J. Microbiol. Biotechnol. (2015), 25(11), 1787–1795
http://dx.doi.org/10.4014/jmb.1503.03042
Research Article
Review
jmb
Targeting the Osmotic Stress Response for Strain Improvement of an
Industrial Producer of Secondary Metabolites S
Octavio Godinez1, Paul Dyson2, Ricardo del Sol2, Javier Barrios-Gonzalez1, Cesar Millan-Pacheco1, and
Armando Mejia1*
1
Biotechnology Department, CBS Division, Metropolitan Autonomous University, 09340, Mexico
Institute of Life Science, College of Medicine, Swansea University, Swansea SA2 8PP, UK
2
Received: March 13, 2015
Revised: July 1, 2015
Accepted: July 1, 2015
First published online
July 2, 2015
*Corresponding author
Phone: +52-55-5804-4611;
Fax: +-52-55-5804-4712;
E-mail: [email protected]
S upplementary data for this
paper are available on-line only at
http://jmb.or.kr.
pISSN 1017-7825, eISSN 1738-8872
Copyright © 2015 by
The Korean Society for Microbiology
and Biotechnology
The transition from primary to secondary metabolism in antibiotic-producing Streptomyces
correlates with expression of genes involved in stress responses. Consequently, regulatory
pathways that regulate specific stress responses are potential targets to manipulate to increase
antibiotic titers. In this study, genes encoding key proteins involved in regulation of the
osmotic stress response in Streptomyces avermitilis, the industrial producer of avermectins, are
investigated as targets. Disruption of either osaBSa, encoding a response regulator protein, or
osaCSa, encoding a multidomain regulator of the alternative sigma factor SigB, led to increased
production of both oligomycin, by up to 200%, and avermectin, by up to 37%. The mutations
also conditionally affected morphological development; under osmotic stress, the mutants
were unable to erect an aerial mycelium. In addition, we demonstrate the delivery of DNA
into a streptomycete using biolistics. The data reveal that information on stress regulatory
responses can be integrated in rational strain improvement to improve yields of bioactive
secondary metabolites.
Keywords: Antibiotic production, Streptomyces, stress responses, avermectin
Introduction
Bacteria belonging to the genus Streptomyces are responsible
for producing the majority of known antibiotics, together
with some immunosuppressant compounds, antitumor
agents, and anthelmintics such as avermectin [8]. These
non-motile actinobacteria most commonly inhabit the soil,
and in this habitat they frequently encounter and adapt to
a variety of environmental stresses. Their secondary
metabolism, and hence antibiotic production, is normally
linked to morphological development and on solid media
coincides with the development of an aerial mycelium that
subsequently produces spores. Both processes can be
triggered in response to environmental factors, such as
osmotic stress [1, 18]. Moreover, stress responses are integral
to reprogramming the physiology of Streptomyces as they
transition from primary to secondary metabolism. For
example, proteomic analyses of Streptomyces coelicolor cultures
sampled at successive time points during fermentations
have identified elevated expression of stress proteins,
including regulatory proteins such as the response regulator
OsaB controlling the osmotic stress response, in the so-called
transition phase immediately preceding the upregulation
of secondary metabolism [14, 19]. Similarly, a proteomic
study of S. avermitilis identified several stress proteins that
were highly expressed specifically at the onset of avermectin
production [22].
Key components of regulation of the osmotic stress
response that impact on both development and antibiotic
production in the model streptomycete S. coelicolor have
been described [2, 6]. Loss of function of either OsaB or
OsaC, respectively a response regulator and a multidomain
regulator of the alternative sigma factor SigB, has
contrasting effects on differentiation and secondary
metabolite production. Under osmotic stress conditions,
mutants in S. coelicolor cannot erect aerial hyphae but
produce up to 5-fold greater antibiotic yields (actinorhodin
and undecylprodigiosin) compared with the wild-type
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Godinez et al.
strain [2]. To examine if this regulatory paradigm is generally
applicable and can be translated to improve production of
commercially relevant secondary metabolites, we have
investigated disruption of orthologous regulatory genes in
S. avermitilis, a commercial producer of avermectins and
oligomycin. The approach we adopted to construct one of
the mutants was to introduce DNA by biolistics. There
have been few reports of bacterial transformation by
biolistics and none to date with Streptomyces; indeed, this
method has been used primarily for DNA delivery into
eukaryotic cells, notably into plant or animal tissue [3, 13].
Materials and Methods
Microorganisms and Culture Conditions
Microorganisms and plasmids used are indicated in Table S1.
E. coli strains were cultivated on LB agar plates [17]. Antibiotics
used were kanamycin (25 µg/ml), ampicillin (100 µg/ml),
chloramphenicol (25 µg/ml), hygromycin (100 µg/ml), spectinomycin
(10 µg/ml) and apramycin (100 µg/ml). The sporulation medium
was MS agar [9]. For S. avermitilis genomic DNA isolation, a
Wizard Genomic DNA Purification kit (Promega, USA) was used.
For that purpose, cultures were grown on LB agar for 36 h, and
100 mg of mycelium was scraped off and then processed as per the
manufacturer’s instructions.
Cloning of SAV2511 and Derivation of a Tn5062 Insertion
A 5.1 kb DNA fragment containing SAV2511 was released from
cosmid CL-236-G10 by digestion with EcoRI and BglII enzymes;
pME6 was simultaneously digested with the same enzymes. The
purified restricted DNA fragments were ligated together and the
recombinant plasmid pMBSa1 was obtained after electroporation
of E. coli JM109. To reduce the size of the inserted fragment to
contain just the SAV2511 gene, pMBSa1 was digested with KpnI to
release a 2.1 kb fragment, which was then ligated with pME6
digested with the same enzyme. The recombinant plasmid
pMBSa2L was recovered. To disrupt SAV2511, pMBSa2L was cut
with AatII that recognizes a single site within the open reading
frame. The restricted plasmid was subsequently treated with T4
DNA polymerase enzyme to generate blunt ends and ligated with
a 3.4 kb PvuII fragment containing the transposon Tn5062. The
plasmid pMBTn1 with a Tn5062 insertion in SAV2511 was
recovered. To construct the corresponding S. avermitilis SAV2511
mutant, intergeneric conjugation of pMBTn1 from E. coli ET12567/
pUZ8002 was used [7]. Apramycin-resistant, kanamycin-sensitive
exconjugants were recovered. The identity of S. avermitilis mutants
was confirmed by Southern hybridization [17] using a 3.4 kb PvuII
fragment derived from Tn5062 as a probe.
Cloning of SAV2513 and Derivation of a Tn5062 Insertion
SAV2513 was amplified from S. avermitilis genomic DNA using
oligonucleotides with the following sequences:
J. Microbiol. Biotechnol.
(Forward) 5’-TGA ATT CTT CCA CGA ACC GGA CAT ACC-3’
(Reverse) 5’-TTC TAG ATA CCT CCA GCT CCG TCT CGT-3’
DNA amplification was performed in a PTC-200 thermocycler
(Bio-Rad, UK), with an initial 95°C denaturation temperature for 3
min, followed by 30 amplification cycles (95°C, 1 min; 60°C, 1 min;
72°C, 2 min), using high-fidelity Pfu polymerase (Agilent Technologies
Inc./Stratagene, USA). Subsequently, a 2.1 kb amplicon corresponding
to SAV2513 was purified. For cloning, the SAV2513 amplicon and
pME6 were digested with EcoRI and Xbal enzymes. They were
then ligated together and the recombinant plasmid pMCSa1 was
subsequently recovered after electroporation of E. coli JM109. To
disrupt SAV2513, pMCSa1 was digested with EcoRV that has a
single recognition site within the open reading frame. The
digested plasmid was combined with the 3.4 kb PvuII fragment
containing Tn5062 and the recombinant plasmid pMCTn2 containing
the insertion subsequently recovered in E. coli JM109.
S. avermitilis Transformation Employing Biolistics
Instead of employing intergeneric conjugation, we investigated
using biolistics as an alternative method to introduce pMCTn2
into S. avermitilis. MS-sorbitol (0.75 M) plates were spread with
1 × 106 spores and incubated for 24 h at 30°C until a bacterial
background lawn was obtained. Vector introduction was
performed using a Biolistic PDS-1000/He Particle Delivery System
(Bio-Rad, Mexico), selecting a 9 cm bombardment distance and a
1,200 psi pressure. The microcarriers of tungsten were prepared in
50% glycerol (30 mg/ml) mixed for 5 min on a platform vortexer to
resuspend and disrupt agglomerated particles. Then 50 µl (3 mg)
of microcarriers was transferred to a 1.5 ml microcentrifuge tube.
Continuous agitation of the microcarriers was needed for uniform
DNA coverage. While vortexing vigorously, the following were
added in order: 5 µl of DNA (1 µg/µl), 50 µl of 2.5 M CaCl2, and
20 µl of 0.1 M spermidine (free base, tissue culture grade).
Vortexing was continued for 2–3 min. The microcarriers were then
allowed to settle for 1 min and pelleted by spinning for 2 sec in a
microfuge. The liquid was removed and discarded and 140 µl of
70% ethanol (HPLC or spectrophotometric grade) was added.
This washing was repeated twice and then 48 µl of 100% ethanol
was added. The final pellet was gently resuspended by tapping
the side of the tube several times, and then by vortexing at low
speed for 2–3 sec. The DNA-coated microcarriers were fired into
the bacterial lawn and the plates then incubated at 30°C for 16 h
prior to overlaying lawns with a solution of apramycin (100 µg/
ml). They were incubated under the same conditions for 7 days.
Single colonies were subsequently isolated and tested for
sensitivity to kanamycin. The identity of the mutants was
confirmed by Southern hybridization [17] using a 3.4 kb PvuII
fragment derived from Tn5062 as a probe.
Oligomycin Production
For pre-cultures, 25 ml of ϕ medium containing MgSO4·7H2O
(0.5 g/l, J.T. Baker, Mexico), CaCl2·2H2O (0.7 g/l, J.T. Baker), glucose
(10 g/l, J.T. Baker), tryptone (5 g/l, Bioxon), yeast extract (5 g/l,
The Osmotic Stress Response for Strain Improvement
1789
Bioxon), and Lab Lemco Powder (5 g/l, Oxoid), adjusted to pH 7
in a 250 ml Erlenmeyer flask was inoculated with 1 × 106 spores.
Cultures were incubated at 30°C with 225 rpm shaking for 17 h or
before production of melanin was evident. Subsequently, 5 ml of
the pre-culture was inoculated into a 250 ml Erlenmeyer flask
with 20 ml of the production medium (AP-5), the composition of
which was L-glutamic acid (0.6 g/l, J.T. Baker), FeSO4·7H2O (0.01 g/l,
J.T. Baker), corn starch (80 g/l, Sigma), yeast extract (5 g/l,
Bioxon), CaCO3 (7 g/l, J.T. Baker), MgSO4·7H2O (1 g/l, J.T. Baker),
and K2HPO4 (1 g/l, J.T. Baker), adjusted to pH 6.9 and supplemented
with 1 ml of trace metal solution containing ZnCl2 (20 mg/l, J.T.
Baker), FeSO4·7H2O (20 mg/l, J.T. Baker), MnCl2·4H2O (20 mg/l,
J.T. Baker), and distilled H2O (20 ml). Cultures were incubated at
28°C with 225 rpm shaking for 10 days. For the experiments under
osmotic stress conditions, KCl (J. T. Baker) was added to the
production medium (AP-5) at a final concentration of 250 mM.
biomass by mixing with an equal volume of methanol for 30 min.
A 10 µl sample of methanol extract supernatant was analyzed by
HPLC. A Nova-pak C18 column (Waters Ltd, USA: 3.9 mm in
inner diameter, 150 mm in length) was developed with methanol
water (85:15 (v/v)) at a flow rate of 0.8 ml/min in ambient
conditions. The quantities of total avermectins (AVMs) were
calculated from the integration value at 246 nm using an authentic
sample of avermectin B1a (SigmaAldrich, Mexico) as a standard.
The B1a proportion among avermectins was calculated as a ratio
of B1a peak area and the total area at 246 nm.
Oligomycin Bioassay with Aspergillus niger
Samples (400 µl) were obtained at different time points during
the fermentation of strains in the (AP-5) production medium. The
biomass and supernatants were separated by centrifugation, with
the former subsequently dried and weighed. Then, 200 ml of AP-5
agar medium poured in 230 mm × 15 mm Petri dishes was
inoculated with 1 × 109 spores of A. niger (A10). Eighteen wells (10
mm diameter) per plate were subsequently made with a punch, and
200 µl per well of fermentation supernatant or a range of
concentrations of commercial oligomycin (Sigma) was added.
Plates were placed at 4°C for 30 min and subsequently incubated
at 30°C for 24 h or until vegetative growth was observed. The
diameters of inhibition halos were measured.
Comparison of Osmotic Stress Regulation in S. avermitilis
and S. coelicolor
The S. avermitilis genome contains an orthologous gene
cluster of osmoadaptation genes [20] with a similar
architecture to that found in the S. coelicolor genome. Genes
encoding an atypical two-component signal transduction
system are located in one arm of the chromosome, with a
hybrid histidine kinase encoded by osaASa (SAV2512) that
shares 92.3% amino acid identity with the corresponding
S. coelicolor protein. Downstream of osaASa is the response
regulator gene osaBSa (SAV2511), whose protein product
shares 93.8% amino acid identity with its S. coelicolor
ortholog. OsaA and OsaB proteins from both organisms
have very similar domain architectures. Upstream and
divergently transcribed from osaASa is a multidomain
regulator encoded by osaCSa (SAV2513) sharing 87.5% amino
acid identity with OsaCSc. Comparison of the domain
architectures revealed that the RsbW-like N-terminal
kinase domain (HATPase_c domain) of S. coelicolor OsaC is
less well conserved in the S. avermitilis protein; indeed, it is
not predicted by SMART (http://smart.embl-heidelberg.de/)
[12] (Fig. 1A). More specific alignment of the corresponding
regions of the respective OsaC proteins with other better
characterized RsbW-like kinase domains found in Bacillus
subtilis proteins revealed the absence of conserved amino
acid residues adjacent to and within the putative ATPbinding Bergerat fold [5] of the S. avermitilis protein (Fig. 1B).
Of particular note is the substitution of a conserved acidic
aspartate residue by glycine in the Bergerat fold G3 box.
The RsbW-like kinase domain of OsaCSc has a function in
regulating the activity of the alternative sigma factor SigB
[6], which can in turn regulate other alternative sigma factors
Avermectin Production
Seed cultures were grown in 250 ml flasks containing 30 ml of
seed medium (30 g/l soluble corn starch, 15 g/l yeast extract,
(Bioxon), 5 g/l corn syrup solids, 0.4 g/l (Bioxon) KH2PO4 (J. T.
Baker), and 2.5 g/l polyethyleneglycol (PEG 2,000, Sigma) at 28oC
and 200 rpm for 24 h. Then, 4 ml of seed culture was transferred to
a 500 ml Erlenmeyer flask containing 40 ml of production medium
(30 g/l soluble corn starch (Sigma), 14.4 g/l soybean flour
(FloryVida), 21.6 g/l yeast extract, (Bioxon) 7.2 g/l corn syrup
solids (Bioxon), 0.03 g/l CoCl2 (J. T. Baker), and 0.6 g/l KH2PO4
(J. T. Baker), pH 7.2). Fermentations were for up to 5 days at 28oC
and 200 rpm. To measure production under osmotic stress
conditions, KCl (J. T. Baker) was added to the production medium
at a final concentration of 250 mM.
Quantification of Avermectin by HPLC
Samples (1 ml) were obtained at different time points during
the fermentation of strains in the avermectin production medium.
One half of each sample was used to determine the biomass dry
weight. The remainder was centrifuged to separate the mycelia
and supernatant. Avermectins were extracted from the separated
Reproducibility of Results
Experiments were performed in duplicate and repeated at least
twice. The variation coefficient was always lower than 8%.
Results
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Godinez et al.
Fig. 1. Comparison of OsaCSc and OsaCSa.
(A) The Simple Modular Architecture Tool (SMART) was used to predict the domain structures of the respective proteins, indicating the absence of
the N-terminal HATPase_c domain in OsaCSa (HATPase_c, histidine kinase-like ATPase domain; PAS, Per-Arnt-Sim signal sensor domain; GAF,
cAMP/cGMP binding regulatory domain typical of cGMP-specific phosphodiesterases, adenylyl and guanylyl cyclases, and phytochromes; PP2CSIG, Sigma factor PP2C-like phosphatase domain). (B) An alignment of residues 41-197 of OsaCSa with the HATPase-c/RsbW-like kinase domain
of OsaCSc and related kinase proteins from B. subtilis, indicating the conserved N-box and G1, G2, and G3 boxes of the Bergerat ATP-binding fold.
Residues highlighted in black are conserved, those in dark grey share >75% identity, and those in light grey share >50% identity.
involved in the osmotic stress response in S. coelicolor [11].
Comparison of SigBSa and SigBSc revealed that they share
77.8% amino acid identity.
Mutagenesis of SAV2511 (osaBSa)
To disrupt osaBSa, the gene was first subcloned from
cosmid CL-236-G10. A copy of Tn5062 was then introduced
at the single AatII site within the osaBSa coding sequence (at
position 167 within the 687 bp coding DNA sequence) to
create pMBTn1. This plasmid, which cannot replicate in
Streptomyces, was transferred into S. avermitilis from E. coli
by intergeneric conjugation, exploiting the oriT carried by
the transposon. Six apramycin-resistant clones were
J. Microbiol. Biotechnol.
selected for further analysis. Southern hybridization of
genomic DNA isolated from each clone with a Tn5062
probe indicated replacement of the wild-type gene by the
disrupted allele (Fig. 3).
The six mutants, together with the parental strain, were
grown on MS sporulation medium with and without
250 mM KCl (Fig. 2). Whereas all strains could erect an
aerial mycelium on non-supplemented MS, all six mutants,
in contrast to the wild-type, were unable to do so when
challenged by osmotic stress (Fig. 2B), paralleling the
phenotype of an S. coelicolor osaB mutant [2].
Further evidence for a conservation of function between
the OsaB proteins from both species was obtained by
The Osmotic Stress Response for Strain Improvement
1791
Fig. 2. S. avermitilis osaB and osaC mutants are conditionally bald.
(A and B) Six independent osaB mutants (1–6) and the parental wild-type strain (P) were plated on MS medium (A) and MS medium supplemented
with 250 mM KCl (B) and grown for 5 days. The mutants failed to erect an aerial mycelium on the supplemented medium. (C) The parental strain
(P), an osaB mutant (1), and the mutant with pPM04 expressing osaBSc (2) were grown on MS medium supplemented with 250 mM KCl for 7 days.
(D and E) Six independent osaC mutants (1–6) and the parental wild-type strain (P) were plated on MS medium (D) and MS medium supplemented
with 250 mM KCl (E) and grown for 7 days. The mutants failed to erect an aerial mycelium on the supplemented medium. (F) The parental strain
(P), an osaC mutant (1), and the mutant with pSHOsaC1 expressing osaCSc (2) were grown on MS medium supplemented with 250 mM KCl for
7 days.
Fig. 3. Verification of osaBSa mutants.
Southern hybridization of genomic DNA isolated from each clone
with a Tn5062 probe indicated replacement of the wild-type gene by
the disrupted allele. Genomic DNA from the six apramycin-resistant
mutants (lanes 1-6) was digested with NotI, as was pMBTn1 (lane C).
A hybridizing 3.1 kb fragment common to each lane indicated the
presence of the disrupted allele in each mutant. A lambda DNA
HindIII digest was included as a molecular size marker.
genetic complementation of the S. avermitilis osaB mutant
by osaBSc. An integrating plasmid carrying osaBSc, pPM04
[2], was introduced into a representative S. avermitilis osaB
mutant and could restore development of an aerial
mycelium when the strains were grown under osmotic
stress (Fig. 2C).
Mutagenesis of SAV2513 (osaCSa)
The osaCSa gene sequence was amplified from cosmid CL236-G10 and cloned as an EcoRI-XbaI fragment. It was
disrupted by insertion of Tn5062 at the single internal
EcoRV site (at position 358 within the 2,748 bp coding DNA
sequence), generating plasmid pMCTn2. This plasmid,
which is unable to replicate in Streptomyces, was introduced
by biolistics into young hyphae of S. avermitilis growing on
solid agar. This, we believe, is the first report of delivery of
DNA into a streptomycete by this method. From 110
colonies, six apramycin-resistant mutants were selected for
further analysis. PCR analysis of genomic DNA confirmed
the replacement of osaCSa by the mutated allele in each
mutant. Phenotypic analysis also indicated that the
mutants were unable to erect an aerial mycelium when
grown under osmotic stress (Fig. 2E), reproducing the
phenotype of the corresponding S. coelicolor osaC mutant
[6]. However, unlike the example of interspecific
complementation observed for osaB, when the S. coelicolor
osaC gene on plasmid pSHOsaC1 [6] was introduced into a
representative mutant, there was no evidence for
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Godinez et al.
Fig. 4. Comparison of oligomycin production kinetics of the wild type and osmoadaptation mutants.
Quantification of oligomycin titers (µg/mg dry weight of biomass) produced by the parental strain (open circles), an osaB mutant (solid squares),
and an osaC mutant (solid triangles) fermented in AP-5 medium (A) and in AP-5 medium supplemented with 250 mM KCl (B).
complementation of the developmental phenotype of the
osaCSa mutant (Fig. 2F).
Oligomycin Production Is Increased in Osmoadaptation
Mutants of S. avermitilis
One representative osmoadaptation mutant of each type
was chosen for analysis of antibiotic production. To
monitor oligomycin production, S. avermitilis strains were
grown in submerged culture in AP-5 medium with and
without 250 mM KCl to induce osmotic stress. Culture
supernatants were sampled at different time-points and
their oligomycin contents measured using a bioassay
against Aspergillus niger, [16] comparing with known
concentrations of commercial oligomycin. For the wild
type, production remained at between 0.1 to 0.15 µg/mg
dry weights of biomass from 26 h until 160 h fermentation,
irrespective of osmotic stress (Figs. 4A and 4B). In contrast,
when grown in non-supplemented production medium,
both mutant strains displayed a peak in oligomycin
production at 26 h, with titers 3- to 3.5-times greater than
produced by the wild type at the corresponding time-point.
For longer fermentations, the antibiotic yields from the
mutants subsequently declined but remained consistently
higher than those of the wild type. These production
profiles were reproduced but tended to be amplified when
the osaCSa was grown under osmotic stress. At 26 h, the
oligomycin titer of this mutant reached 0.52 µg/mg dry
weight of biomass, approximately 5 times greater than that
of the wild type. However, after 52 h, production declined
J. Microbiol. Biotechnol.
to levels similar to the wild type. An increase in production
after 26 h fermentation in osmotic stress conditions was
also noted for the osaBSa mutant, and this also declined after
longer fermentations (Fig. 4B).
Avermectin Production Is Increased in Osmoadaptation
Mutants of S. avermitilis
Avermectin production was analyzed using HPLC
separations of culture extracts grown under non-stressed
Fig. 5. Comparison of avermectin production kinetics of the
wild type and osmoadaptation mutants.
Quantification of avermectin B1a titers (ng/mg dry weight of biomass)
produced by the parental strain (open circles), an osaB mutant (solid
squares), and an osaC mutant (solid triangles) fermented in production
medium.
The Osmotic Stress Response for Strain Improvement
conditions. Under these conditions, avermectin production
was increased 37% with respect to the parental strain in
both osmoadaptation mutants, peaking at 285 ng/mg dry
weight of biomass in the mutants compared with 180 µg/
mg dry weight of biomass in the wild type after 120 h
fermentation (Fig. 5). No significant differences in avermectin
production were observed between the strains fermented
in osmotic stress conditions, with yields peaking at 150 ng/mg
dry weight of biomass after 120 h fermentation.
Discussion
Empirical strain improvement programs applied to
antibiotic-producing streptomycetes have exploited random
mutagenesis and selection of overproducing variants [21].
More recently, this approach has been combined with
protoplast fusion to shuffle combinations of useful traits
between genomes [23, 24]. A “semi-rational” approach has
been to isolate mutations resulting in antibiotic resistance
due to changes in ribosome or RNA polymerase function
that, for reasons that are not entirely clear, result in
improved antibiotic production [20]. Rational engineering
of strains has targeted optimizing primary metabolism,
and hence precursor supply, or rate-limiting steps in
secondary metabolic pathways to improve overall flux and
corresponding yields of specific antibiotics [8, 15]. In
addition, pathway-specific regulator genes, encoding either
transcriptional activators or repressors, have been targeted
to increase expression of genes within specific secondary
metabolite biosynthetic gene clusters [4]. Lastly, pathways,
often activated or de-repressed, have been expressed in
heterologous hosts that have been modified to delete
competing secondary metabolite pathways. For example,
using S. avermitilis for heterologous expression of the
streptomycin biosynthetic gene cluster, a 6-fold increase in
streptomycin production, from 3 to 18 µg/ml, was obtained
in a genome-minimized strain compared with the wild
type [10]. Some of these approaches have been applied to
S. avermitilis to increase production of its own antibiotics.
For example, ribosomal mutations and genome shuffling
improved production of an avermectin analog, doramectin,
11.2-fold [23]. Increased expression of aveR, encoding the
transcriptional activator of the avermectin biosynthetic
genes, improved avermectin yield by 50% [25].
To assess whether modifying pleiotropic regulatory
pathways could be another broadly applicable rational
approach to improve antibiotic production, we have created
specific mutants of S. avermitilis impaired in the regulatory
pathways controlling osmoadaptation. A conserved signal
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transduction system, comprising a hybrid histidine kinase,
OsaA, and an atypical response regulator, OsaB, was
disrupted by knocking out the function of the latter. As is the
case of the corresponding osaB null mutant of S. coelicolor,
the S. avermitilis mutant was conditionally “bald,” being
unable to erect an aerial mycelium when grown on
medium supplemented with osmolyte. Moreover, the
mutant overproduced two different antibiotics. In nonsupplemented fermentations, a 3-fold increase in oligomycin
production was obtained after 26 h growth; this was
moderately enhanced further by inclusion of osmolyte in
the fermentation. For avermectin, a 37% increase in yield
was obtained in non-supplemented fermentations. The
OsaB protein consists of a conserved N-terminal receiver
domain, but, unlike most response regulators, it lacks a
C-terminal DNA binding domain. Both S. avermitilis and
S. coelicolor proteins contain a coiled-coil region that is
required for dimerization (unpublished results), but the
mechanism for relaying a signal to effect a response in
conditions of high external osmolarity is unknown. The
observation that expression of OsaBSc can restore aerial
development in an osaB mutant of S. avermitilis implies that
the mechanism is common to both species.
The respective OsaC proteins of both S. avermitilis and
S. coelicolor share less identity than their OsaB proteins.
However, the osaCSa null mutant was, like the corresponding
S. coelicolor mutant, conditionally bald. Antibiotic production
was also elevated: in non-supplemented fermentations,
oligomycin production was increased 3.5-fold and avermectin
production improved by 37%. An even greater increase in
oligomycin production, up to 5-fold, was obtained in
fermentations with added osmolyte. OsaC is the paradigm
for a multidomain regulator protein family specific to
Streptomyces. To date, the function of only one domain has
been investigated in detail: the N-terminal RsbW-like kinase
(HATPase_c) domain. This domain confers on OsaCSc an
anti-sigma factor activity required to modulate the activity
of the alternative sigma factor SigB subsequent to the
osmotic stress response [6]. There is evidence that, under
specific growth conditions that induce the osmotic stress
response, this sigma factor sits at the top of a cascade of
alternative sigma factors, including SigL and SigM [11].
The molecular targets for OsaC’s regulatory functions may
be similar in both species. Even though SMART fails to
predict a HATPase_c domain in OsaCSa, manual alignment
with RsbW-like kinase domains indicates the presence of
the majority of conserved residues. The inability of OsaCSc
to restore aerial development in the osaCSa mutant may be a
consequence of divergence of the molecular targets; for
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Godinez et al.
example, the respective SigB proteins share only 78%
identity. The approach we adopted to construct the osaCSa
mutant was to introduce the non-replicating plasmid
carrying the Tn5062-disrupted allele into S. avermitilis by
biolistics. To the best of our knowledge, this is the first
report of DNA delivery to Streptomyces by biolistics, and
this opens possibilities for manipulation of less genetically
tractable actinobacteria.
In conclusion, we have demonstrated that targeted
mutations to disrupt regulatory pathways involved in the
osmotic stress response lead to antibiotic overproduction
in a species of great importance to biotechnology. An
inference of this study is that these mutations can be
incorporated in rational strain improvement programs to
improve antibiotic yields in other streptomycetes. In
addition, as the effects are pleiotropic, affecting more than
one pathway, they can be utilized in generating genetic
backgrounds in which so-called cryptic pathways may be
activated, expanding the range of bioactive secondary
metabolites produced by the genus.
Acknowledgments
This research was supported by a grant from the European
Commission, reference AML/B7-311/97/0666/II-0313-FAF and O.G. was supported by scholarship No. 163988 from
CONACyT, México.
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November 2015 ⎪ Vol. 25 ⎪ No. 11