scholarly journals Engineering of Primary Carbon Metabolism for Improved Antibiotic Production in Streptomyces lividans

2002 ◽  
Vol 68 (10) ◽  
pp. 4731-4739 ◽  
Author(s):  
Michael J. Butler ◽  
Per Bruheim ◽  
Srdjan Jovetic ◽  
Flavia Marinelli ◽  
Pieter W. Postma ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Lividans ◽  
Pentose Phosphate ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild ◽  
Commercially Important ◽  
Biochemical Product ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Wild Type Control

ABSTRACT Deletions were made in Streptomyces lividans in either of two genes (zwf1 and zwf2) encoding isozymes of glucose-6-phosphate dehydrogenase, the first enzyme in the oxidative pentose phosphate pathway (PPP). Each mutation reduced the level of Zwf activity to approximately one-half that observed in the wild-type strain. When the mutants were transformed with multicopy plasmids carrying the pathway-specific transcriptional activator genes for either the actinorhodin (ACT) or undecylprodigiosin (RED) biosynthetic pathway, they produced higher levels of antibiotic than the corresponding wild-type control strains. The presumed lower flux of carbon through the PPP in each of the Δzwf mutants may allow more efficient glucose utilization via glycolysis, resulting in higher levels of antibiotic production. This appears to occur without lowering the concentration of NADPH (the major biochemical product of the oxidative PPP activity) to a level that would limit antibiotic biosynthesis. Consistent with this hypothesis, deletion of the gene (devB) encoding the enzyme that catalyzes the next step in the oxidative PPP (6-phosphogluconolactonase) also resulted in increased antibiotic production. However, deletion of both zwf genes from the devB mutant resulted in reduced levels of ACT and RED production, suggesting that some of the NADPH made by the PPP is utilized, directly or indirectly, for antibiotic biosynthesis. Although applied here to the model antibiotics ACT and RED, such mutations may prove to be useful for improving the yield of commercially important secondary metabolites.

scholarly journals Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

2006 ◽  
Vol 188 (17) ◽  
pp. 6269-6276 ◽  
Author(s):  
Sofiane Ghorbel ◽  
Aleksey Smirnov ◽  
Hichem Chouayekh ◽  
Brice Sperandio ◽  
Catherine Esnault ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sufficient Conditions ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild

ABSTRACT The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

scholarly journals The Phosin PptA Plays a Negative Role in the Regulation of Antibiotic Production in Streptomyces lividans

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 325
Author(s):  
Noriyasu Shikura ◽  
Emmanuelle Darbon ◽  
Catherine Esnault ◽  
Ariane Deniset-Besseau ◽  
Delin Xu ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Lividans ◽  
Positive Control ◽  
Phosphate Limitation ◽  
Antibiotic Biosynthesis ◽  
Nudix Hydrolase ◽  
Energetic Stress ◽  
Accessory Factor ◽  
Negative Role ◽  
Weak Expression

In Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate.

scholarly journals Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining

2020 ◽  
Vol 61 (5) ◽  
pp. 639-647
Author(s):  
Yan Du ◽  
Yoshihiro Hase ◽  
Katsuya Satoh ◽  
Naoya Shikazono
Keyword(s):  
Gamma Rays ◽  
Wild Type ◽  
End Joining ◽  
Complex Type ◽  
Single Base ◽  
In The Wild ◽  
Non Homologous End Joining ◽  
Dna Ends ◽  
Wild Type Control

Abstract To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

scholarly journals Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis

2006 ◽  
Vol 6 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Michele Saliola ◽  
Gina Scappucci ◽  
Ilaria De Maria ◽  
Tiziana Lodi ◽  
Patrizia Mancini ◽  
...  
Keyword(s):  
Biomass Yield ◽  
Kluyveromyces Lactis ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Respiratory Metabolism ◽  
Wild Type ◽  
Dehydrogenase Gene ◽  
Increased Sensitivity ◽  
Dimeric Form ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield.

Effects of sugars and polyols on basidiocarp formation in a phosphoglucose isomerase mutant of Coprinus cinereus

1984 ◽  
Vol 30 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Hiroshi Nyunoya ◽  
Tsuneo Takemaru ◽  
Tatsuo Ishikawa
Keyword(s):  
Culture Medium ◽  
Complete Recovery ◽  
Biochemical Properties ◽  
Phosphoglucose Isomerase ◽  
Coprinus Cinereus ◽  
Partial Recovery ◽  
Wild Type ◽  
In The Wild ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Basidiocarp Formation

Several biochemical properties of a mutant deficient in phosphoglucose isomerase (pgi) of Coprinus cinereus were examined in connection with its ability to produce basidiocarps. Mycelium of the pgi mutant accumulated glucose-6-phosphate and showed higher glucose-6-phosphate dehydrogenase activity than wild type. A conventional fruiting medium did not support basidiocarp formation by the homozygous dikaryon pgi/pgi, but the addition of a reduced amount of glucose plus a compensatory amount of fructose to the culture medium resulted in partial recovery of fruiting ability. This modification of the culture medium decreased the intracellular glucose-6-phosphate to almost the same level as in the wild type. The addition of polyols such as mannitol resulted in complete recovery of fruiting ability by the dikaryon pgi/pgi without affecting the level of glucose-6-phosphate. Mycelium of the mutant showed an elevated activity of NAD-linked polyol dehydrogenase and an elevated intracellular NAD level, irrespective of whether the mycelium was grown in the presence or absence of polyol.

scholarly journals Estrogen receptor-α mediates estrogen-inducible abnormalities in the developing penis

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 1057-1067 ◽  
Author(s):  
H O Goyal ◽  
T D Braden ◽  
P S Cooke ◽  
M A Szewczykowski ◽  
C S Williams ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Levator Ani ◽  
Estrogen Receptor Α ◽  
Control Group ◽  
Wild Type ◽  
Penile Length ◽  
In The Wild ◽  
Os Penis ◽  
Harmful Effects ◽  
Wild Type Control

Previously, we reported an association between estrogen receptor-α (ERα) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERα knockout (ERαKO) mouse model to test the hypothesis that ERα mediates DES effects in the developing penis. ERαKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 μg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80–240 days of age and tissues were examined at 96–255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERαKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wild-type/DES group. ERαKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERαKO mice when compared with controls, although testosterone concentration was much higher in the ERαKO mice. Hence, the resistance of ERαKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERα in mediating the harmful effects of neonatal DES exposure in the developing penis.

scholarly journals EshA Accentuates ppGpp Accumulation and Is Conditionally Required for Antibiotic Production in Streptomyces coelicolor A3(2)

2006 ◽  
Vol 188 (13) ◽  
pp. 4952-4961 ◽  
Author(s):  
Natsumi Saito ◽  
Jun Xu ◽  
Takeshi Hosaka ◽  
Susumu Okamoto ◽  
Hiroyuki Aoki ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Molecular Signaling ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
In The Wild ◽  
Actinorhodin Production ◽  
Cyclic Nucleotide Binding Domain

ABSTRACT Disruption of eshA, which encodes a 52-kDa protein that is produced late during the growth of Streptomyces coelicolor A3(2), resulted in elimination of actinorhodin production. In contrast, disruption of eshB, a close homologue of eshA, had no effect on antibiotic production. The eshA disruptant accumulated lower levels of ppGpp than the wild-type strain accumulated. The loss of actinorhodin production in the eshA disruptant was restored by expression of a truncated relA gene, which increased the ppGpp level to the level in the wild-type strain, indicating that the reduced ppGpp accumulation in the eshA mutant was solely responsible for the loss of antibiotic production. Antibiotic production was also restored in the eshA mutant by introducing mutations into rpoB (encoding the RNA polymerase β subunit) that bypassed the requirement for ppGpp, which is consistent with a role for EshA in modulating ppGpp levels. EshA contains a cyclic nucleotide-binding domain that is essential for its role in triggering actinorhodin production. EshA may provide new insights and opportunities to unravel the molecular signaling events that occur during physiological differentiation in streptomycetes.

scholarly journals Comparative Proteomic Analysis of Streptomyces lividans Wild-Type andppkMutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

2013 ◽  
Vol 79 (19) ◽  
pp. 5907-5917 ◽  
Author(s):  
Pierre Le Maréchal ◽  
Paulette Decottignies ◽  
Christophe H. Marchand ◽  
Jeril Degrouard ◽  
Danièle Jaillard ◽  
...  
Keyword(s):  
Streptomyces Coelicolor ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
Content Type ◽  
Storage Lipids ◽  
Storage Lipid ◽  
Mutant Strains ◽  
Layer Chromatography

ABSTRACTStreptomyces lividansTK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of theppkgene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of theppkmutant, the proteomes of the wild-type (wt) andppkmutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in theppkmutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt andppkmutant strains ofS. lividansas well as that ofStreptomyces coelicolorM145, a strain that produces antibiotics at high levels and is closely related toS. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis inStreptomyces.

Effect of protease mutations on the production of xylanases in Streptomyces lividans

2007 ◽  
Vol 53 (6) ◽  
pp. 695-701 ◽  
Author(s):  
Éliana Arias ◽  
Haiming Li ◽  
Rolf Morosoli
Keyword(s):  
Protein Secretion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Catalytic Domain ◽  
Streptomyces Lividans ◽  
Wild Type ◽  
Secretion Systems ◽  
Binding Domains ◽  
Tat Pathway ◽  
In The Wild

Three protease mutants — 7 (tap–), 12 (tap–, ssp–), and 17 (multiple mutations) — of Streptomyces lividans were tested for their influence on protein secretion. Streptomyces lividans grown in xylan secretes 3 xylanases (A, B, and C). Xylanases A (XlnA) and B (XlnB) are secreted by the Sec pathway, whereas xylanase C (XlnC) is secreted by the Tat pathway. The production of XlnA and XlnC was affected in the mutants, suggesting that the mutations interfered with both Sec- and Tat-secretion systems. However, the processing rate for the Sec and Tat precursor was similar to the wild-type strain, indicating that the mutations had no direct effect on secretion. Streptomyces lividans naturally produced 2 forms of XlnB: XlnB1, which contains the catalytic and the xylan-binding domains, and XlnB2, which contains the catalytic domain only. There was no change from the wild-type strain in the ratio of XlnB1/XlnB2 produced by the mutants, indicating that these proteases are not involved in this process. Although XlnA1, partially truncated in its xylan-binding domain, was rapidly degraded to its catalytic domain (XlnA2) in the wild-type strain, the rate of conversion was reduced in the 3 mutants, indicating that the proteases participated to some extent in this proteolytic process.

The G243D mutation (afsB mutation) in the principal sigma factor σ HrdB alters intracellular ppGpp level and antibiotic production in Streptomyces coelicolor A3(2)

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2384-2392 ◽  
Author(s):  
Guojun Wang ◽  
Yukinori Tanaka ◽  
Kozo Ochi
Keyword(s):  
Sigma Factor ◽  
Parent Strain ◽  
Wild Type Strain ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Fine Tuning ◽  
Wild Type ◽  
New Class ◽  
In The Wild ◽  
Amino Acid Depletion

Deficient antibiotic production in an afsB mutant, BH5, of Streptomyces coelicolor A3(2) was recently shown to be due to a mutation (G243D) in region 1.2 of the primary sigma factor σ HrdB. Here we show that intracellular ppGpp levels during growth, as well as after amino acid depletion, in the mutant BH5 are lower than those of the afsB+ parent strain. The introduction of certain rifampicin resistance (rif) mutations, which bypassed the requirement of ppGpp for transcription of pathway-specific regulatory genes, actII-ORF4 and redD, for actinorhodin and undecylprodigiosin, respectively, completely restored antibiotic production by BH5. Antibiotic production was restored also by introduction of a new class of thiostrepton-resistance (tsp) mutations, which provoked aberrant accumulation of intracellular ppGpp. Abolition of ppGpp synthesis in the afsB tsp mutant Tsp33 again abolished antibiotic production. These results indicate that intracellular ppGpp level is finely tuned for successful triggering of antibiotic production in the wild-type strain, and that this fine tuning was absent from the afsB mutant BH5, resulting in a failure to initiate antibiotic production in this strain.

Sign in / Sign up

Export Citation Format

Share Document