It was thought that a
conserved histidine motif was responsible for the catalysis (HHXXXDG), but mutational
studies shown that catalysis in C domains is based on electrostatic interactions instead
of an acid/base mechanism 62 63. The V-shape of the domain facilitates the extension of
the pantetheinyl arm of the up and downstream of the PCP. It also been shown that
the C-face of the V is non-selective against any substrate whilst the N-face shows strict
stereoselectivity.64

17
 Figure 315 A) Detail of how acyl groups are joined together.The carbonyl group gets
protonated so it submits easily a nucleophilic attack by the primary amine forming a
carbinolamine. In non enzymatic systems a posterior dehydration would lead to the form of an
imino group.

B)Condensation domain with conserved His motif in black, N-terminus in red and C-
terminus in blue, joined by a linker region in purple

18
 However the C domain does not only accept aminoacyl and peptidyl substrates
but is also able to condensate polyketide intermediates and interestingly non
proteinogenic amino acids.15 Their modular nature makes them suitable and
interesting for engineering, since making changes in its structure could lead to the
incorporation of alternative substrates 65. There have been different approaches to
reach this objective to produce a library of new NRP, those strategies were centered in
fusion of modules or domains66 . Nevertheless, so far there has been no great success
due to non-specific binding of substrates67 and discordant protein-protein
interactions68

19
 Figure 4. NRPS PCP protein domain 15, in blue, red ,orange and purple can be seen the -helix
I,II,III IV that aquires different folding conformations depending of the state of the enzyme

A-apo form

B-apo/holo form

C-holo form

20
Figure 5a. Adenylation reaction carried out by the adenylation domain in an NRPS

Figure 5b. Reaction between a PCP and its cognate activated amino acid

21
 Figure 6. Examples of non ribosomal peptides69 ( A: destruxin. B: amanitin.C: cyclosporin. D:
ergotamine. E: HC-toxin. F: AM-toxin. G: ferrichrome. H: enniatin. I: victorin. J: ACV. K: peptaibol.)

Figure 7. Kirromycin , a MGB antibiotic produced by Streptomyces collinus70

22
 Figure 8. Linear NRPS and example metabolite vancomycin 15

Figure 9.Iterative NRPS and example metabolite enniatin15

Figure 10. Non Linear NRPS and example metabolite vibriobactin15

23
 2.3 Congocidine

Congocidine was discovered in the research laboratories of Pfizer by A.C. Finlay

and colleagues in 1950 71. It was isolated from an undescribed actinomycete, later
named Streptomyces netropsis 71. Congocidine (Fig. 11) has two pyrrole rings with an
amide moiety at the start and the end of the molecule, this features gives the molecule
a particular spectra with a max=295 nm and makes it a member of the pyrrole-amide
antibiotic family, like TAN-868 A72, Lynamicins A-E73 or Pyrrolomycin A&B74.

It has been shown that congocidine specifically binds to DNA in its minor
groove75, however the chance of a successful bond is most likely to be feasible when
four or more pairs of AT nucleotides are placed in the minor groove75. It has also been
shown that the bond occurs in a non-covalent manner; the amide groups in the
extremes of congocidine form van de Waals close forces with the N-3 in adenine or O-2
when the nucleotide is thymine75. Recent work has also revealed that congocidine
shows at least two different complexes when binding the AATT sequence76. These
properties make congodicine and others DNA binding agents very interesting research
targets since minor groove binders interfere in the transcription of genes 77-78, making
them suitable not only for antibiotics purposes but also antitumor and antiviral
drugs77-78. An example of this potential is the work carried out to improve the survival
from endotoxemia disrupting a NO2 promoter79. Some proteins acts as transcription
factors, and some of them recognices and bind its cognate DNA promoter in the minor
groove. HMGA type I TF, one of these proteins has three DNA binding domains named
as TA hooks. Congodicine binds avidly this TA hooks competing with HMGA I TF,
resulting in a decrease of NOS production. However this same study showed that
congodicine may target other promoters due to its small binding sequence.

Even though more than 60 years have passed since congocidine was first
isolated, there is not much known about its biosynthesis. However it is known that S.
ambofaciens produces congocidine80, so when in 2006 the chromosome of the strain
ATCC23877 of this bacterium had been sequenced in a comparative study with S.
coelicolor and S. avermilitis 81, the biosynthetic cluster encoding the synthesis for
congocidine was identified18. Juguet et al., (2006)18 discovered that congocidine is
assembled by an unusual NRPS, which is formed from various single domains and a
stand alone NRPS module 18. They claim that this is the first work characterising the
biosynthesis of any pyrrole amide product. The cluster directing congocidine synthesis
has been narrowed to the genes

SAM0898-0921, as shown below: The 22 contiguous genes have been named (Fig. 12),
yet they are not all characterized experimentally.

24
Figure 11.Chemical structure of congocidine22

25
Figure 12.Genetic organisation of the congocidine gene cluster18

26
 2.4 Distamycin
Distamycin A was discovered in 1964, isolated for the first time from S.
distallicus among a mixture of antibiotic substances with butanol 82. Distamycin A (Fig.
13) is structurally related to congodicine with pyrrole amide rings. As a result of this,
the ultra violet and infra-red spectra of these two molecules are very similar82, with
max=303 nm for distamycin. Similar to congodicine, distamycin also binds to AT rich
regions in DNA 22. This antibiotic has been shown to be too toxic for use as an
antitumor drug83 yet has led to the synthesis of synthetic derivatives based in natural
aminopyrrols called lexitropsins. Lexitropsins and distamycin both show antiviral and
antibiotic activities84.

27
Figure 13.Chemical structure of Distamycin22

28
3.Project
Congocidine and distamycin have a narrow DNA selectivity among AT rich
secuences85, yet some synthetic minor groove binders called polyamides had been
developed with the ability to bind to AT and also GC rich sequences 86 making them
suitable modulators of gene expression, no great efficiency has been achieved in their
synthesis.

Recently the congocidine coding cluster has been identified18 showing the
presence of a stand-alone PCP and an A-PCP NRPS that could be manipulated to
incorporate pyrrole amide and imidazole building blocks to the molecule or using a
combinatorial approach, in to the related compound distamycin. Congocidine
represents an easy linear platform to modify and distamycin has a very similar
structure that could accept congodicine intermediates.

Aims
This PhD project is focussing on introducing non-natural compounds into the
backbone of existing antibiotics such as congocidine and distamycin, and also creates
hybrids from the two molecules,in order to do so, this work will be carried:

Create potential novel congodicine derivates using a congodicine coding

biosynthetic cluster in a heterologous Streptomyces bacterial host through
incorporation of non-natural amino acids.

Create potential novel congodicine-distamycin derivates using a

congodicine coding biosynthetic cluster or parts of it in a distamicyn
producing Streptomyces bacterial strain.

Awaken silent secondary metabolite clusters of Streptomyces using a

random peptide aptamer approach.

Sequence other Streptomyces strains in order to characterize the cluster

coding for known antibiotics and use it as another platform to produce
related compounds following the feeding strategy or the combinatorial one
(in example the pair Kirramicin-Aurodox)

29
Figure 1487.a)Congocidine gene cluster.b)Proposed pathway to congocidine

30
 A)

B)

1. R=H;R1=N 3. R=H;R1,R2=N
2. R=Me;R1=N 4. R=H;R1=CH,R2=N
3. R=CD3;R1=CH 5. R=H;R1=N,R2=CH

6. R1=CH,R2=N,R3=CH 8.
7. R1=N,R2=CH,R3=N

Figure 15.a) Pyrrolamide b)Pyrrolamide derivates to feed to Streptomyces Congodicine producer to

produce novel compounds

31
4.Materials and Methods
4.1 Bioinformatics
The congodicine cluster and its nucleotide sequence were obtained from
GenBank trough the NCBI search website:

http://www.ncbi.nlm.nih.gov/nuccore

The translation into protein sequence was carried out using software:

CLC Workbench (CLC Bio, v 6.9)

Protein sequences and searches for conserved regions among related catalytic
centres both online and software tools were used:

ClustalW2: http://www.ebi.ac.uk/Tools/msa/clustalw2/

MEGA 5 ( Mega software, v 5.2.1 )

BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi

For the biobrick engineering of the congocidine cluster the online NEBuilder
assembly tool was used:

http://nebuilder.neb.com/

Also for all the digestions and clonings, and for the annotation of the new
congocidine engineered cluster the next software was used:

Clone Manager (Sci Ed Central , v 6)

CLC sequence viewer (CLC Bio, v 6.9)

4.2 Media

The media used for all the experiments carried out during the project are listed
in this next section. If not specified, the water used in each medium was reverse
osmosis ultrafiltered water. To see the suppliers see annex. They were sterilized by
autoclaving or through 0.22 m filtration. The media was prepared with the
compositions given in glass bottles. The chemicals were measured and mixed before
autoclaved. For solid media, once solidified they were re-melted using a microwave
oven.

32
 4.2.1 Mannitol Soya Flour Medium4

MS agar is prepared with 1 L of tap water and 16 g of the following compounds:

Agar, Mannitol and Soya flour.

4.2.2 Mannitol Soya Flour Medium4+10 mM MgCl2

Its composition and preparation is as MS medium but added MgCl 2 to a 10mM

final concentration ( filtered from a stock solution in a 0.22 m filter).

4.2.3 Mannitol Soya Flour Medium4+R2 salt solution

It was used in order to promote the sporulation of some strains. Its

composition and preparation is as MS medium but added 200 l R2 solution/L of
medium. The R2 solution was prepared with the following components and then
autoclaved:

Table 1 .R2 salt solution composition

Component Amount (mg/L)

ZnCl2 40
FeCl36H2O 200
CuCl22H2O 10
MnCl24H2O 10
Na2B4O710H2O 10
(NH4)6Mo7O244H2O 10

4.2.4 Glucose Yeast Malt Medium (GYM) 88

The composition of the medium per liter of water is 2 g of CaCO3, 4 g of glucose

and yeast extract,10 g of malt extract and 12 g of agar.

The pH is adjusted to 7.2 before the addition of agar. For liquid medium agar
and calcium carbonate are omitted.

4.2.4 Malt Yeast Medium (MYM)

The composition of the medium per liter of water is 4 g of maltose and yeast
extract, 10 of malt extract and 20 g of agar.

The water used should be half volume of it from the tap, the other half from
reverse osmosis . After autoclaving 0.4 ml of R2 solution/200 ml MYM is added.

33
 4.2.5 Nutrient Agar Medium (NA) 89

The composition of the medium per liter of water is 3 g of Yeast/Meat extract,

5 g of Peptone and NaCl and 15 g of Agar.

The pH should be adjusted to 7. If the Nutrient broth desired instead the agar,
it is made identically but removing the agar.

4.2.6 2xYeast Tryptone Medium (2x YT)4

The composition of the medium per liter of water is 5 g of NaCl , 10 g of yeast

extract and 16 g of Tryptone.

The solid version of this media was obtained after adding 10g/L of agar to the
broth recipe.

4.2.7 YEME4

The composition of the medium per liter of water is 3 g of malt extract and
yeast extract , 5 g of peptone and 10 g of glucose.

To prepare solid media 10 g/L of agar is added .

If the medium is used for cultivation of a strain designated for DNA extraction ,
sucrose should be added to a final concentration of 34% w/w.

After autoclaving, MgCl26H2O is added to a final concentration of 5mM.

4.2.8 Luria Bertani (LB)90

The composition of the medium per liter of water is 1 g of glucose , 5 g of NaCl

and yeast extract and 10 g of tryptone.

34
4.3 Cultivation of Streptomyces strains
4.3.1 Isolation and cultivation of the strains of interest

Sterile tooth picks were used to pick a single colonies from a stock plate to
streak onto fresh plates. Cultivation at 30C for 6 days.

4.3.2 Preparation of a spore stock

The sporulating plates of the strain of interest were flooded with 3 ml of a 20 %

glycerol (v/v). In order to facilitate the removal of the spores from the solid media into
the liquid, a sterile cotton bud was used to rub softly over the surface of the plate.
After the release of the spores into the liquid, the spore suspension was collected into
1.5 ml tubes and stored at -20C.

4.3.3 Fermentation of the strains

The fermentations were carried as follows:

For the production of antibiotics at determined time points, 50 ml of YEME

medium in 250 ml flask was inoculated with 30 l of the spores of the strains of
interest. After 12 , 24 , 48 and 72 hours cells were harvested by centrifugation (2518 g
4C 10min).

For the growth curves, 500 ml of sterile YEME medium was poured into 2 L flask
and inoculated with spores of the strain of interest. The inoculum always came from
the same spore glycerol stock in order to have a better reproducibility among the same
strain.

When bioactive samples were detected, 6 L of culture were fermented until the
maximum antibiotic activity point to purify the compounds of the sample.

4.3.4 Growth curves

35
 The cell pellet obtained in each time point during the fermentation after the
centrifugation , was filtered in 0.22 m cellulose filters. The biomass on the filter was
dried by heating with a microwave oven at 750 W power for 10 minutes. After that the
filters were measured and the biomass calculated by subtraction of the weight of the
blank filter (previously measured).

4.4 MGB extraction and bioassays

4.4.1 MGB extraction from supernatant in small scale.

Samples (45 ml) from the fermentation broth were centrifuged at 2518 g for 15
min at 4C. After this, the cell pellet was removed from the supernatant and kept to
measure dry biomass subsequently.

The supernatant obtained was shaken to mix with 20 ml of chloroform and

centrifuged at 2518 g for 2 min at 4C. Whilst the organic phase was kept ,the aqueous
phase was mixed again with another 20 ml of chloroform with the later centrifugation
at 2518 g for 2 min at 4C. The aqueous phase was saved to check bioactivity.

The two organic phases were pooled and the chloroform was evaporated using
a water bath at 56C under 0.4 atm vacuum. After getting the dry extract, it was
suspended in 750 l of DMSO.

4.4.1 MGB extraction in large scale

Following 96 h of cultivation the cultures were centrifuged at 9,000 g to remove

the biomass. Part of the supernatant was frozen as the extraction takes 2 days. The 6 L
were subdivided into 40 x 150 ml aliquots. Each one of them were mixed with 40 ml of
chloroform and then decanted twice into a separation funnel. All the organic phases
were pooled and filtered with 0.22 m cellulose filters. After filtration, concentration
of the sample was carried in a rotary evaporator at 35C under vacuum. The
concentration was stopped before drying the extract (approximately 5 ml of
chloroform left) and transferred to the final container, in which the sample was dried
with pressurised air. Samples were resuspended in water in an ultrasonic bath.

4.4.2 Bioassay: activity of extracts/supernatant against B.

subtilis.

36
Bioassays were carried out in order to test the bioactivity of the extracted MGB to
avoid HPLC useless work.

4.4.2.1 Single plate bioassay.

Aliquots (300 l) of an overnight culture of the indicator organisms Bacillus

subtilis were spread on LB plates and left to dry. When the plate was dry, 4 mm
absorbent disk were laid onto the plates. To these disk 8 l of non-extracted
supernatant were added.

The plates were incubated at 30 for 24 h.

4.4.2.2 96 well plate bioassay.

The 96 wells in the plate were filled with 200 l of LB medium. One column of
the plate was used as a control with only medium. All the other columns were
inoculated with 4 l of an overnight culture of indicator organism Bacillus subtilis.
Furthermore the culture extracts were added at different dossages(1 , 2 , 5 l).

The extracts were added in three different wells and the negative controls
were LB+ B.subtilis , LB+ B.subtilis +DMSO , whilst the positive control was the addition
of pure congodicine. In order to determine the bioactivity of the supernatants
produced during the fermentation, we needed to validate it against the known
standard.

In order to do this we reproduced the same experiment using different

concentrations of congodicine standard instead of supernatant. In this assay we made
dilutions of pure congodicine in DMSO and added it into a 96 well plate containing LB
medium inoculated with B.subtilis. The range of concentrations used was 20,10 ,5 ,4 ,3
,2 ,1 ,0.5 ,0.4 ,0.3 ,0.2 ,0.1 mg/ml ,5 , 2.5 , 1.2 ,0.6 l/ml.

The plates were incubated in an orbital incubator at 180 rpm - 28C for 24h.

Growth of the indicator organisms were followed through UV-Vis absorbance at

255 ,450 ,580 and 650 nm, in order to check variances in the absorbance caused by the
microorganism growth and discard the absorvance of the extract added (maximum
absorbance of congodicine is 295 nm)

4.4.4 Stabilisation of the samples for HPLC

37
 The samples that were showed antimicrobial activity in the bioassay were
freeze dried to remove the DMSO of them and stored till the moment of their analysis.

To do this, the samples, which were approximately 700 l of DMSO extract

were diluted into approximately 5.5 ml of water. After the dilution the samples were
frozen to -80C using liquid nitrogen. Once frozen the samples were placed in a flask
under vacuum with a pressure of 0.1 millibar. After 24 h the majority of the samples
were converted to a powder ready to be storage. For the ones that were not ,the
procedure was repeated. To the samples from the large scale extraction procedure
MeCN was added to a final concentration of 5% because they were partially water
insoluble.

Before injecting the sample to the column, samples were centrifuged and
filtered with 0.22m filters to avoid precipitation of solids into the resin.

4.4.5 HPLC detection of congodicine.

In order to detect congodicine in the samples, it was first necessary to measure

a congodicine standard to optimise the detection procedure. To elute the column 3
different buffers were used. The reason for using them is to check the stability of the
analyte at different pH and also to choose among the one which gives the narrowest
peak.

The column used was a Phenomenex, model SphereClone 5u (250 x 4.6mm)

onto which the congodicine standard was loaded.

The elution buffers were:

1. H2O/CH3CN (pH 7.00)

2. CF3CO2H 0.1 % in H2O / CF3CO2H 0.1 % in CH3CN (pH 2.00 )
3. HCO2H 0.1 % in H2O / HCO2H 0.1 % in CH3CN (pH 3.00 )

The method was the same with each buffer, consisting in a linear gradient 95:5
to 0:100 over 28 min (Flow = 1ml/min).

38
4.5 Molecular Biology
4.5.1 Strains and plasmid

Strains:
E. coli
BL21(DE3) F- ompT hsdSB (rB-mB-) gal dcm (DE3).9193

DH5 F- 80dlacZ M15 (lacZYA-argF) U169 recA1 endA1hsdR17(rk-, mk+) phoAsupE44 -

thi-1 gyrA96 relA1 .94

et12567 puz8002 95

Streptomyces
S. coelicolor ATCC BAA-471 / A3(2) / M14529

S. coelicolor CH999 (act red cpk cda-) 96

S. coelicolor M1146 (act red cpk cda-) 39

S. coelicolor M1152 (act red cpk-cda- rpoB[C1298T])39

S. coelicolor M1154 (act red cpk cda- rpoB[C1298T] rpsL[A262G])39
S. netropsis DSM 4084671

Bacillus subtilis 168 97

Plasmids:
pET-15 b vector possessing an HisTag at the N-terminus, a subsequent [thrombin cleavage site] and
three cloning sites.Ori pBR322, T7 promoter, Ampicillin resistant Novagen

pBeloBAC11 Ori s (Ori2), T7 promoter, SP6 promoter, Higromycin resistant

pEX-A T7 promoter,CMV promoter Ampicillin resistant Eurofins Mwg Operon

39
4.5.2 Preparation of Chemical Competent Cells and
Transformation 98.

A 5ml culture was inoculated with a single colony from a plate of the respective
E.coli strain and grown overnight at 37 C at 250 rpm. 150 l overnight cultures was
used to inoculate 50 ml LB medium in a 250 ml flask and were grown at 37 C until an
OD600 of 0.3-0.4 was reached. The cells were harvested by centrifugation for 10 min at
4,000 rpm and were resuspended carefully using a pipette in 12. 5 ml ice cold 100 mM
MgCl2. Cells were centrifuged for 10 min at 4,000 rpm. Cells were resuspended
carefully in 3 ml of ice cold 100 mM CaCl2, another 22ml were added and the
suspension was kept on ice for at least 20 min. The cell suspension was centrifuged at
4,000 rpm for 10 min and was resuspended in 1 ml sterile 100 mM CaCl2 in 20%
glycerol. 100 l were aliquoted and frozen at -80 C.

Table 2. Buffer composition

Substance Amount (g) H2O volume (ml) Glycerol volume (ml)

CaCl2H2O 3.68 250 0
MgCl2H2O 5.09 250 0
CaCl2H2O 3.68 200 50

A 50 l aliquot of competent cells were mixed with plasmid DNA (1-5 l) and
were incubated on ice for 30 min. The mixture was heat shocked for 30 s at 42 C and
placed immediately on ice for 2 min, then 1ml of LB or 2xYT medium was added to the
cells and were incubated at 37 C in a shaking incubator for 60 to 180 min (depending
on the strain). The cells were then spread on LB agar containing the appropriate
antibiotics and incubated overnight at 37 C.
Colonies were picked to inoculate 5 ml LB medium with the antibiotics. The
cultures were used for conjugation of Streptomyces or they were used to prepare
glycerol stocks.

4.5.3 Intergenic Conjugation of Plasmids and Cosmids from E.

coli to Streptomyces99

An overnight culture of the E. coli strain ET12567/puZ8002 transformed with

the respective cosmid and was grown to an OD600 of 0.4 to 0.6 and cells were
harvested by centrifugation for 5 min at 4,000 rpm. The cells were washed twice with
LB medium without any antibiotics and meanwhile a spore suspension of S. coelicolor
was centrifuged and diluted to a spore number of 2 x 108 spores per ml using 2xYT. The
40
mixture was heated to 50 C for 10 min. 500 l of each organism were mixed and
plated onto MS agar and incubated for 14-18 h after that the plates were overlaid with
nalidixic acid (25 g/ml) and the selective antibiotic (apramycin). The plates were
incubated or two days at 30 C and the Streptomyces colonies were transferred onto
nutrient agar (one on an apramycin containing plate and one to apramycin plus
kanamycin containing plate) to screen for double crossover strains.

4.5.4 Screening for Double Crossovers in Streptomyces99

Primary exconjugant were transferred onto nutrient agar (one on an apramycin

containing plate and one to apramycin plus kanamycin containing plate) and were
incubated for two days. Colonies growing on apramycin (selection marker for insertion
cassette) but not on kanamycin and apramycin (cosmid backbone selection marker)
were assumed to be double crossovers. The colonies were streaked on MS agar for the
preparation of spore stocks for further characterisation.

4.5.5 Isolation of Plasmid and Cosmid DNA from E.coli Cultures

using the Alkaline Lysis Method 100

Aliquots (1.5ml) of an overnight culture of the strain of interest was harvested

by centrifugation at 13,000 rpm for 2 min and the pellet was resuspended in 100 l of
ice cold solution 1 (glucose 50 mM, Tris HCl 25 mM , EDTA 10 mM, pH 8.0), then 200 l
of solution 2 (NaOH: 0.2 mol , SDS 1%) is added and mixed by inverting the tube.
Finally 150 l of solution 3 (60 ml of 5 M potassium acetate, 11.5 ml of glacial acid,
28.5 ml of dH2O) is added The contents were mixed by vortexing shortly and were
stored on ice for 5 min. The mixture was centrifuged at 13,000 rpm for 5 min and the
supernatant was transferred to a fresh tube, two volumes of ethanol were added and
was mixed by inversion and allowed to stand at room temperature for 2 min. The tube
was centrifuged for 5 min at 13,000 rpm and the supernatant was removed, the pellet
was rinsed with 1ml 70% ethanol and allowed to air dry at room temperature for 10
min. The pellets were resuspended in 50 l of TE buffer (10mM Tris-HCl, 1mM EDTA,
pH 8.0)

41
4.5.6 Restriction Digest

The digestion of DNA molecules has been planned in silico in order to

determine the fragment sites and enzymes to use before performing the digestion. The
restriction digests were performed as the supplier specifications (NEB, Promega).

4.5.7 PCR

All the PCRs in the project were carried out using Q5 Hot Start polymerase
from NEB using the protocol recommended from the supplier

The programm used on the PCR machine was:

1. 95C for 1 minute

2. 95C for 15 seconds
3. 60C for 30 seconds
4. 72C for 30 seconds
5. Go to 2 , 29 times
6. 72C for 10 minutes
7. Cool down to 12C.

4.5.8 Cloning

The genes of interest were taken from the congodicine cluster and cloned with
EcoRI-NdeI sites into pET15b. The insert and the vector were ligated using T4 DNA
ligase for 12 h starting at room temperature decreasing to 4C at a constant gradient
using the buffer recommended by the supplier.

4.5.9 Overexpression of proteins

Aliquots (20 ml) of an overnight culture in LB and selective antibiotic

(carbenicillin 100g/ml) of the E. coli BL21 strain with the plasmid of interest were
used to inoculate 1 L of fresh LB containing the selective antibiotic (carbenicillin
100g/ml) in a 2 L Erlenmeyer flask. The strain was cultivated in a rotary incubator
(210 rpm at 37 C) until it reached an OD600 of 0.4-0.6. At this point a sample of 1 ml
was taken for SDS-PAGE and the rest of the culture was induced with 1 ml of 1M

42
solution of IPTG (1mM final concentration) with further incubation for 2 hours. 1ml
sample was taken for SDS-PAGE and then the cells were harvested by centrifugation at
12,000 g. The supernatant was discarded and the cell pellet resuspended in A buffer
(50 mM Tris-HCl pH 7.5, 7.2mM MgCl2, 7 mM KCl, 5% glycerol and 20 mM imidazole).
This can be kept at -80 C until protein purification is carried out.

4.5.10 SDS PAGE

The composition of the gel varies according with the size of the protein. High
percentage acrylamide gels are used for lower sized proteins. The composition of the
resolving gel is on the table 3:

Table 3. Acrylamide gel composition per given concentration

% of the Gel 15 14.4 14.25 13.5 12.75 12.45 12 11.4

1.5 M Tris-HCL, 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
0.4% SDS,pH 8.8
H2O 2.5 2.7 2.75 3 3.25 3.35 3.5 3.7
30 % Acrylamide 5 4.8 7.75 4.5 4.25 4.15 4 3.8
The stacking gel where the samples are loaded is always the same: 2.5 ml of
0.5 M Tris-HCL 0.4% SDS pH 6.8, 5.5 ml of H2O and 2 ml of 30% acrylamide solution.

Before purification of the protein , samples were taken before induction of

IPTG and after are processed to determine the presence of the protein. Both 1 ml
samples are centrifuged at 11,000 g, afterwards 900 l of the supernatant was
removed and the cell pellet resuspended in 100 l. 100 l SDS 1x buffer (63mM Tris-
HCl, 2% SDS w/v, Glycerol 10% v/v , B-mercaptoethanol 5% v/v, Bromophenol blue
0.001% w/v) is added and incubated at 95 C for 5 min after mixed.

If the samples come from a purificated protein, 10 l are taken and mixed with
equal volume of SDS 1x buffer and boiled for 5 min at 95 C.

4.5.11 Protein purification

Cell pellet from protein overexpression are resuspended in A buffer (50mM

TRIS-HCl pH7.5, 7.2mM MgCl2, 7mM KCl, 5% Glycerol and 20mM Imidazole). After
thawing of the pellet the cells were lysed sonication (Branson Sonifier with Tappered
microtip 1/8). Afterwards the cell lysate was centrifuged at 15,000 rpm for 30 min at
4 C. The supernatant of the lysate was then manually loaded with a 10 ml syringe onto

43
1ml HisTrapTM column (GE Healthcare). Once loaded , the column was placed on Akta
purifier. The elution were performed with two buffers, A and B (composition as in
section xy) ,starting with 100 %A : 0% B (50mM TRIS-HCl pH7.5, 7.2mM MgCl2, 7mM
KCl, 5% Glycerol and 500mM Imidazole) a linear gradient was used to 0%A:100%B over
20 min.

44
5.Results
5.1 Transformation of host strains

The congodicine cluster were included in the peloBAC cosmid with a C31
integration site resulting PCG002.

To integrate the cluster in the genome of the host Streptomyces strains, they
were conjugated with E coli ET12567/ pUZ8002/ pCG002. Higromycin was used as the
selection marker and nalixidic acid to eliminate E.coli. It was observed that the
successfully transformed strains struggled to sporulate and some of them did not
sporulate at all (S. netropsis/pCG002, M1152/pCG002, M1154/pCG002). Single
colonies from the non-sporulating strains were picked and streaked into MS medium
without hygromycin, leading to sporulation to being able to prepare spore stocks.

To double check the retention of the cluster in the genome, from this spore
preparation plates were streaked for single colonies without the selection marker, and
from this plates 20 different CFU were picked and patched onto a plate with the
selection marker. All of the colonies of each strain growth in the plate with the
selection marker, proving a successfully genome integration event.

5.2 Optimisation of growth in different media .

As some of the strains struggled to sporulate, their cultivation was performed

on various media. One of the strains can be appreciated in the fig. 16.

45
Figure 16. Streptomyces M1146 plated in MS medium (left) vs MS medium+R2 salt solution (right)

46
5.3 Overexpression of the cgc19 peptidyl carrier protein in E.coli

The cgc19 gene encoding the stand alone PCP enzyme in the congodicine
cluster was chemically synthesized by MWG Biotech and was cloned in to their
standard plasmid pEX (Fig. 17).

In order to determine the presence of the gene the digestion of pEX containing
the cgc19 gene was performed with the restriction enzymes NdeI / EcoRI. For that
purpose two different colonies were picked resulting from the transformation of DH5
with the plasmid, which were cultivated overnight. The plasmid from these cultures
was extracted and digested. 366 bp and 2450 bp bands were expected. However
running the products on an agarose gel showed a non-expected band pattern.
Therefore primer (Fw: CGAGTCGATCGACGTACAC, Rv:
GAGTGGCGTTCTCGAAGAG,product size 164 bp) were designed that were
inside the gene in order to check the presence of the gene in the plasmid by PCR.

The PCR product was revealed in an agarose gel, (fig.18) confirming the presence of
the gene in the plasmid.

47
Figure 17. Map of pEX(. T7 promoter,CMV promoter Ampicillin resistant eurofins mwg operon)
containing the cgc19 gene

48
Figure 18. PCR amplification of a region of the cgc19 gene a 166 bp size was expected, and the product
obtained appeared to be of the expected size.

49
 After confirmation the presence of the correct sequence, the cloning of the
fragment into pET15b for overexpression of the protein was done. To that end, fresh
isolation of both plasmid were performed from an overnight culture in LB with the
appropriate selection markers. Those plasmids were digested with the same pair of
enzymes EcoRI / NdeI to obtain the vector and the insert with a PCR / GEL isolation kit
from Promega. As before the digestion were simulated in silico using clone manager
(fig 19). The gene insert was expected to be 419 bp long whilst the vector 5375 bp (Fig
20).

Once isolation of insert and vector, an overnight ligation was performed at 1:2
ratio. The resulting plasmid was transformed into DH5 and then spread on a plate
with the appropriate selection markers. Subsequently 6 of the resulting colonies were
cultivated overnight to make fresh preparation of the assembled pET15bcgc19. A
digestion of this plasmid was carried to check the presence of cgc19 in the ligation. The
digestion consisted in EcoRI/NdeI to check but also NcoI/PstI which were cutting inside
the insert.

Colonies 1, 2 and 3 showed a correct band pattern but the size of the bands
were not clear as it can be seen in the fig. 21, for that reason pET15bcgc19 was sent
for sequencing to Eurofins Operon.

The sequencing data showed a successful cloning without any mutation, and so
the protein overexpression was settled. Before proceeding to do the purification a
SDS-PAGE with the samples before and post induction were run. (fig. 22).

Aliquots (0.5 ml) collected during the purification were loaded on to an SDS
PAGE gel (Fig. 23). A band of the expected size (14KDa) can be observed in addition to
a possible dimer at 24 KDa (Fig. 23).

50
Figure 19. a)pET15b cut with NdeI/EcoRI vector expected band of 5375bp,b)pEX cut with NdeI/EcoRI
insert expected band of 419 bp,c) final assembly .

51
Figure 20. Gel purification of vector:insert before cloning. Lanes 1,2 containing the digestion of pET15b
with EcoRI/NdeI, the 5375bp corresponds to open vector suitable for cloning. Lanes 3,4 containing the
digestion of pEXcgc19 with EcoRI/NdeI, the 419 bp band corresponding to the cgc19 insert.

52
Figure 21. Digestion of potential clones containing pET15b-cgc19. NcoI/PstI cutting the inside of cgc19
on the left part of the gel , three band expected if cgc is in the plasmid, 4569bp,834bp,393bp . Colonies
1,2,3 shown expected bands. Digestion with EcoRI/NdeI shows correct size of vector but no insert,
bands at 5347bp and 421bp were expected.

53
Figure 22. Overexpression of cgc19 protein in whole cells

54
Figure 23. Cgc19 protein post purification by AKTA. From left to right, sequential eluted fractions from
the column. It can be appreciated a small dimer band at 24 kDa when looking to the right.

55
5.4 Antibiotic production and isolation

5.4.1.Growth curves

The strains were fermented to different time points and then the culture was
centrifuged and the supernatant was ready for antibiotic extraction. The biomass was
measured to analyse the growth to calculate the antibiotic yield per biomass. A first
growth curve was performed using 200 l of spores as inoculum to YEME medium. The
assay was stopped at 72 h.The fig.24 , shows the first growth curve.

A second fermentation was carried out for 108 hour, the amount of inoculum
was reduced to 80 l.

56
 400

350
S.Netropsis
300
S.Netropsis002
Biomass (g/L)

250
WTM145

200 WTM145002
CH999
150
M1146002
100 M1152002
M1154002
50

0
6 12 18 24 30 36 48 66 72 Time (h)

Figure 24. Growth curve of Streptomyces heterologous host and Streptomyces netropsis in a 72 hours
run. Only one sample per timepoint.

57
 450

400

350
S.Netropsis
300
S.Netropsis002
250 WTM145
Biomass (g/L)

WTM145002
200
CH999
150 M1146002
M1154002
100
M1152002
50

0
18 24 30 66 72 78 90 96 108
-50 Time (h)

Figure 25. Growth curve of Streptomyces heterologous host and Streptomyces netropsis in a 108 hours
run. Only one sample per timepoint.

58
5.4.2.Congodicine standard bioassay

All concentrations from 20 mg/ml to 5 g/ml showed a complete inhibition of

the growth of the indicator microorganism. The next concentration 2.5 g/ml also
showed a strong inhibitory effect, close to 95% of the inhibition. However after that, a
concentration of 1.2 g/ml and lower, could not be differentiated from the wells
containing no antibiotics.

5.4.3. Crude supernatant and supernatant extract bioassay

Some of the extracts/aqueous phase coming from the fermentation of the

strains showed complete, partial or no effect in the growth of the indicator
microorganism.

The percentage of inhibition in the growth of Bacillus subtilis can be seen in the
tables 5,6,7.

59
 Tables 5,6,7. Showing the inhibition percentage of Bacillus subtilis growth with different dosage of
culture extract. Meaning 100% for complete inhibition

1l/Well of 1 2 3 4 5 6 7 8 9
S.netropsis 214% 240% 990% 00% 134% 174% 80% 163% 164%

S.netropsis002 515% 333% 254% 163% 125% 195% 294% 406% 216%
WTM145 116% 311% 282% 161% 362% 982% 242% 121% 1521%
WTM145002 154% 309% 962% 89% 412% 992% 202% 113% 227%
CH999002 225% 972% 183% 282% 373% 983% 141% 241% 228%
M1146002 991% 991% 331% 371% 414% 254% 163% 241% 235%
M1152002 261% 3711% 317% 3511% 3610% 2210% 107% 242% 1912%
M1154002 294% 990% 296% 990% 349% 379% 206% 224% 191%
timepoint(hours) 18 24 30 66 72 78 90 96 108

2l/Well of 1 2 3 4 5 6 7 8 9
S.netropsis 175% 352% 382% 323% 376% 3515% 295% 344% 257%
S.netropsis002 364% 368% 416% 381% 331% 348% 443% 521% 242%
WTM145 376% 572% 991% 211% 281% 8734% 151% 06% 281%
WTM145002 299% 624% 990% 2317% 279% 8736% 246% 364% 312%
CH999002 479% 593% 991% 413% 4310% 8539% 1813% 343% 332%
M1146002 5615% 613% 4616% 445% 4510% 8931% 2512% 336% 355%
M1152002 5314% 569% 444% 453% 448% 371% 181% 346% 365%
M1154002 1000% 5410% 567% 10540% 8246% 3714% 344% 274% 431%
timepoint(hours) 18 24 30 66 72 78 90 96 108

5l/Well of 1 2 3 4 5 6 7 8 9
S.netropsis 214% 243% 990% 014% 131% 1712% 87% 165% 165%
S.netropsis002 51% 338% 259% 167% 129% 194% 292% 401% 213%
WTM145 117% 319% 285% 166% 368% 981% 249% 128% 154%
WTM145002 1516% 309% 961% 86% 412% 990% 209% 1130% 2225%
CH999002 2210% 970% 188% 285% 374% 980% 148% 2414% 221%
M1146002 991% 9936% 333% 3725% 416% 250% 1610% 2416% 236%
M1152002 2612% 376% 311% 354% 362% 226% 108% 244% 19%6
M1154002 2943% 9943% 296% 9940% 349% 372% 2011% 224% 196%
Timepoint(hours) 18 24 30 66 72 78 90 96 108

60
5.4.4.HPLC detection of congodicine standard

HPLC analysis of the extracts show a congodicine peak at approximately 10

minutes retention time, although this is buffer dependent (Fig. 26-28).

61
 Figure 26. Congodicine elution in H2O/CH3CN (pH 7.00), congodicine peak at 9.872 min
(red arrow pointing)

Figure 27. Congodicine elution in CF3CO2H 0.1 %/CH3CN (pH 2.00 ) Congodicine peak at
10.922 min(red arrow pointing)

62
Figure 28. Congodicine elution in HCO2H 0.1 % /CH3CN (pH 3.00 ) Congodicine peak at
9.663 min(red arrow pointing)

63
6.Discussion and future work
6.1 Discussion
Transformation of host strains
All of the colonies of each strain growth in the plate with the selection marker,
proving a successfully genome integration event.

Optimisation of growth in different media

The conclusion of this series is that the best medium to cultivate Streptomyces
strains is MS + R2 salt solution.

Overexpression of the cgc19 peptidyl carrier protein in E.coli

A band of the expected size (14KDa) can be observed in addition to a possible dimer at
24 KDa (Fig. 23).

Growth curves
It can be appreciated the same tendency in both assays from the beginning to
the 72 h point.

The transformated S. netropsis containing the congodicine cluster starts

growing faster than the nontransformed from the 66 h point, and specially after 90 h
(fig. 25). This can be explained as an increased resistance to congodicine since the
cosmid contain the resistance mechanism.

However due to the lack of consistency and poor data yield more growth curves
has to be performed in order to give any conclusion.

Congodicine standard bioassay

This result allowed us to conclude the presence of at least 2.5 g/ml of
congodicine in the bioactive samples obtained from the supernatant of the cultures
from the strains M1146/PCG002,M1152/PCG002,M1154/PCG002,CH999/PCG002
which were the ones producing no antibiotics before the inclusion of the congocidine
cluster.

64
Crude supernatant and supernatant extract bioassay
The results may be inaccurate since the measurements went out of the linear
range that Lambert Beer Law allows. However the aim of the experiment was to
determine production of congocidine in an heterologous host and to point which
samples were worth to analyse by HPLC in S. netropsis/PCG002.

This allowed to determine which were should be analysed by HPLC.

HPLC detection of congodicine standard

The background peaks can be attributed to the equilibration of a new
HPLCcolumn. Elution with water resulted in a higher concentration of congocidine
because the sample was less diluted than in the runs using TFA and formic acid. We
observed that congodicine is stable under all three separation conditions and no major
changes in the retention time occur. Also no degradation of the compound was
observed.

6.2 Future work

To achieve the aims stated, the congocidine gene cluster will be engineered in
order to try and force a host containing it to incorporate non-natural compounds to its
synthesis (fig.15).

Those non-natural compounds would be well known DNA specific targeting

molecules (i.e. GC pairs, seen on figure 15b) just to determine proof of concept.

The first step in that direction will be to determine which host strains are able
to produce congocidine and/or distamycin in a most productive manner.

The next thing to do will be to search for highly conserved sequences in related
NPRS to determine which residues should be mutated to change Km of the PCP or A
domains.

As the non-natural compounds to feed will be amino-pyrrol derivatives, we can

investigate modifying cgc18 and cgc19 if we pay attention to the proposed congodicine
pathway (Fig. 14):

Also the cgc2 and cgc16 genes could be engineered since they are as well
independent NRPS modules to incorporate other chemicals in an earlier stage.

In order to have an easy work platform, the whole cluster will be modified placing
unique restriction enzymes between specific genes, in order to easily remove certain
parts of the cluster and add modified genes.

65
6.2.1 Congodicine cluster biobrick like assembly

Since the ultimate essence of the project involves the engineering of the
standalone enzymes responsible of the recognition and assembly of the precursors of
congodicine, an easy platform to work with has to be established.

Subdividing the cluster encoding the congodicine synthesis in easy

exchangeable parts like lego bricks that can be combined in many different ways. For
this unique restriction sites will be inserted between the genes encoding the NRPS
modules present in the cluster. Doing so, it will be able to switch between different
engineered parts in just one cloning reaction.

To achieve this the Gibson Cloning technology 101 will be used. This cloning
method was developed in 2009 and allows to combine simultaneously (up to 10) DNA
fragments that join due to overlaps in their sequence (20bp overlap as a minimum),
through a mixture of enzymes.

The enzymes used to link the DNA are:

T5 exonuclease : to chew back 5 ends, resulting in one stranded DNA ready to

anneal
DNA polymerase: that will incorporate nucleotides to fill the gaps
Taq ligase: that will join the DNA overlapped

This cloning strategy (figure 29) has been used to the chemical synthesis and
assembly of the mitochondrial DNA of a mice 102

The genes in the congodicine cluster with the flanking restriction sites will be
produced individually by PCR. After this, the individually synthetized genes will be
assembled using a Gibson Cloning kit.

6.2.2 Enzyme characterisation and mutation

Once isolated the stand alone enzymes some feeding experiments can be done
with radiolabeled precursors or modificated precursors that will be synthetized at the
Strathclyde Organic Chemistry department to understand how the protein uptake and
assembly them either in vitro or in vivo. Also a library of mutants can be performed
using the Bio brick like platform.

66
Figure 29. Gibson cloning strategy overview, adapted from https://www.neb.com/

67
6.2.3 Random Aptamers Library

A library of random peptide aptamers will be used to screen for mutants. These
small protein peptides could bind some DNA sequences in Streptomyces, which in turn
could activate the expression of confirmed but silent secondary metabolite clusters.

The strategy to follow is to produce a library of plasmid containing the aptamer

DNA . The aptamer gen will have an EcoRI/NdeI site for cloning into pET15b and 16
random amino acid encoding triplets. Regarding this triplets, the last base always will
be G or C since the probability of producing a stop codon in Streptomyces drops.

Once the library is constructed, congujation in to Streptomyces coelicolor A3(2)

will be performed. Exconjugants will be cultured and screened for novel metabolite
production.

6.2.4 HPLC analysis of bioactive samples

After developing a method for congodicine elution, analysis of the bioactive

samples from the fermentation experiments will be carried out. This will allow to pick
the best super host in terms of stability, reliability and antibiotic production. Also it
could be lead to discover a hybrid in the S.netropsis transformed strain.

68
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