The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene

Pérez‐Redondo, Rosario; Santamarta, Irene; Bovenberg, Roel A. L.; Martı́n, Juan F.; Liras, Paloma

doi:10.1099/mic.0.035840-0

Cited by 21 publications

(15 citation statements)

References 25 publications

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“…This gene is recognized as a key factor that may contribute to "glucose repression" of nonpreferred sugars (1,6,44). However, defective and suspended D-glucose consumption had previously been observed in E. coli, B. subtilis, and S. clavuligerus when genes responsible for glucose transport were inactivated or were expressed at low levels (13,34,35). Thus, it was possible that glcG would not be an ideal gene target for engineering to relieve "glucose repression."…”

Section: Discussionmentioning

confidence: 99%

Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose

Xiao

Ning

et al. 2011

Appl Environ Microbiol

127

100

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Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.

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Section: Discussionmentioning

confidence: 99%

Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose

Xiao

Ning

et al. 2011

Appl Environ Microbiol

127

100

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“…A 550-bp fragment of S. coelicolor M145 that contains the rpoZ gene with its promoter and terminator sequences was amplified with primers RPOZ-Cu1 (5Ј-GGGCCTCTAGATAAGTCAGCGCA) and RPOZ-PD (5Ј-ACGAGCTGGATCCGCAGGCTCA), which had been modified with XbaI and BamHI restriction sites, respectively. The PCR product was cloned into pRA (36), an integrative conjugative plasmid. In order to introduce the complementation plasmid in the apramycin-resistant ⌬rpoZ mutant strain, a 1.4-kb fragment containing the Tn5/Neo resistance gene was cloned in the EcoRV site of pRA.…”

Section: Methodsmentioning

confidence: 99%

The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

Santos-Beneit

Barriuso-Iglesias

Fernández-Martínez

et al. 2011

Appl Environ Microbiol

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The RNA polymerase (RNAP) omega factor () forms a complex with the ␣ 2 ␤␤ core of this enzyme in bacteria. We have characterized the rpoZ gene of Streptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the rpoZ gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ⌬rpoZ strain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ⌬rpoZ mutant with the wild-type rpoZ allele restored both phenotype and antibiotic production. Interestingly, the rpoZ gene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed that rpoZ promoter activity was increased in a ⌬phoP background, it can be concluded that rpoZ is controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation in Streptomyces.In bacteria, the RNA polymerase (RNAP) complex plays a central role in transcription and is a target for regulation of primary metabolism (6,7,44). The rpoZ gene encodes the RNAP omega () subunit, which forms a complex with the ␣ 2 ␤␤Ј core of this enzyme. The subunit has been identified in the RNAPs of most free-living bacteria. This protein is functionally homologous to the RpoK subunit of the archaeal RNA polymerase complex and the RPB6 subunit of the eukaryotic RNA polymerases I, II, and III (32). In Escherichia coli the subunit interacts with the ␤Ј subunit and promotes assembly of the RNA polymerase complex (14, 32), although it is not essential for survival in this bacterium (13).Streptomyces spp. are soil-dwelling bacteria that are notorious for their ability to produce thousands of antibiotics, pigments, antitumor agents, immunomodulators, and a variety of other bioactive secondary metabolites (1, 2, 8). Differential expression of secondary metabolism genes occurs following nutrient depletion (34), but the transcriptional control mechanisms that govern the onset of secondary metabolites are still obscure (29). The rpoZ gene of Streptomyces kasugaensis has been shown to be required for antibiotic production and morphological differentiation but is not essential for growth (21). A DNA fragment containing the rpoZ gene was shown to complement an S. kasugaensis pleiotropic mutant deficient in aerial mycelium formation and kasugamycin biosynthesis. Although sigma factors in Streptomyces have received considerable attention in relation to the expression of antibiotic biosynthetic genes (9,19,20), the role of the RNAP subunit is still obscure.The expression of many genes involved in antibiotic biosynthesis is negatively controlled by the phosphate concentration in the medium (reviewed in references 28 and 30). Limitati...

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“…1a). Moreover, this position is not included in the PhoP core operator (28), so it should not be involved in PhoP binding. Our results with the M1 mutant confirmed that the AfsR and PhoP binding sequences start at positions displaced in 1 nucleotide.…”

Section: Analysis Of the Phop And Afsr Binding Sequences In The S Comentioning

confidence: 99%

Complex Transcriptional Control of the Antibiotic Regulator afsS in Streptomyces: PhoP and AfsR Are Overlapping, Competitive Activators

2011

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The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene

Cited by 21 publications

References 25 publications

Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose

Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose

The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

Complex Transcriptional Control of the Antibiotic Regulator afsS in Streptomyces: PhoP and AfsR Are Overlapping, Competitive Activators

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