Transcriptome analysis of wild-type and afsS deletion mutant strains identifies synergistic transcriptional regulator of afsS for a high antibiotic-producing strain of Streptomyces coelicolor A3(2).

Most secondary metabolism in Actinobacteria is controlled by multi-layered, gene-regulatory networks. These regulatory mechanisms are not easily identified due to their complexity. As a result, when a strong transcriptional regulator (TR) governs activation of biosynthetic pathways of target antibiotics such as actinorhodin (ACT), additional enhancement of the biosynthesis is difficult in combination with other TRs. To find out any "synergistic transcriptional regulators (sTRs)" that show an additive effect on the major, often strong, transcriptional regulator (mTR), here, we performed a clustering analysis using the transcriptome datasets of an mTR deletion mutant and wild-type strain. In the case of ACT biosynthesis in Streptomyces coelicolor, PhoU (SCO4228) and RsfA (SCO4677) were selected through the clustering analysis, using AfsS (SCO4425) as a model mTR, and experimentally validated their roles as sTRs. Furthermore, through analysis of synergistic effects, we were able to suggest a novel regulation mechanism and formulate a strategy to maximize the synergistic effect. In the case of the double TR mutant strain (ΔrsfA pIBR25::afsS), it was confirmed that the increase of cell mass was the major cause of the synergistic effect. Therefore, the strategy to increase the cell mass of double mutant was further attempted by optimizing the expression of efflux pump, which resulted in 2-fold increase in the cell mass and 24-fold increase in the production of ACT. This result is the highest ACT yield from S. coelicolor ever reported.

The role of NdgR in glycerol metabolism in Streptomyces coelicolor.

Streptomyces, which produces many pharmaceutical antibiotics and anticancer agents, is a genus of soil-dwelling bacteria with numerous regulators that control both primary and secondary metabolism. NdgR is highly conserved in Streptomyces spp. and is known to be involved in antibiotic production, tolerance against shock and physical stress, nitrogen metabolism, leucine metabolism, and N-acetylglucosamine metabolism. As another function of NdgR, we report the involvement of NdgR in glycerol metabolism in S. coelicolor. Initially, a glycerol utilization operon containing gylCABX was found to be up-regulated in an ndgR deletion mutant (BG11) grown in N-acetylglucosamine solid minimal media compared with wild-type strain (M145). BG11 produced more antibiotics with a small amount of glycerol and increased glycerol utilization, yielding higher concentrations of lactate and acetate per cell. Moreover, fatty acid production was also changed in BG11 to produce longer chain fatty acids, phenolic compounds, alkanes, and fatty alcohols. Using a gel retardation assay, NdgR was found to bind the upstream region of gylC, working as a repressor. NdgR is a second regulator of a glycerol utilization operon, for which only one regulator, GylR was previously known.

Genome-wide primary transcriptome analysis of H2-producing archaeon Thermococcus onnurineus NA1.

In spite of their pivotal roles in transcriptional and post-transcriptional processes, the regulatory elements of archaeal genomes are not yet fully understood. Here, we determine the primary transcriptome of the H2-producing archaeon Thermococcus onnurineus NA1. We identified 1,082 purine-rich transcription initiation sites along with well-conserved TATA box, A-rich B recognition element (BRE), and promoter proximal element (PPE) motif in promoter regions, a high pyrimidine nucleotide content (T/C) at the -1 position, and Shine-Dalgarno (SD) motifs (GGDGRD) in 5' untranslated regions (5' UTRs). Along with differential transcript levels, 117 leaderless genes and 86 non-coding RNAs (ncRNAs) were identified, representing diverse cellular functions and potential regulatory functions under the different growth conditions. Interestingly, we observed low GC content in ncRNAs for RNA-based regulation via unstructured forms or interaction with other cellular components. Further comparative analysis of T. onnurineus upstream regulatory sequences with those of closely related archaeal genomes demonstrated that transcription of orthologous genes are initiated by highly conserved promoter sequences, however their upstream sequences for transcriptional and translational regulation are largely diverse. These results provide the genetic information of T. onnurineus for its future application in metabolic engineering.

Medium engineering for enhanced production of undecylprodigiosin antibiotic in Streptomyces coelicolor using oil palm biomass hydrolysate as a carbon source.

In this study, a biosugar obtained from empty fruit bunch (EFB) of oil palm by hot water treatment and subsequent enzymatic saccharification was used for undecylprodigiosin production, using Streptomyces coelicolor. Furfural is a major inhibitor present in EFB hydrolysate (EFBH), having a minimum inhibitory concentration (MIC) of 1.9mM, and it reduces utilization of glucose (27%), xylose (59%), inhibits mycelium formation, and affects antibiotic production. Interestingly, furfural was found to be a good activator of undecylprodigiosin production in S. coelicolor, which enhanced undecylprodigiosin production by up to 52%. Optimization by mixture analysis resulted in a synthetic medium containing glucose:furfural:ACN:DMSO (1%, 2mM, 0.2% and 0.3%, respectively). Finally, S. coelicolor was cultured in a fermenter in minimal medium with EFBH as a carbon source and addition of the components described above. This yielded 4.2µg/mgdcw undecylprodigiosin, which was 3.2-fold higher compared to that in un-optimized medium.

Biomass-derived molecules modulate the behavior of Streptomyces coelicolor for antibiotic production.

Various chemicals, i.e., furfural, vanillin, 4-hydroxybenzaldehyde and acetate produced during the pretreatment of biomass affect microbial fermentation. In this study, effect of vanillin, 4-hydroxybenzaldehyde and acetate on antibiotic production in Streptomyces coelicolor is investigated. IC 50 value of vanillin, 4-hydroxybenzaldehyde and acetate was recorded as 5, 11.3 and 115 mM, respectively. Vanillin was found as a very effective molecule, and it completely abolished antibiotic (undecylprodigiosin and actinorhodin) production at 1 mM concentration, while 4-hydroxybenzaldehyde and acetate have little effect. Microscopic analysis with field emission scanning electron microscopy (FESEM) showed that addition of vanillin inhibits mycelia formation and increases differentiation of S. coelicolor cells. Vanillin increases expression of genes responsible for sporulation (ssgA) and decreases expression of antibiotic transcriptional regulator (redD and actII-orf4), while it has no effect on genes related to the mycelia formation (bldA and bldN) and quorum sensing (scbA and scbR). Vanillin does not affect the glycolysis process, but may affect acetate and pyruvate accumulation which leads to increase in fatty acid accumulation. The production of antibiotics using biomass hydrolysates can be quite complex due to the presence of exogenous chemicals such as furfural and vanillin, and needs further detailed study.

Determination of single nucleotide variants in Escherichia coli DH5α by using short-read sequencing.

Escherichia coli DH5α is a common laboratory strain that provides an important platform for routine use in cloning and synthetic biology applications. Many synthetic circuits have been constructed and successfully expressed in E. coli DH5α; however, its genome sequence has not been determined yet. Here, we determined E. coli DH5α genome sequence and identified genetic mutations that affect its phenotypic functions by using short-read sequencing. The sequencing results clearly described the genotypes of E. coli DH5α, which aid in further studies using the strain. Additionally, we observed 105 single nucleotide variants (SNVs), 83% of which were detected in protein-coding regions compared to the parental strain E. coli DH1. Interestingly, 23% of the protein-coding regions have mutations in their amino acid residues, whose biological functions were categorized into two-component systems, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis. These results underscore the advantages of E. coli DH5α, which tolerates the components of transformation buffer and expresses foreign plasmids efficiently. Moreover, these SNVs were also observed in the commercially available strain. These data provide the genetic information of E. coli DH5α for its future application in metabolic engineering and synthetic biology.

Increased vulnerability to physical stress by inactivation of NdgR in Streptomyces coelicolor.

The antibiotic production and spore formation process in Streptomyces coelicolor need complex decision making processes by several regulatory units. These regulatory units are involved in both primary and secondary metabolism. As a result, most regulators have several functions, and those are worthwhile themes to study about different functions of a known regulator. In this study, a deletion mutant of ndgR, which encodes the nitrogen-dependent growth regulator, was examined by the cell viability test, TEM, and growth in N-acetylglucosamine/asparagine (GlcNAc/Asn) liquid medium. The results of the study show that NdgR is also involved in the structure of the cell membrane affecting survival under physical shocks. Deletion of ndgR leads to abnormal cell membrane resulting in the vulnerable cells to physical stress caused by shaking with beads in liquid culture condition. This empirical observation is the first meaningful explanation to why ndgR mutant could not grow well in a liquid minimal medium due to the defect of N-acetylglucosamine (GlcNAc) utilization and phospholipid synthesis.

Emerging tools for synthetic genome design.

Synthetic biology is an emerging discipline for designing and synthesizing predictable, measurable, controllable, and transformable biological systems. These newly designed biological systems have great potential for the development of cheaper drugs, green fuels, biodegradable plastics, and targeted cancer therapies over the coming years. Fortunately, our ability to quickly and accurately engineer biological systems that behave predictably has been dramatically expanded by significant advances in DNA-sequencing, DNA-synthesis, and DNA-editing technologies. Here, we review emerging technologies and methodologies in the field of building designed biological systems, and we discuss their future perspectives.

Deletion of an architectural unit, leucyl aminopeptidase (SCO2179), in Streptomyces coelicolor increases actinorhodin production and sporulation.

Several reports state that three architectural units, including integration host factor, leucyl aminopeptidase (PepA), and purine regulator, are involved in transcriptional process with RNA polymerase in Escherichia coli. Similarly, Streptomyces species possess the same structural units. We previously identified a protein, Streptomyces integration host factor (sIHF), involved in antibiotic production and sporulation. Subsequently, the function of PepA (SCO2179) was examined in detail. PepA is highly conserved among various Streptomyces spp., but it has not yet been characterized in Streptomyces coelicolor. While it is annotated as a putative leucyl aminopeptidase because it contains a peptidase M17 superfamily domain, this protein did not exhibit leucyl aminopeptidase activity. SCO2179 deletion mutant showed increased actinorhodin production and sporulation, as well as more distinct physiological differences, particularly when cultured on N-acetylglucosamine (GlcNAc) minimal media. The results of two-dimensional gel analysis and reverse transcription PCR showed that the SCO2179 deletion increased protein and mRNA levels of ftsZ, ssgA, and actinorhodin (ACT)-related genes such as actII-ORF4, resulting in increased actinorhodin production and spore formation in minimal media containing GlcNAc.

Rational design of modular allosteric aptamer sensor for label-free protein detection.

An aptamer can be redesigned to new functional molecules by conjugating with other oligonucleotides. However, it requires experimental trials to optimize the conjugating module with the sensitivity and selectivity toward a target. To reduce these efforts, we report rationally-designed modular allosteric aptamer sensor (MAAS), which is composed of coupled two aptamers and the regulator. For label-free protein detection, the protein-aptamer was conjugated with the malachite green (MG) aptamer for signaling. The MAAS additionally has the regulator domain which is designed to hybridize to a protein binding domain. The regulator makes MAAS to be inactive by destructing the original structure of the two aptamers. However, its conformation becomes active by dissociating the hybridization from the protein recognition signal, thereby inducing the binding of MG emitting the enhanced fluorescence. The design of regulator is based on the thermodynamic energy difference by the RNA conformational change and protein-aptamer affinity. Here we first demonstrated the MAAS for hepatitis C helicase and replicase. The target proteins were detected up to 250nM with minimized blank signals and displayed high specificities 10-fold greater than in non-specific proteins. The MAAS provides valuable tools that can be adapted to a wide range of configurations in bioanalytical applications.

In vitro selection of sialic acid specific RNA aptamer and its application to the rapid sensing of sialic acid modified sugars.

Sialic acids (SAs) are located on the terminal positions of glycan on a cell surface, which play important role in the spread and metastasis of cancer cells and infection of pathogen. For their detection and diagnosis, the finding of SA specific ligand is an essential prerequisite. Here, RNA aptamer for N-acetylneuraminic acid (Neu5Ac), a representative of SAs, with the high affinity of 1.35 nM and the selectivity was screened by in vitro selection method. The strong binding of the screened aptamer was enough to protect the hydrolysis of Neu5Ac by neuraminidase with the stoichiometry of 1:1 molar ratio. For the rapid detection of SAs, the RNA aptamer was further engineered to the aptazyme sensor by conjugating with a ribozyme following the characterization of selected aptamer by RNase footprinting assay. Without additional desialylation, modification, or/and purification processes, the aptazyme indicated high catalytic activities in the presence of Neu5Ac over 20 µM in several minutes. Also, we observed that the aptazyme sensor shows high sensitivities to Neu5Ac-conjugated sugars as well as Neu5Ac monomer, but not in non-Neu5Ac modified sugars. The aptamer for Neu5Ac can support valuable tools in a wide range of bioanalytical applications as well as biosensors.

Increased sensitivity to chloramphenicol by inactivation of manB in Streptomyces coelicolor.

Phosphomannomutase (ManB) is involved in the biosynthesis of GDP-mannose, which is vital for numerous processes such as synthesis of carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins. Here, we discovered that a deletion mutant of manB (BG101) in Streptomyces coelicolor (S. coelicolor) showed higher sensitivity to bacteriostatic chloramphenicol (CM) than the wild-type strain (M145), along with decreased production of CM metabolites. Deletion of manB also decreased the mRNA expression level of drug efflux pumps (i.e., cmlR1 and cmlR2) in S. coelicolor, resulting in increased sensitivity to CM. This is the first report on changes in antibiotic sensitivity to CM by deletion of one glycolysis-related enzyme in S. coelicolor, and the results suggest different approaches for studying the antibiotic-resistant mechanism and its regulation.

NdgR, a common transcriptional activator for methionine and leucine biosynthesis in Streptomyces coelicolor.

We show here that NdgR, a known transcriptional activator of isopropylmalate dehydratase in actinomycetes, may have other targets in the cell. An in-frame deletion mutant of ndgR showed unexpectedly poor growth in defined minimal medium even in the presence of leucine. To our surprise, it was supplementation of cysteine and methionine that corrected the growth. Based on this, we propose that NdgR induces cysteine-methionine biosynthesis. Direct involvement of NdgR in the very last steps of methionine synthesis with methionine synthase (metH) and 5,10-methylenetetrahydrofolate reductase (metF) was examined. From a pulldown assay, it was seen that NdgR was enriched from crude cell lysates with a strong affinity to metH and metF upstream sequences. Direct physical interaction of NdgR with these targets was further examined with a gel mobility shift assay. ndgR, leuC, metH, and metF were inducible in M145 cells upon nutrient downshift from rich to minimal medium but were not induced in the ndgR knockout mutant. Taking these observations together, NdgR-dependent metH-metF expression would account for the abnormal growth phenotype of the ndgR mutant although there may be additional NdgR-dependent genes in the Cys-Met metabolic pathways. As the first transcriptional factor reported for regulating Cys-Met metabolism in Streptomyces, NdgR links two disparate amino acid families, branched-chain amino acids (BCAAs) and sulfur amino acids, at the transcriptional level. Considering that Cys-Met metabolism is connected to mycothiol and one-carbon metabolism, NdgR may have broad physiological impacts.

Bead affinity chromatography in a temperature-controllable microsystem for biomarker detection.

This paper describes a temperature-controllable bead affinity chromatography (BAC) in a microsystem for biomarker detection, and preparing samples for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Cancer marker proteins were captured in the microsystem by BAC with RNA aptamer-immobilized microbeads. The captured proteins were then denatured and released from the microbeads by controlling temperature. The microsystem consists of a microreactor for trapping microbeads and a temperature control unit for thermal treatment of the trapped beads. We used polymethylsilxoane or single crystalline silicon in fabricating two different types of reaction chamber to compare the differences in performance originated from the materials. Carcinoembryonic antigen was concentrated and purified from human serum using the microsystem and detected by MALDI-TOF MS to demonstrate the usefulness of the microsystem. The microsystem simplifies a sample preparation process required for protein analysis and cancer biomarker detection, which will accelerate the process of cancer research.

A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome.

Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3' region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor.

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor.

γ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.

A novel function of Streptomyces integration host factor (sIHF) in the control of antibiotic production and sporulation in Streptomyces coelicolor.

Bacterial integration host factors (IHFs) play important roles in site-specific recombination, DNA replication, transcription, genome organization and bacterial pathogenesis. In Streptomyces coelicolor, there are three putative IHFs: SCO1480, SCO2950 and SCO5556. SCO1480 or Streptomyces IHF (sIHF) was previously identified as a transcription factor that binds to the promoter region of redD, the pathway-specific regulatory gene for the undecylprodigiosin biosynthetic gene cluster. Here we show that production of the pigmented antibiotics actinorhodin and undecylprodigiosin is strongly enhanced in sihf null mutants, while sporulation was strongly inhibited, with an on average 25% increase in spore size. Furthermore, the sihf mutant spores showed strongly reduced viability, with high sensitivity to heat and live/dead staining revealing a high proportion of empty spores, while enhanced expression of sIHF increased viability. This suggests a major role for sIHF in controlling viability, perhaps via the control of DNA replication and/or segregation. Proteomic analysis of the sihf null mutant identified several differentially expressed transcriptional regulators, indicating that sIHF may have an extensive response regulon. These data surprisingly reveal that a basic architectural element conserved in many actinobacteria such as mycobacteria, corynebacteria, streptomycetes and rhodococci may act as a global regulator of secondary metabolism and cell development.

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor.

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor∆manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor∆manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K(cat)/K(m) (mM(-1) s(-1) = 0.41 for D-mannose-6-phosphate), but failed to show GDP-D-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor∆manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.

Rapid functional screening of Streptomyces coelicolor regulators by use of a pH indicator and application to the MarR-like regulator AbsC.

To elucidate the function of an unknown regulator in Streptomyces, differences in phenotype and antibiotic production between a deletion mutant and a wild-type strain (WT) were compared. These differences are easily hidden by complex media. To determine the specific nutrient conditions that reveal such differences, we used a multiwell method containing different nutrients along with bromothymol blue. We found several nutrients that provide key information on characterization conditions. By comparing the growth of wild-type and mutant strains on screened nutrients, we were able to measure growth, organic acid production, and antibiotic production for the elucidation of regulator function. As a result of this method, a member of the MarR-like regulator family, SCO5405 (AbsC), was newly characterized to control pyruvate dehydrogenase in Streptomyces coelicolor. Deletion of SCO5405 increased the pH of the culture broth due to decreased production of organic acids such as pyruvate and alpha-ketoglutarate and increased extracellular actinorhodin (ACT) production in minimal medium containing glucose and alanine (MMGA). This method could therefore be a high-throughput method for the characterization of unknown regulators.

Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal.

The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal.