The spontaneous mutation frequency is in the range 10-4 to 10-2 (~ 3 - 4 folds higher than the typical one)

The genus Streptomyces is prone to a remarkable genetic instability

The lost characters are associated to very large deletions on the chromosome, involving tens of bp thousands

The instability can cause the loss of the ability to sporulate, or to produce the desiderd products or both, and it is a serious problem for the industrial strains

This peculiar feature derives from the very structure of the chromosome

The unusually large chromosome is linear

Characterized by the presence of Terminal Inverted Repeats (TIR)

TIR

TIR

Telomeric proteins bind the TIRs

GENOME STRUCTURE

The chromosome has a unique A/T rich replication origin (oriC), located in the central region

OriC interacts with DnaA in the same way observed in E. coli

The replication proceeds from oriC towards the ends

ORI C

During replication the 5' end of the two strands remains incomplete, resulting in a stretch of single stranded DNA (250 - 300 nt) at each extremity

In the single strands there are several repeats that double over to form hairpin structures at each end of the chromosome, forming protective telomeres

Several Tpg proteins have been identified, and they are highly conserved in size (184 to 185 amino acids) and sequences

The archetypal telomeres in both the chromosome and the linear plasmids, are capped by telomeric proteins encoded by tpg genes

The TPG proteins bind covalently to the 5′ ends of the DNA

the telomere sequences are highly conserved for about 200 bp in essentially all the linear chromosomes and most of the linear plasmids

of Streptomyces spp.

ORI C

The TPG proteins have different functions

acting as primers for the DNA synthesis at the two ends

favouring the interaction of telomeres to form circular structures

anchoring the chromosome to the membrane during replication

A peculiar feature of streptomycetes is the presence of giant linear plasmids, the best studied among them is SCP1 (S. coelicolor)

juxtaposition proteins bring together the plasmid termini by binding to specific regions of palindromic symmetry

SCP1 contains atypical telomere sequences and no tpg homologues, and can replicate in the absence of tpg, as it carries a novel TP gene

Justaposition prot.s

The tpc gene on SCP1 encodes the 259-amino-acid TP that caps the plasmid telomeres and shares no homology with Tpgs. The tac gene upstream of tpc is

also required to replicate the SCP1 telomeres

The chromosome has a central CORE

And lateral arms

The genes related to the primary functions of the cell are clustered in the chromosomal CORE

In the arms are located mainly the genes associated with the production of secondary metabolites and hydrolytic enzymes, or to the response to environmental stimuli

The arms differ among the species and are probably more recent

This is the more ancient region, conserved among the sequenced Streptomyces species, and shows

similarities also with other actinobacteria

The presence of transposons is also frequent

Variability, in streptomycetes, is observed

Within the same species

In the same strain, in a time course (i.e. some features are not stable)

Of course, instability is a serious problem for the industrial

strains

It relates to the frequent appearance of deletions on the chromosome

It has been studied thoroughly

oriC

S. lividans chromosome (8 Mb)

AUD1 AUD2 argG cmr

deletions are frequently focussed on both ends, suggesting that, timely, the chromosome spontaneously circularizes

The deletions are often accompanied by tandem amplifications of up to several hundred copies of specific DNA sequences: the Amplifiable Units of DNA (AUD) of about 3 to 100 kb

The most frequently involved genes share the external localization, near the telomeres

The mutations mainly affect the genes related to the secondary metabolism, and the argG (argininosuccinate synthetase) causing an auxotrophic state for Arg

Where both the secondary metabolism related genes, and argG are found

An attempt to artificially achieve the circularization of the chromosome with a targeted recombination process was done

the telomeres were removed and replaced by a kanamycin resistance gene (aphII).

Until the cells were kept under kanamycin selection, the chromosome was very much stable as demonstrated by

the decreased loss frequency of the cmr gene

K K

However, the cultures harbouring the circularized chromosomes grew and sporulated poorly

cmr

Instead of the simple replacement (bracketed)

By relieving the kanamycin selection, all the amplified DNA was deleted together with long stretches of the neighboring regions

the circularized chromosome contained tandem amplifications (bracket with the

subscript n) of the aphII gene together with one of the flanking DNAs

Possibly, the stable circularization could interfere with the chromosome duplication, by hampering the recognition of the telomeres

Otherwise, the telomeres of Streptomyces could be essential to the anchoring and/or the mobility and distribution of chromosomes in the cells

By now, the artificial circularization is not regarded as an expedient tool to improve the stability of the strains

Further strategies have been proposed

TO MOVE THE BIOSYNTHETIC TARGETED GENE CLUSTERS TO THE

CORE, IN ORDER TO PREVENT THEIR DELETION

TO MOVE ESSENTIAL GENES TO THE ARMS TO PREVENT

DELETIONS TO OCCURR

Such an approach may be useful for genetics studies, while manipulating aploid spores

TO MOVE THE ESSENTIAL GENES FROM THE CORE TO THE ARMS, DOES NOT ADDRESS THE PROBLEM

So, even genomes which have lost essential traits can persist, as the deleted functions are provided by the

intact copies located in the same cytoplasm

Many nucleoids coexist within the syncytial cells of the streptomycetes mycelium

But the industrial cultures, grow in

liquid media, and do not sporulate

TO MOVE THE GENE(s) OF INTEREST IN THE CORE REGION MAY HELP: IT HAS BEEN OBSERVED THAT

The OXYTETRACYCLIN biosynthetic cluster

(S. rimosus)

Core localization

To place the genes in plasmid vectors could be expedient and, moreover, allow a higher expression level

Arms localization

Very high loss frequency

ACTINORHODIN biosynthetic cluster

(S. coelicolor)

Very stable

This approach could offer a solution but it implies the need to create a dedicated engineered strain for each product

GENE CLONING IN STREPTOMYCES

Benefits: high secretion ability Manipulation techniques are available

Their filamentous nature makes it difficult both to set and control the growth conditions in the fermenters

The presence of very efficient restriction systems limits the choice of the E. coli strains suitable to prepare the DNA

Only a few Streptomyces strains have been studied and are commercially available; the cloning systems are not yet well characterized

The regulation patterns are very complex

DRAWBACKS

The diversity, even among the strains of the same species is larger than the one encountered with other bacteria

The secretion of aminopeptidases can damage the products, in a sequence-dependent manner

Possible strategies aimed to limit such events are:

Purposefully placing specific signal sequences upstream to the ORFs to be expressed

Such a problem was observed, for example, while expressing TNFa: the first 4 N-terminal AAs were removed

So to provide an unambiguous substrate for the already known Streptomyces extracellular aminopeptidases

Creating aminopeptidase deficient strains

S. coelicolor (“of the color of the sky”) is the model organism for genetic and morphological analysis

WHICH SPECIES TO CHOOSE?

Streptomyces is a very large genus so to choose the best possible host is very important

However, as most streptomycetes, its powerful restriction systems form an almost invincible barrier for the shuttle

plasmids propagated in E. coli

for cloning purposes S. lividans (strain 66), a close relative of the model organism, is the host of choice

S. lividans (producing a a livid colour; as a word pun: the one who provokes bruises)

As all the other species within the Streptomyces genus, it produces a very large amount of extracellular proteins

And due to its limited restriction–modification system that avoids the need of non-methylated DNA for transformation or conjugation

Two S. lividans mutant strains (TK64 and 3104) have been obtained, by curing their natural plasmids SLP2 and SLP3

TK64 and 3104 are endowed of two useful chromosomal markers: both of them share a proline auxotrophy (pro-2) and they are resistant to streptomycin (str-6) or spectinomycin (spc-1), respectively

Streptomyces lividans is routinely employed as a host for gene cloning,

largely because this species recognizes a large number of promoters, has a low

endogenous protease activity

Grow and sporulate very well on solid media

Are able to grow in liquid media (without sporulating)

Are easily converted to quickly regenerating protoplasts

S. lividans 66 and the derived strains

The protoplasts are easy to transform and to transfect with the φC31 phage

S. lividans 66 and the derived strains are deprived of replicons able to interfere with the replication and/or the analysis of plasmids or phages used as cloning vectors

They accept the DNA from many donors, including many other Streptomyces species

Unlike many other streptomycetes, such as S. coelicolor

SELECTION MARKERS

the main resistance marker used to select the recombinant S. lividans colonies is the resistance to thiostrepton

The streptomycetes are among the main producers of antibiotics, hence they are insensitive to most of them

Thiostreptons (cyclic oligopeptides) bind to ribosomes, blocking the A site

related functions

The choice of selection markers is less straightforward than for E. coli or Bacillus

The resistance gene is derived from S. azureus, one out of the thiostreptons

producing species

Apramycin (Nebramycin II) a broad-spectrum aminocyclitol antibiotic produced by Streptoallotheichous tenebrarius

It is used in veterinary medicine, and employed as a marker for the lateral gene transfer in both mycobacteria and streptomycetes

Neomycin: a narrow-spectrum and quite toxic aminoglycoside antibiotic Produced by Lechevalieria (ex Streptomyces) fradiae

PROMOTERS The DNA regions acting as promoters in Streptomyces are classified in two groups

the first group partly conserves the structure of the σ70 promoters of E. coli, with regularly spaced and well defined -10 e -35 consensus sequences upstream the +1

Eσ70-like Different from Eσ70

Several promoters, within this group are functional in both S. lividans and E. coli

Eσ70-like Consensus -35: T-T-G-A-C-(Pu) -10: T-A-g-(Pu)-(Pu)-T

THE PROMOTION SEQUENCES DIFFERENT FROM Eσ70

Are the most frequently found in Streptomyces and E. coli does not recognize them

These sequences are characterized by several motives, overlapping or in tandem, which are recognized by the σ factors which transcribe genes regulated by

timing, development or nutrition

ermEp is the constitutive promoter of the erythromycin resistance gene of Saccharopolyspora erythrea (the producing species)

In streptomycetes, only a limited number of promoters have been shown to be functional for the expression of heterologous genes

the one-base-pair mutated form ermEp* is regarded as one of the strongest Streptomyces promoters

Constitutive Inducible

Another constitutive promoter is the STI-IIp derived from S. longisporus where it drives the expression of a serine protease inhibitor. It has been successfully used

to express soluble derivatives of the human CD4 receptor

Constitutive promoters are used in metabolic engineering and synthetic biology

constitutive promoters with different strengths are useful for fine-tuning gene expression levels, which can facilitate the pathway optimization of the desired

chemicals for higher production

Strong constitutive promoters also can be used to trigger expression of some cryptic clusters, resulting in the discovery of novel natural products

Sequencing cryptic clusters cloning under constitutive promoters

Usually, constitutive promoters are obtained by looking for essential genes whose transcript levels are presumed to be constant

Streptomyces species have long and complex life cycles, including sophisticated metabolism shifts and morphological differentiations

Unlikely other bacteria, the streptomycetes growth curve is divided in two Rapid Growth phases (on solid media corresponding to vegetative and aerial mycelium, respectively)

RG1 and RG2 are separated by a Transition step when growth stops, macromolecular syntheses dramatically drop and the antibiotics biosynthetic genes activate

the traditional approaches to identify constitutive promoters in microbes with short growth periods (e.g. E. coli and B. subtilis), may be not reliable for

streptomycetes, due to their complex developmental life cycles

As a consequence, the expression levels of essential genes are not always constant and might be appropriately modulated to adapt to physiological changes

For example, hrdB encodes the essential principal sigma factor HrdB that is supposed to have stable expression in streptomycetes

However, the transcription of hrdB is regulated by several factors and its transcript level is not constant along the whole life cycle

For industrial applications of streptomycetes several approaches have been tried, among these:

A thoroughly search of constitutive promoters

Engineering of sigma factors

Searching for promoters (e.g. a study of Luo, Zhang, Barton, Zhao 2015)

RNA-seq analysis of Streptomyces albus J1074

32 candidate promoters

Cloning as fused with the reporter gene xilE

10 selected, checked with qPCR 4 characterized

200-1300% folds stronger than ermEp*

used in a plug-and-play platform to activate a cryptic gene cluster from

Streptomyces griseus

Success expression of the target pathway in some other industrial Streptomyces strains

Proposal for use in current synthetic biology platforms to activate and characterize silent natural product biosynthetic pathways as well as to optimize the pathways for the synthesis of important natural products in actinobacteria

The importance of HrdB for the industrial production of the antibiotic Actinorhodin has been demonstrated by disrupting the encoding gene in S. coelicolor

M145(hrdB+) + pSET152 (empty vector)

M760(hrdB-) + pSET152 (empty vector)

M760(hrdB-) + pIJ8760 (a derivative of pSET152 containing hrdB)

S. coelicolor has 75 recognized sigma factors (E. coli has 7)

Engineering of sigma factors

teicoplanin is a glycopeptide antibiotic playing a key role in the treatment of serious infections caused by drug-resistant bacteria: the industrial producing strain is

Actinoplanes teichomyceticus L-27

A. teichomyceticus L-27 has been improved by many

strategies to optimize the production, but it has a

poor choice of endogenous transcription factors

Exogenous principal sigma factor σHrdB (coded by hrdB gene), a kind of global transcription factors, have been therefore selected and engineered to

refine this valuable cell factory.

Have been rationally selected and engineered by

σHrdB genes from

random mutagenesis DNA shuffling

point mutation

Salinispora arenicola CNS-205

Micromonospora aurantiaca ATCC27029

Actinoplanes missouriensis 431

. . .

http://journal.frontiersin.org/article/10.3389/fbioe.2014.00033/full#B20

the resulting library has been transferred in A. teichomyceticus L-27.

The mutants showed significant diversity in the region 1.1 of σHrdB, apparently generated by DNA shuffling

a recombinant strain with a twofold increase in teicoplanin production in a pilot scale

fermentation (5.3 mg L−1) was obtained

The PtipA promoter can be both induced with thiostreptone and regulated by modulating the osmotic pressure

The transcriptional activator is TipAL that binds an IR, localized between the -10 and -35 hexamers, spaced by 19 bp

-35 -10

19 bp

In its natural context, this promoter drives the synthesis of an mRNA that encodes both the TipAL (31kDa) and TipAS (17kDa) proteins

INDUCIBLE PROMOTERS

TipAS modulates the activation by substracting the inductor to TipAL

The bond increases the affinity for the recognition sequence in PtipA

The two peptides, indeed, share the C-term binding thiostrepton domain, but TipAS is produced in molar excess to TipAL

TipAL is a transcriptional activator auto-regulated, which drives the expression of tipA

The thiostreptone binds TipAL already at doses in the range 10-7M-10-9M, much lower than those needed for the antibiotic activity

By increasing the osmolarity of the culture medium, therefore

However, when the osmolytes concentration in the culture medium increases, the structure of the recognition domain changes conformation

It is possible to get a strong and long lasting expression even without adding

thiostrepton

the link with TipAL is strengthened

Making irrelevant the action of TipAS

!

PtipA PtipA

PtipA

PtipA O S M

Thiostrepton increases the strength of the bond between typAL and the promoter

TipAS is more abundant of TipAL and competes successfully

for the inductor

By raising the osmolarity, the binding between PtipA and TipAL strengthens and thiostrepton is no more needed

SUMMARIZING..

Another regulable system is used in an expression cassette based on the Ptra promoter

In its natural context Ptra trascribes the operon needed for the conjugative trasfer of the SN22 plasmid of S. nigrifaciens

This is a very strong promoter and is repressed by the TraR protein, encoded by the corrispondent gene, located

on the same plasmid. TraR regulates also its own transcription

To be used in the expression system, the repressor has been modified to be heat-sensitive

A temperature increase inactivates the repressor protein, so indirectly obtaining the activation of the

promoter

kasO (cpkO) encodes an activator of the cryptic type I polyketide synthase (cpk) gene cluster in Streptomyces coelicolor

kasO/kasOp* promoters

A constitutive, strong form of this promoter has also be obtained by removing the binding sites of ScbR and ScbR2 with a combined

deletion/random mutagenesis approach

The modified form is recognized by the main Streptomyces sigma factor HrdB

it is temporally expressed and strictly controlled by ScbR (the y-butyrolactone receptor) and ScbR2 (the antibiotic receptor), acting

as repressors

ScbR2 activates after the QS-dependent derepression of ScbR. Both of them can act as homodimers, but are also able to form an heterodimer, which

partecipates to the complex regulative cascades of S. coelicolor

+ +

Hyper-inducible expression system PnitA-NitR

in the actinobacterium Rhodococcus rhodochrous, which is used for the industrial production of acrylamide and nicotinamide, the nitrilase is strongly

induced by adding ε-caprolactam to the culture medium

The transcriptional positive regulator protein, NitR forms a complex with ε-caprolactam, and activates nitA by binding a specific site

To suppress unwanted transcriptional read-through, a terminator is located just upstream of each PnitA

This expression system has been demonstrated to function in several streptomycete strains, and the induced expression level is very high

A peculiar promoter is the 167nt region detected within the streptomyces archetypal telomeres, with

a G+C content equal to 70%

The END167 sequence lacks of both the typical −35 and −10 RNA polymerase binding sequences and shows no similarity to other known Streptomyces promoters

It was detected while “fishing” for proteins able to bind the telomeres

As one of them was the RNA polymerase holoenzyme

The hairpins are made mainly of GCA (3′ strands) and TGC (5′ strands) loops

possibly, under negative superhelicity, the END167 palindromic sequences form hairpin loops, which provide a ready access for the RNA polymerase

The GCA loops are proposed to be closed by highly stable G·A pairing, resulting in a single nucleotide (C) loop that is resistant to single-strand-specific nucleases

In an in vitro transcription system using purified Streptomyces RNA polymerase holoenzyme (3 and 6 picomoles)

Even greater than the promoters of the ribosomal operon rrnD

the End167 DNA displayed strong promoter activity

DNA fragments of various sizes, containing the END167 or the different rrnD

promoters, were used as the template

The telomere DNA acts as a promoter in Streptomyces

End167 has been cloned, in both the orientations (+ and -), upstream to a promoterless neo gene, encoding kanamycin resistance

neo ter

The recombinant plasmids were used to transform

M145

3456 (SCP1 integrated)

S. coelicolor

WT 1326 (linear

TK64 (a plasmidless

d i ti )

S. lividans

The efficiency has been tested on gradient kanamycin plates

Prepared by first stratifying a slanted agar layer with the antibiotic

K K

K

And applying a second layer, without antibiotic, after the first slant has solidified

In this way, the concentration of kanamycin, forms a gradient through the plate

Interestingly, the telomere also functioned as a promoter in E. coli and as an enhancer of transcription in Saccharomyces cerevisiae

the telomere promoter was active in vivo on the circular plasmids

Regardless of the orientation of the inserts, all the transformants exhibited an high resistance to kanamycin as compared to the control transformants containing pIJ487

The expression of the neo gene was tested by streaking 100 μl of spore suspensions on an LB plate containing a kanamycin gradient, followed by incubation

at 30°C for 7 days

The telomeric DNA acts as a promoter in E. coli, driving the transcription of GFP in both the orientations

promoterless EGFP gene END167 (+) EGFP pLUS889 (-) EGFP

In contrast, the END167 sequence does not function as a promoter in yeast

It acts however as an enhancer, eliminating the need of an activator for initiation at the pCyc-1 promoter

It is possible that the hairpin structures facilitate the opening up of the downstream promoter

END167 does not provide an activating function in mammalian cells, probably due to of the more complex regulation of gene transcription in higher eukaryotes

The LacZ expression is high only if both END167 and the CYC1 promoter are present

By deleting the promoter the expression ceases (i.e. END167 cannot vicariate it)

The transcriptional terminators of Streptomyces are quite similar to those of other bacteria

the rho-independent terminators of Streptomyces are similar to the E. coli ones, other than they often lack of the poliU sequences

3 4

3 4 mRNA

TERMINATORS

Their palindromes are long, often imperfect, an form hairpins

3 4

UUUUUU

E. coli

3 4

Streptomyces

TRANSLATION SIGNALS

The structure of the Streptomyces mRNAs analysed till now, differ from those of both E. coli and Bacillus

Unlike other bacteria, in Streptomyces some genes are translated from mRNAs where the transcription start point and the first base

of the translation start codon coincide

Among these genes, there are also aph and ermE, whose strong promoters are often used for the expression systems

When it exists, the untranslated region, in Streptomyces mRNAs, has a very variable length, anyway greater than that of E. coli

The spacing between the start of transcription and the coding region, ranges 9 to 345 nts, with an average of about 100, against the average of 23 in E. coli

The longer regions so far observed are 298, 335 and 345 bp

+1 AUG

+1 AUG

in these cases the sequence spanning between +1 and AUG shows many secondary structures, involved in the regulation, and / or anti-terminators

SD sequences are located 5-12 nucleotides upstream the initiation codon (average 8.5)

The SD consensus of the streptomycetes genes is quite similar to E. coli:

Yet, heterologous genes with weak SDs are expressed in a satisfactory manner in Streptomyces (eg. ampC: TATGGAA)

This is due to the permissiveness of the Streptomyces ribosomes that do not require a strict complementarity between the SD and the 3’ end of the 16S rRNA

(A/G)GGAGG

Some more than E. coli (5-9) and some less than Bacillus subtilis (7-14)

The most popular expression systems designed for Streptomyces employ some SPs from endogenous, highly expressed genes such

those encoding:

SECRETION SIGNALS

The secretion signal peptides in Streptomyces are consistent with the general rules described for the Gram-positive bacteria

But the Streptomyces SPs are even longer than the Bacillus ones

The merging with particularly expressed genes has not been systematically attempted in Streptomyces but some

preliminar attempts appear encouraging

Subtilisin repressing proteins of S. albogriseolus, S. venezuelae and S. longisporus

The B protease of S. griseus

The Tendamistat of S. tendae (alpha-amylase inhibitor )

The Streptomyces genome has an high GC% content (70%), related to a codon usage that is biased towards the GC rich codons

TTA and CTA are especially rare, and found only in a few genes

TTA

CTA

LEU Other codons In Streptomyces coelicolor, bldA encodes the

only tRNA for the rare leucine codon, UUA

CODON USAGE

This tRNA is not essential to the growth

Information from the sequenced genomes, shows that only approximately 2-3% of the genes contain a TTA codon

most out of them have been acquired through species-specific horizontal gene transfer

Yet, it is required for some aspects of secondary metabolism and morphological development, as revealed by the phenotypes of bldA mutants in several streptomycetes

the pleiotropic regulator adpA is one of the few widely conserved TTA-containing genes

bldA mutants are defective in both aerial growth and sporulation; secondary metabolism is partially biased

bldA inactivation

Failure to translate adpA

In S. coelicolor, AdpA targets several genes essential to the action of some extracellular proteases (needed to convert the primary biomass into spores)

The TTA codon in adpA is largely responsible for the phenotype of bldA mutants

In S. coelicolor, the bld mutants are blocked for synthesis of several antibiotics, including the blue compounds actinorhodin (ACT) and undecylprodigiosin (RED)

The extent of impairment in secondary metabolism varies in different Streptomyces species

The effects of bldA mutations on secondary metabolism are mostly attributable to the presence of TTA codons in some pathway-specific

genes, particularly in transcriptional activators encoding genes

This is not confined to S. coelicolor: it is true for about half of all known antibiotic biosynthetic gene sets from streptomycetes

transcriptomic & proteomic analysis of the ΔbldA cellular processes during submerged culture. (i. e. the conditions in the industrial production of antibiotics)

At the end of RG1, a co-ordinated transient up-regulation of about 100 genes, including many for ribosomal proteins, was seen in the parent strain but not the

ΔbldA mutant

genes or gene clusters involved in secondary metabolite production and biased by the ΔbldA mutation

a further 147 genes were bldA-influenced; most of them were expressed late in the WT

Many of these genes were "function unknown", and may represent undiscovered

aspects of Streptomyces biology

Only two contained a TTA codon, but some effects could be traced to TTA codons in regulatory genes or polycistronic operons

Several proteins were affected post-translationally by the bldA deletion

In Streptomyces (and the related actinobacteria) even small multi-copy plasmids of <10kb in size are normally self-transmissible and able to mobilize

chromosomal resistance genes and auxotrophic markers

Efficiency of transfer reaches nearly 100% and between 0.1% and 1% of the transconjugants obtain chromosomal fragments during mating

STREPTOMYCES CONJUGATION

The transfer of chromosomal markers, in Streptomyces exhibits several differences as compared with other bacteria

In other bacteria, the conjugative plasmid integrates into the chromosome at specific positions by homologous recombination between IS elements

generating a HFR strain

Then the plasmid-encoded relaxase initiates rolling-circle replication at the oriT and drives the ssDNA molecule to the plasmid-encoded type IV protein secretion system

oriT

relaxase

TraB recognizes also several clt-like sequences scattered all over the chromosome

In Streptomyces, the translocase TraB binds noncovalently to the clt locus and drives the transfer of a double-stranded plasmid molecule

There is no need to rely on a previous plasmid integration.

So, it can directly drive the transfer of chromosomal markers

Most of the vectors aimed to recombinant expression in Streptomyces are shuttle plasmid Streptomyces /E. coli, based on the pIJ101 replicon

pIJ101 is a natural conjugative plasmids purified from the S. lividans ISP5434 strain and with a wide host spectrum within Streptomyces

It is extremely stable and maintains a high number of copies, even in the absence of selection, in several Streptomyces species

Rep introduces a nick in the DSO (dsDNA Origin) to start the ssDNA replication

pIJ101 replicates in a rolling circle manner, and depends from the rep protein encoded by the plasmid

(SSO or sti) is the ssDNA from which the synthesis of the second strand originates

rep

DSO

kilB

spdB spdA tra

korA

korB

SSO(sti)

pIJ101

traB is involved in plasmid transfer and is repressed by TraR, it is however able to mediate efficient conjugal transfer, in response to an unknow, most

probably low molecular weight, signal

DNA transfer takes place only on solid surfaces in the substrate mycelium growth phase

the developmental inhibition in turn promotes the transfer process, perhaps by prolonging the growth

period during which transmission can occur

when individual spores containing a plasmid germinate within a dense lawn of plasmidless potential recipient mycelia

subsequent to the plasmid transfer from donors to surrounding recipients, finite circular regions form, where aerial hypha development and sporulation are transiently

delayed or prevented

Such zones or “pocks” correspond to cells that have received plasmid copies

spdA and spdB genes are included on a transcript that initiates upstream of the tra gene and terminates downstream of orf66, a small open reading frame (ORF) of unknown function

rep

DSO

kilB

spdB spdA tra

korA

korB

SSO(sti)

pIJ101

spdA, spdB, and kilB are required for the plasmid to spread

the kilB gene is expressed separately from its own promoter, and is negatively controlled by korB

korB encodes a repressor protein that regulates also its own transcription

a KorB independent transcription of kilB is ensured by a read through from the tra promoter, when also TraB is expressed

kilB encodes a toxic protein lethal in minimal replicons, where korB is not present

Expression of traB fusion of the peptidoglycan layers

TraB mediates the partial fusion of donor and recipient membranes

Apical cell growth

A multiprotein complex mediates the spreading of the plasmid

Pore formation and TraB-mediated DNA translocation

Sometimes, a transgeneric conjugation can be performed

Toward the end of the 1980s, indeed, it was demonstrated that DNA could be transferred from E. coli to several Gram-positive bacteria by conjugation

But not Streptomyces

Streptococcus Enterococcus Bacillus

Listeria Lactococcus

The plasmid vectors employed for the first experiment revealed unsuitable, but, once designed more suitable

shuttle vectors, good results were obtained already in 1989

Since then, the procedure has been applied to many Streptomyces species and also with Saccharopolyspora spinosa and other Actinobacteria strains

Many plasmid vectors have been obtained: they do not replicate in Streptomyces, but integrate in the chromosome at the integration

site of the φC31 bacteriophage

All of these vectors can be mobilized by helper plasmids:

pRK2: it is a E. coli criptic plasmid with a broad host range oriT

pUZ8002: it has a mutated oriT: its own transfer efficiency is 1000 folds lower that RK2, but it allows to mobilize other plasmids very well

Intergeneric conjugation has been used to obtain the integration of antibiotic biosynthetic clusters in several Streptomyces and

Saccharopolyspora erythraea species

Two main factors hamper the conjugation transfer of a plasmid or a a cosmid in S. coelicolor

1) The intergeneric conjugation strictly depends upon the presence of a RK2 derived OriT

2) DNA must be propagated in a HsdM E. coli strain

So to circumvent the methylation specific restriction systems of this species

DNA methylated (N6-methyladenine or 5-methylcytosine) indeed is destroyed in S. coelicolor and other Streptomyces species

If the Streptomyces species is a non-restricting one (e.g. S. lividans) or the strain has been modified, other E. coli strains, such as DH5α

can be used, instead

The E. coli donor must always bear the plasmid Puz8002, so to enable the in trans mobilisation of tra defective vectors

A popular strain for the intergeneric conjugations is the E. coli ET12567 (dam-13::Tn9 dcm-6 hsdM CmR)

A drawback of ET12567 is its high generation time, longer than the DH5α one

E. coli ET12567

Not properly an Extra-Terrestrial as the Spielberg character..

x3

E. coli cells are prepared for the conjugation by centrifuging a 18-24 broth culture at 4 °C

The cells are washed 3 times in ice-cold LB/LB-NaCl and kept on ice

Streptomyces spores are induced to germinate with a termic shock (10’ at ~ 50 °C per 10’) and placed on ice

~ 108 E. coli cells are mixed with about 107 Streptomyces spores, and ten fold

dilutions are prepared

A 100 µl volume of each dilution is plated on SFM (soy-mannitol medium) + MgCl2

After an incubation of 18-24h at 30 °C, an agar overlay with the suitable antibiotic is poured on the plate

Nalidixic acid for E.coli (if the strain is sensitive) Apramycin or neomycin for

Streptomyces

Then the cultures are incubated for 3-4 giorni at 30 °C

E F F I C I E N C Y

The conjugation procedure is underperforming in liquid media (an intrinsic feature of the IncP conjugative plamids used for the gene transfer)

the efficiency decreases of about one order of magnitude if the procedure is performed on

filters placed upon the plates

The physiological state of the recipient is critical

The pre- germination of the spores usually increases the transfer efficiency by 5-10 times

However, it turned out to be counterproductive with S. noursei spores, so it should be experimentally checked for each recipient

Trials to directly transform the mycelia have been unsuccessful

Not all the Streptomyces species can be successfully transformed by intergeneric conjugation:

S. pristinaespiralis (ATCC 25486) and S. viridochromogenes (DSM 40736) have

confirmed the results obtained with S. lividus

S. noursei has been transformed but some modification to the usual protocol were required

However, the same procedures have failed to transform S. parvullus or S hygroscopicus

pIJ702 was obtained from a larger autonomous plasmid and is often used as a cloning vehicle to be used together with S. lividans

The selective marker is the resistance to thiostrepton

NON-CONJUGATIVE PLASMIDS

The differential marker by is provided by the mel operon of S. antibioticus, encoding a

tyrosinase involved in melanin formation

The host range of vector pIJ702 extends beyond Streptomyces spp., and its high copy number has been exploited for the overproduction of heterologous genes

This vector/host combination has been successfully used

However, the secretion levels depend also upon the promoter: by cloning the tendamistat encoding gene in the pIJ702/S. lividans system, variable results

were obtained:

to investigate antibiotic biosynthesis, gene structure and expression

to map several Streptomyces mutants

promoter origin Tendamistat secretion

Escherichia coli-like consensus synthetic 10 mg/l

aphI promoter of the neomycin resistance gene

S. fradiae 10 mg/l

The ermEp* Saccharopolyspora erythraea

500 mg/l

melC promoter of the tyrosinase operon

Streptomyces antibioticus 200 mg/l

tipA promoter (non induced) S. lividans

less than 0.5 mg/l

tipA promoter (induced)

S. lividans

up to 40 mg/l

Tendamystat: a specific inhibitor of mammalian alpha-amylase

Some low copy vectors have been obtained, based on the natural plasmid SP1_2

The most popular is pIJ61, a conjugative plasmid with a narrow host range

pIJ61 is endowed of many unique restriction sites, but there are no suitable marker for insertional inactivation

Multicopy plasmids do not always ensure good results due to

TOXICITY DELETIONS

REARRANGEMENTS

pIJ61 encodes the neomycin resistence and reaches up to 5 copies in S. lividans

The intact Streptomyces cells cannot be transformed

The transformation efficiency is satisfying for plasmids up to 20 kb, it lowers with increasing plasmid mass and it is quite low with

chromosomal DNA

Instead, the protoplasts are trasformed, by the same procedures used to manipulate B. megaterium

However, chromosomal DNA can be trasferred by transformation by trapping it inside liposomes and perform protoplast-fusion techniques

one of recombinant pharmaceuticals derivatives of the Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced in S. lividans

The (GM-CSF) is a monomeric glycoprotein, with two disulfide bonds, found at low levels in human cells, where it exists in five differently glycosylated forms

It has been produced in E. coli by adding only the N-terminal methionine

As a pharmaceutical, however, the unglycosylated form, is 20 folds more active than the natural ones

Prokaryotes are the most suitable hosts

Cytoplasmic localization, inclusion bodies, difficult purification

It has been produced in S. lividans, by adding an endogenous signal peptide

the product is secreted in the culture medium

CULTIVATION

Streptomycetes can be easily grown on rich culture media

Agar plates should be prepared somewhat thicker than usual to prevent from drying out due to the Actinobacteria long growth times

The SFM medium (soy bean flour + mannitol)

And YEME (Yeast Extract, glucose and Malt)

Are among the most popular ones

Spores preparations are a very expedient way to store the well sporulating isolates

LONG-TERM STRAINS CONSERVATION: SPORE PREPARATION

If the strain bears a non-integrative plasmid, the culture aimed to collect the spores must be added with the selection factor

The stock suspension can be stored in 20% glycerol for many years, avoiding frequent freezing and thawing

The strain is grown for 4-5 days at 30 °C

A 10 mL volume syringe is placed in a 50 mL tube

the plunger is removed and a sterile gauze pad is introduced into the syringe

the culture is washed with 5 ml of sterile H2O to collect the spores

the plunger is placed again in the syringe to force the spores in the tube

The suspension is centrifuged and the pellet collected,

suspended in sterile glycerol and freezed at -20 °C

avoiding to form agar fragments that could be taken up with the spores

Actinobacteria, notably Streptomyces, are the richest prokaryotic source of natural products, especially antibiotics, antimetabolites and antitumor agents

ACTINOBACTERIA AND NATURAL PRODUCTS

Natural isolates usually produce only small amounts of antibiotic (µg/l),

The production rates needed to set up a cost-effective production process are in the range of g/l

To increase the industrial yield of products, different strategies can be adopted

γ-butyrolactones

Secondary metabolism

Biosynthetic genes

Activators Repressors Resistance

Media composition

Carbon source Phosphate Nitrogen

Amino Acids

Primary metabolism

Metabolic engineering Ribosome engineering

IMPROVEMENT OF SECONDARY METABOLITE PRODUCTION

On the basis of the effect of rpoB and rpsL on antibiotic biosynthesis, the ribosome engineering as been proposed as a means to activate Streptomyces cryptic gene clusters

The strains were cultured on sublethal concentrations of antiribosomal antibiotics to select antibiotic-resistant mutants, which were then tested for secondary metabolite

profiles

The feasibility of this concept was first exemplified by the activation of ACT production in S. lividans, in

which the act genes are usually not expressed

Introduction of rpsL and rpoB mutations into strains of S. coelicolor has proved useful to maximize the expression of introduced heterologous sets of biosynthetic genes

RIBOSOME ENGINEERING

Amplification of the resistance genes

the antibiotic producing bacteria need resistance genes to avoid the suicide

In natural conditions, a very low amount of antibiotics is produced, and low resistance expression is sufficient to protect the cell

So enhancing the self-resistance levels in producing organisms has been tried

A

However, if the antibiotic synthesis increases, the cells could be damaged by their own products

A A

A A

The 6'-N-acetyltransferase encoding gene derived from S. kanamyceticus was cloned into the high copy plasmid vector pIJ702

In both cases, increased resistance levels to a number of aminoglycoside antibiotics were observed, so as a substantially

increased production of kanamycin and neomycin

Similar results were described in several antibiotic overproducing organisms and different resistance mechanisms

and transferred in S. kanamyceticus (kanamycin) and S. fradiae (neomycin) producers

acetyl-CoA + kanamycin-B CoA + N6'-acetylkanamycin-B

γ-butyrolactones (GBLs), produced by several Streptomyces species, have been shown to serve as quorum-sensing signalling molecules for activating antibiotic production

four such systems with a known GBL and receptor, are: A-factor/ArpA (S. griseus) SCB1/ScbR (S. coelicolor) IM-2/FarA (S. lavendulae) VB/BarA (S. virginiae)

they share high sequence similarity, but the effects of GBL systems on secondary metabolism are different from species to species

γ-butyrolactones : exploiting the Quorum sensing signals

γ-butyrolactones are similar to AHLs except for the carbon side-chain

However, the γ-butyrolactone receptors do not bind to AHL, and viceversa

The γ-butyrolactone (GBL) system of Streptomyces, typically consist of a GBL synthase and a cognate receptor

GBL receptor GBL synthase

The γ-butyrolactones bind to cytoplasmic receptor proteins and inhibit their binding to specific DNA targets

Each receptor protein is highly specific for its cognate γ-butyrolactone

Most of these receptor proteins act as repressors, so that binding to GBLs induces the expression of the target genes

Biosynthesis of GBLs is still unclear but it seems to require a member of a protein family, the archetype of which is AfsA (S. griseus)

Notwithstanding the recent intensive studies, little is known about GBLs biosynthesis and its regulation

It has been recently demonstrated that the «A» factor is obtained by the

condensation of DHAP and a β-keto acid derivative, catalyzed by AfsA,

O O

S ACP-

AfsA

HO OPO3H2

O

dihydroxyacetone phosphate (DHAP)

(2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone)

followed by two reduction steps and one dephosphorylation

The deep blue colonies (Actinorhodin expressing) are the distinctive character of S. coelicolor, differentiating it from the related species S. lividans

However, multiple copies of the two regulatory genes, afsR and afsR2, activate Actinorhodin

(ACT) production in S. lividans, indicating that it encodes a functional ACT biosynthetic pathway

in S. coelicolor, AfsR and AfsR2(AfsS) are the proteins which regulate the Actinorhodin (ACT) production

the phenotypic distinction between S. lividans and S. coelicolor is nullified by simply changing the culture condition

Glucose and other carbon sources suppress the production of many secondary metabolites, included ACT, in S. lividans

by repressing the transcription of afsR2 that encodes a global regulator of the

secondary metabolite biosynthesis

Morphogenesis and actinorhodin genes NOT

transcribed

With glucose

INACTIVATION OF ADENYLATE CYCLASE

ATP cAMP

CAP

Morphogenesis and actinorhodin

Without glucose

ADENYLATE CYCLASE ACTIVE

ATP cAMP

CAP

Regulation of Antibiotic Production by N-Acetylglucosamine

Morphological differentiation and secondary metabolite biosynthesis are supported by nutrients released by autolysis of substrate mycelium

downstream targets include the cluster-situated regulators (CSRs) that activate the pathways for ACT and RED

N-Acetylglucosamine (GlcNAc) released from the cell wall during this process is reimported, phosphorylated and deacetylated

GlcN_P accumulates in the cytoplasm and binds to the GntR-like regulator DasR, relieving the repression of its target genes

The addition of GlcNAc to solid minimal medium cultures stimulates antibiotic

production and development

On opposite, a inhibitory effect is observed on rich culture media

+GlcNac -GlcNac

+GlcNac -GlcNac

This observation does not fit well with the known activities of DasR

It has been suggested that DasR could activate the transcription of a reductase believed to play a role in modification of some particular GBLs (the SCBs)

the production of antibiotics and other secondary metabolites is affected by several nutrients, mainly PHOSPHATE and NITROGEN compounds

microorganisms sense the lack of nutrients through complex signaling pathways, that are often the key to understand the regulatory processes underlying the

synthesis of secondary metabolites

Like most other bacteria, the streptomycetes react to the phosphate starvation trough the

PhoR-PhoP signal transduction system

In S. coelicolor, PhoR-P controls also the antibiotic biosynthesis, so that primary and secondary metabolisms are interconnected

PhoR

PhoP PhoP

PhoR

It binds to the operators of several promoters, activating or repressing them

Pho regulon

Some of these are pathway specific regulators involved in differentiation and secondary metabolism patterns

Secondary metabolism

PhoP

Positive regulators

PhoP is globally involved in the orchestration of primary and secondary metabolism in streptomycetes

Most probably, PhoP suppresses central metabolism, secondary metabolism and developmental pathways until sufficient phosphate is salvaged to support further growth and, ultimately, morphological development

To demostrate the dependence of the antibiotic synthesis upon phosphate starvation some S. lividans were modified

a phoR-P probe demostrated the absence of the WT region in the mutants (3-4) and

of the replacing kr gene in the WT

Both the enginereed mutants were also complemented with the corresponding genes, cloned

on a PHZ plasmid as controls

unlike S. coelicolor, the wild-type S. lividans produces very low levels of actinorhodin and undecylprodigiosin

P conc. (10 μM) P conc. (5 mM)

The complemented (+) transformants grow well at both and

low phosphate concentrations

at very low (10 μM) phosphate concentration the ΔphoP and

ΔphoRP mutants aren’t able to grow

At high (5 mM) phosphate concentration, the deletion mutants

overproduce red pigment

the negative control exerted by phosphate on the secondary metabolism biosynthesis, is restored

Sola-Landa A, Moura RS, Martín JF. PNAS. 2003;100(10):6133-6138. doi:10.1073/pnas.0931429100

P conc. (0.37 mM) P conc. 1.85 mM

The deleted mutants produce very high amounts of antibiotics

The overexpression of the actinorhodin is suppressed in

both deletion mutants by complementation with the phoR-phoP cluster

By adding excess phosphate to the medium, the production of

actinorhodin is still controlled in the ΔphoP and ΔphoRP mutants

phoR-phoP are actually involved in the regulation of secondary biosynthesis

their inactivation increases the yield

At high P concentrations the deletion effect is bypassed

Nitrogen Regulation of Antibiotic Biosynthesis

The effects of mutations in the regulatory on antibiotic production are discernible only under nitrogen excess

directly represses the

AfsQ1

activates

Primary nitrogen assimilation genes

some developmental genes regulates

several antibiotic biosynthetic regulatory and

structural genes

AfsQ1 maintains C, N, and P balance as it

At the promoters of some nitrogen assimilation genes, the repressor AfsQ1 and the activator GlnR partially share the recognition sequence, competing

for the binding site

directly represses the

AfsQ1

Binds to promoters of

PTS (Phosphate uptake)

Gap1 (G3Pase) xysA (xylanase)

When the supply of amino acids becomes rate limiting for protein synthesis, bacteria produce the alarmones

guanosine tetraphosphate (ppGpp)

guanosine pentaphosphate (pppGpp)

which inhibit the synthesis of rRNA and tRNA and activate expression of other genes by altering the RNA polymerase core-binding competitiveness of

the different sigma factors

β β α α

σ70

σ70

σ70

σ70 σ38

σ54

σ32

AMINOACID LIMITATION EFFECTS

The (p)ppGpp synthase enzyme RelA is activated whenever the shortage of amino acids leads to an uncharged tRNA molecule entering the A site of the ribosome

REL-A

GTP ATP

ppGpp

In the transition phase, more than half of the ribosomes are degraded

the cleavage of several tRNAs leads to the accumulation of 30-35 nt RNA species

Like the ribosome and RNA degradation, the onset of secondary metabolites

biosynthesis occurs during the transition as

It is possible that these events are related

Moreover, the 30-35-nt RNA species could act as signalling molecules for secondary metabolism and morphological differentiation

In S. coelicolor, a relA disruption mutant did not produce ACT

Conflicting results have been obtained for the production of clavulanic acid and cephalomycin in Streptomyces clavuligerus

(p)ppGpp overaccumulation increased ACT and RED production

REL-A (p)ppGpp

(p)ppGpp

(p)ppGpp (p)ppGpp

(p)ppGpp (p)ppGpp

(p)ppGpp

(p)ppGpp

it is therefore possible that different antibiotic clusters respond in a different way to (p)ppGpp levels

CHASING THE CRYPTIC GENE CLUSTERS

It has become necessary to devise methods and strategies to identify physiological signals and regulatory mechanisms that can activate these “cryptic” pathways

The massive sequencing of several Streptomyces genomes revealed the

presence of a large number of secondary metabolic gene clusters for previously

UNSUSPECTED products

Has been that of the Gaburedins, a family of γ-aminobutyrate (GABA)-derived ureas, after the deletion of gbnR, an arpA-like putative transcriptional repressor in Streptomyces venezuelae

the first discovery of novel natural products via rational deletion of a putative pathway-specific regulatory gene.

Comparison of metabolite profiles in the wild type and mutant strains revealed six metabolites, in the mutant, that are lacking from the wild type

Natural product biosynthetic genes

GbnR repressor protein

HO2C N H

N H

CO2H

O R

Coculture with Organisms Sharing Common Ecology

Sometimes, the signals or nutrients required for the activation of cryptic gene clusters reflect the presence of interacting microorganisms in the natural environment

Coculture can be effective for activating the production of cryptic metabolites

S. rapamycinicus triggers fungal histone acetylation modifications, causing the specific induction of silent biosynthetic gene clusters in the mold Aspergillus

nidulans, which shares the same habitat

E.g.: coculturing a Streptomyces strain with the mycolic acid-containing Tsukamurella pulmonis from soil samples induced the Streptomyces to produce a novel antibiotic

mycolic acid A

antibiotic

understanding of the cross talk between Streptomyces and other species provides insights and opportunities for the discovery of novel natural products

Although effective this approach requires much time and work miniaturized high-throughput screening methods need to be used

Recently, a very promising method to identify and quantify the small molecules in and around a living bacterial colony by nanoscale mass spectrometry, has been

developed (Nano_DESI)

a dynamic droplet (liquid bridge) is generated and held between: a solvent input capillary and an outlet capillary leading to a

nanospray mass spectrometry (MS) system (nano-DESI)

The method allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation

The method rapidly generated profiles of complex interorganism signal responses at the adjacent edges of pairs of different organisms, such as B. subtilis and S. coelicolor

A colony is brought into contact with the droplet, and the solvent-extractable molecules are drawn off and subjected to MS-MS

However, it has been estimated that only about 10% of the natural products that can be synthesized by

these organisms have been discovered

Amycolatopsis, Actinoplanes, Micromonospora and strains are also a source of clinically significant antibiotics, whereas members of less well-known genera, such as Salinispora and Verrucosispora, are showing promise in this respect

It seems likely that filamentous actinobacteria belonging to such taxa will remain a source of new chemical entities

Filamentous actinobacteria account for ≈ 45% of all microbial bioactive secondary metabolites; out of these (about 7600)

compounds, nearly 80% is produced by streptomycetes 10%

UNDISCOVERED

the whole-genomes of representatives of these bacteria contain 20 or more natural-product biosynthetic gene clusters for the production of known or

predicted secondary metabolites

Both the sea and the deep ocean are regarded as the promising new frontiers for the research on Actinobateria