Streptomyces Lividans Research Articles

Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C.saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed.

Fast and reliable strain characterization of Streptomyces lividans through micro-scale cultivation.

Filamentous organisms of the genus Streptomyces play an important role in industrial production processes, due to their extensive secondary metabolism variability, as well as their ability to secrete efficiently large amounts of (heterologous) proteins. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. This is due to the lack of reliable parallelized and accelerated cultivation techniques for morphologically challenging organisms. To address this challenge, we developed an integrated cultivation workflow for Streptomyces lividans based on a parallelized shaken 48-well microtiter-plate (MTP) cultivation device. In a first step, a feasible pre-culture method was identified and validated, revealing high comparability in subsequent main cultivations (coefficient of variation of 1.1% for in-plate replicates and 3.2% between different pre-cultures). When validating the growth performance in 1 mL MTP cultivation against an established 1,000 mL lab-scale cultivation system, highly comparable cultivation patterns were found for online (pH, dissolved oxygen), as well as for offline derived parameters (glucose uptake, cell-dry-weight, and pellet size). Additionally, the two cultivation regimes were compared with respect to transcriptional and protein secretion activity of Streptomyces, showing overall good comparability with minor, but well explainable discrepancies, most probably caused by different energy dissipation (shaking vs. stirring) and adaption effects due to different illumination conditions. Embedded within the presented cultivation workflow, the 1 mL MTP-based parallelized cultivation system seems to be a suitable screening tool for filamentous and industrial relevant organisms like Streptomyces. This can contribute to widen the field of application for these organisms and facilitate screening and early-stage bioprocess development. Biotechnol. Bioeng. 2017;114: 2011-2022. © 2017 Wiley Periodicals, Inc.

Heterologous production of kasugamycin, an aminoglycoside antibiotic from Streptomyces kasugaensis, in Streptomyces lividans and Rhodococcus erythropolis L-88 by constitutive expression of the biosynthetic gene cluster

Kasugamycin (KSM), an aminoglycoside antibiotic isolated from Streptomyces kasugaensis cultures, has been used against rice blast disease for more than 50years. We cloned the KSM biosynthetic gene (KBG) cluster from S. kasugaensis MB273-C4 and constructed three KBG cassettes (i.e., cassettes I-III) to enable heterologous production of KSM in many actinomycetes by constitutive expression of KBGs. Cassette I comprised all putative transcriptional units in the cluster, but it was placed under the control of the P neo promoter from Tn5. It was not maintained stably in Streptomyces lividans and did not transform Rhodococcus erythropolis. Cassette II retained the original arrangement of KBGs, except that the promoter of kasT, the specific activator gene for KBG, was replaced with P rpsJ , the constitutive promoter of rpsJ from Streptomyces avermitilis. To enhance the intracellular concentration of myo-inositol, an expression cassette of ino1 encoding the inositol-1-phosphate synthase from S. avermitilis was inserted into cassette II to generate cassette III. These two cassettes showed stable maintenance in S. lividans and R. erythropolis to produce KSM. Particularly, the transformants of S. lividans induced KSM production up to the same levels as those produced by S. kasugaensis. Furthermore, cassette III induced more KSM accumulation than cassette II in R. erythropolis, suggesting an exogenous supply of myo-inositol by the ino1 expression in the host. Cassettes II and III appear to be useful for heterologous KSM production in actinomycetes. Rhodococcus exhibiting a spherical form in liquid cultivation is also a promising heterologous host for antibiotic fermentation.

Establishing a high yielding streptomyces-based cell-free protein synthesis system.

Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc.

Discovery of a Phosphonoacetic Acid Derived Natural Product by Pathway Refactoring.

The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the discovery rate of new molecules, this unknown chemical space is rapidly growing, as too are the rewards for developing technologies to exploit it. One class of natural products that has been underrepresented is phosphonic acids, which have important medical and agricultural uses. Hundreds of phosphonic acid biosynthetic gene clusters have been identified encoding for unknown molecules. Although methods exist to elicit secondary metabolite gene clusters in native hosts, they require the strain to be amenable to genetic manipulation. One method to circumvent this is pathway refactoring, which we implemented in an effort to discover new phosphonic acids from a gene cluster from Streptomyces sp. strain NRRL F-525. By reengineering this cluster for expression in the production host Streptomyces lividans, utility of refactoring is demonstrated with the isolation of a novel phosphonic acid, O-phosphonoacetic acid serine, and the characterization of its biosynthesis. In addition, a new biosynthetic branch point is identified with a phosphonoacetaldehyde dehydrogenase, which was used to identify additional phosphonic acid gene clusters that share phosphonoacetic acid as an intermediate.

Erratum to: Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Subsequent Fermentation for Optimized Production.

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40 % of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60 %, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 15 years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans has a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR4-based system is explained. Finally a basic protocol for benchtop bioreactor experiments which can form the start in the production process optimization and upscaling is provided.

Kinetic characterisation of a dye decolourising peroxidase from Streptomyces lividans: new insight into the mechanism of anthraquinone dye decolourisation.

Dye decolourising peroxidases are the most recent family of haem peroxidases to be discovered. The oxidising potential of these enzymes is driven by the formation of ferryl intermediates that enables them to oxidise synthetic dye molecules that are widely used in the textile industry. We have investigated the catalytic cycle of a dye decolourising peroxidase (DtpA) from a biotechnologically important bacterium Streptomyces lividans. Using a combination of steady-state and stopped-flow kinetic investigations, we have determined the rate constants for all steps in the catalytic cycle with a range of substrate molecules. For most substrates, the value of kcat/Km measured by steady-state kinetics is equal to the slowest step in catalysis measured by stopped-flow spectroscopy, namely the decay of the ferryl FeIV[double bond, length as m-dash]O species (compound II) to form the ferric species. With the anthraquinone-based dye, reactive blue 19 (RB19) unusual steady-state kinetic behaviour is observed, which we propose through kinetic modelling of the catalytic cycle is due to a disproportionation mechanism of the dye. At low RB19 concentrations, the rate of disproportionation is slower than that of the rate determining step in DtpA, whereas at higher concentrations of RB19 the rate of disproportionation is faster. This mechanism obviates the need to postulate secondary sites for substrate binding on the enzyme which has been previously proposed for other dye decolourising haem peroxidases.

The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner.

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application.

Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Subsequent Fermentation for Optimized Production.

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40 % of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60 %, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 15 years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans has a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR4-based system is explained. Finally a basic protocol for benchtop bioreactor experiments which can form the start in the production process optimization and upscaling is provided.

Cloning and identification of saprolmycin biosynthetic gene cluster from Streptomyces sp. TK08046.

Saprolmycins A-E are anti-Saprolegnia parasitica antibiotics. To identify the gene cluster for saprolmycin biosynthesis in Streptomyces sp. TK08046, polymerase chain reaction using aromatase and cyclase gene-specific primers was performed; the spr gene cluster, which codes for angucycline biosynthesis, was obtained from the strain. The cluster consists of 36 open reading frames, including minimal polyketide synthase, ketoreductase, aromatase, cyclase, oxygenase, and deoxy sugar biosynthetic genes, as defined by homology to the corresponding genes of the urdamycin, Sch-47554, and grincamycin biosynthetic gene clusters in Streptomyces fradiae, Streptomyces sp. SCC-2136, and Streptomyces lusitanus, respectively. To establish the function of the gene cluster, an expression cosmid vector containing all 36 open reading frames was introduced into Streptomyces lividans TK23. The transformant was confirmed to express the biosynthetic genes and produce saprolmycins by liquid chromatography-mass spectrometry analysis of the extract.

Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase.

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm.

DNA Phosphorothioate Modification Plays a Role in Peroxides Resistance in Streptomyces lividans.

DNA phosphorothioation, conferred by dnd genes, was originally discovered in the soil-dwelling bacterium Streptomyces lividans, and thereafter found to exist in various bacterial genera. However, the physiological significance of this sulfur modification of the DNA backbone remains unknown in S. lividans. Our studies indicate that DNA phosphorothioation has a major role in resistance to oxidative stress in the strain. Although Streptomyces species express multiple catalase/peroxidase and organic hydroperoxide resistance genes to protect them against peroxide damage, a wild type strain of S. lividans exhibited two-fold to 10-fold higher survival, compared to a dnd− mutant, following treatment with peroxides. RNA-seq experiments revealed that, catalase and organic hydroperoxide resistance gene expression were not up-regulated in the wild type strain, suggesting that the resistance to oxidative stress was not due to the up-regulation of these genes by DNA phosphorothioation. Quantitative RT-PCR analysis was conducted to trace the expression of the catalase and the organic hydroperoxide resistance genes after peroxides treatments. A bunch of these genes were activated in the dnd− mutant rather than the wild type strain in response to peroxides. Moreover, the organic hydroperoxide peracetic acid was scavenged more rapidly in the presence than in the absence of phosphorothioate modification, both in vivo and in vitro. The dnd gene cluster can be up-regulated by the disulfide stressor diamide. Overall, our observations suggest that DNA phosphorothioate modification functions as a peroxide resistance system in S. lividans.

Styrene production from a biomass-derived carbon source using a coculture system of phenylalanine ammonia lyase and phenylacrylic acid decarboxylase-expressing Streptomyces lividans transformants

To produce styrene from a biomass-derived carbon source, Streptomyces lividans was adopted as a host strain. The gene encoding ferulic acid decarboxylase from Saccharomyces cerevisiae (FDC1) was introduced into S.lividans, and the resulting S.lividans transformant successfully expressed FDC1 and converted trans-cinnamic acid (CA) to styrene. A key factor in styrene production using microbes is the recovery of volatile styrene. In the present study, we selected polystyrene resin beads XRD-4 as the absorbent agent to recover styrene produced using S.lividans transformants, which enabled recovery of styrene from the culture broth. For styrene production from biomass-derived carbon sources, S.lividans/FDC1 was cultured together with S.lividans/p-encP, which we previously reported as a CA-producing S.lividans strain. This coculture system combined with the recovery of styrene using XAD-4 allowed the production of styrene from glucose, cellobiose, or xylo-oligosaccharide, respectively.

Development of nitrilase promoter-derived inducible vectors for Streptomyces.

An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes.

Characterization of a novel highly thermostable esterase from the Gram-positive soil bacterium Streptomyces lividans TK64.

A novel esterase gene (estW) from soil bacterium Streptomyces lividans TK64 was successfully cloned using a pair of homologous primers. The estW gene encoded a protein (EstW) of 289 amino acid residues with a predicted molecular weight of 31.43 kDa. Sequence alignment revealed that EstW show relatively high levels of homology to other lipolytic enzymes characterized from Streptomyces and phylogenetic analysis suggested EstW belongs to the bacterial lipase/esterase family I. The estW gene was expressed at a high level in Escherichia coli and the recombinant enzyme was purified to homogeneity. The purified EstW was characterized via hydrolysis of various p-nitrophenyl esters and the best substrate was found to be p-nitrophenyl acetate (pNPA). Maximal activity of the recombinant protein was observed at pH 8.0 and 50°C with pNPA as the substrate. The calculated activation energy (Ea ) of the esterase reaction was 9.12 kcal/mol. Half-life of EstW at 95°C was approximately 12.5 H, making it the most thermostable esterase among all of the known lipolytic enzymes from Streptomyces, and the thermostability of EstW was similar to those of some enzymes characterized from the thermophilic bacteria. EstW exhibited relatively high tolerance to several detergents and required no cations for its maximal activity. The unique properties of EstW, namely its high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.

Metabolites from the Fungal Endophyte Aspergillus austroafricanus in Axenic Culture and in Fungal-Bacterial Mixed Cultures.

The endophytic fungus Aspergillus austroafricanus isolated from leaves of the aquatic plant Eichhornia crassipes was fermented axenically on solid rice medium as well as in mixed cultures with Bacillus subtilis or with Streptomyces lividans. Chromatographic analysis of EtOAc extract of axenic cultures afforded two new metabolites, namely, the xanthone dimer austradixanthone (1) and the sesquiterpene (+)-austrosene (2), along with five known compounds (3-7). Austradixanthone (1) represents the first highly oxygenated heterodimeric xanthone derivative. When A. austroafricanus was grown in mixed cultures with B. subtilis or with S. lividans, several diphenyl ethers (8-11) including the new austramide (8) were induced up to 29-fold. The structures of new compounds were unambiguously elucidated using 1D- and 2D-NMR spectroscopy, HRESIMS, and chemical derivatization. Compound 7 exhibited weak cytotoxicity against the murine lymphoma L5178Y cell line (EC50 is 12.6 μM). In addition, compounds 9 and 10, which were enhanced in mixed fungal/bacterial cultures, proved to be active against Staphylococcus aureus (ATCC 700699) with minimal inhibitory concentrations (MICs) of 25 μM each (6.6 μg/mL), whereas compound 11 revealed moderate antibacterial activity against B. subtilis 168 trpC2 with an MIC value of 34.8 μM (8 μg/mL).

Exploring Peptide Ligase Orthologs in Actinobacteria-Discovery of Pseudopeptide Natural Products, Ketomemicins.

We recently identified a novel peptide ligase (PGM1), an ATP-grasp-ligase, that catalyzes amide bond formation between (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid and ribosomally supplied oligopeptides in pheganomycin biosynthesis. This was the first example of an ATP-grasp-ligase utilizing peptides as nucleophiles. To explore the potential of this type of enzyme, we performed a BLAST search and identified many orthologs. The orthologs of Streptomyces mobaraensis, Salinispora tropica, and Micromonospora sp. were found in similar gene clusters consisting of six genes. To probe the functions of these genes, we heterologously expressed each of the clusters in Streptomyces lividans and detected novel and structurally similar pseudotripeptides in the broth of all transformants. Moreover, a recombinant PGM1 ortholog of Micromonospora sp. was demonstrated to be a novel dipeptide ligase catalyzing amide bond formation between amidino-arginine and dipeptides to yield tripeptides; this is the first report of a peptide ligase utilizing dipeptides as nucleophiles.

A Streptomyces lividans SipY deficient strain as a host for protein production: standardization of operational alternatives for model proteins

AbstractBACKGROUNDExtracellular protein production by Gram‐positive bacteria, such as Streptomyces, may be complementary to current established protein production processes. The performance of a Streptomyces lividans mutant strain, deficient in the major signal peptidase (SipY) is investigated for the production of proteins secreted via the secondary Tat pathway.RESULTSThe SipY deficient strain has shown advantages over the wild type strain, in terms of extracellular productivity, specific activity and rheological behaviour. Two operational modes, batch and fed‐batch, have been studied using mannitol as carbon source. The results showed that two successive mannitol additions in fed‐batch mode led to improved secretory protein production using Streptomyces agarase as a model protein. This production process was also explored for the Tat secretory protein S. lividans laccase. The predicted sequence for the pre‐laccase coding sequence has been cloned into the mutant strain under the control of the agarase promoter. Batch and fed‐batch laccase production, using either mannitol or glucose as carbon source, have been developed and quantified.CONCLUSIONSThe usefulness of a Streptomyces lividans SipY deficient strain as protein producer has been demonstrated. A proposed operating mode with substrate additions has been employed for the optimisation of Tat proteins production, although some adjustments might be necessary depending on the secretory protein. © 2016 Society of Chemical Industry

New approaches to achieve high level enzyme production in Streptomyces lividans.

BackgroundActinomycetes are saprophytic soil bacteria, and a rich source of industrial enzymes. While some of these enzymes can be produced using well-characterized production platforms such as Escherichia coli or Bacillus subtilis,Streptomyces lividans may be the preferred host for proper folding and efficient secretion of active enzymes. A combination of promoters, signal peptides and hosts were tested in order to obtain the best protein expression in this actinomycete. The xylanase, Xys1, from S. halstedii, the α-amylase, Amy, from S. griseus and the small laccase, SLAC, from S. coelicolor were used as reporters.ResultsThe promoters xysAp from S. halstedii JM8 and pstSp from S. lividans were the most efficient among those tested. An improvement of 17 % was obtained in xylanase activity when the signal peptide of the α-amylase protein (Amy) of S. griseus IMRU3570 was used to direct its secretion. Enhanced expression of SsgA, a protein that plays a role in processes that require cell-wall remodelling, resulted in a improvement of 40 and 70 % of xylanase and amylase production, respectively. Deletion of genes SLI7232 and SLI4452 encoding putative repressors of xysAp provided improvement of production up to 70 % in the SLI7232 deletion strain. However, full derepression of this promoter activity was not obtained under the conditions assayed.ConclusionsStreptomyces lividans is a frequently used platform for industrial enzyme production and a rational strain-development approach delivered significant improvement of protein production by this host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0425-7) contains supplementary material, which is available to authorized users.

Kinetic model of metabolic network for xiamenmycin biosynthetic optimisation.

Xiamenmycins, a series of prenylated benzopyran compounds with anti‐fibrotic bioactivities, were isolated from a mangrove‐derived Streptomyces xiamenensis. To fulfil the requirements of pharmaceutical investigations, a high production of xiamenmycin is needed. In this study,, the authors present a kinetic metabolic model to evaluate fluxes in an engineered Streptomyces lividans with xiamenmycin‐oriented genetic modification based on generic enzymatic rate equations and stability constraints. Lyapunov function was used for a viability optimisation. From their kinetic model, the flux distributions for the engineered S. lividans fed on glucose and glycerol as carbon sources were calculated. They found that if the bacterium can utilise glucose simultaneously with glycerol, xiamenmycin production can be enhanced by 40% theoretically, while maintaining the same growth rate. Glycerol may increase the flux for phosphoenolpyruvate synthesis without interfering citric acid cycle. They therefore believe this study demonstrates a possible new direction for bioengineering of S. lividans.