Weishan Wang

Angucyclines as signals modulate the behaviors of Streptomyces coelicolor

Significance This work addresses the molecular basis for interspecies signaling effects of antibiotics, which have been a controversial but potentially significant emerging topic over the last few years. The “pseudo” gamma-butyrolactone (GBL) receptor (i.e., those GBL receptor homologues often found in Streptomyces genomes, but apparently not binding or responding to GBLs), ScbR2, was identified as the receptor of JdB. It has an extraordinary ability to bind and respond to exogenous angucyclines, as well as to be able to directly regulate the biosynthesis of different endogenous antibiotics and the morphological development of Streptomyces. Our findings significantly extend understanding of antibiotic-mediated signaling mechanisms and the ecological impact of antibiotics. The angucycline antibiotic jadomycin B (JdB) produced by Streptomyces venezuelae has been found here to induce complex survival responses in Streptomyces coelicolor at subinhibitory concentration. The receptor for JdB was identified as a “pseudo” gamma-butyrolactone receptor, ScbR2, which was shown to bind two previously unidentified target promoters, those of redD (redDp) and adpA (adpAp), thus directly regulating undecylprodigiosin (Red) production and morphological differentiation, respectively. Because AdpA also directly regulates the expression of redD, ScbR2, AdpA, and RedD together form a feed-forward loop controlling both differentiation and Red production phenotypes. Different signal strengths (i.e., JdB concentrations) were shown to induce the two different phenotypes by modulating the relative transcription levels of adpA vs. redD. The induction of morphological differentiation and endogenous antibiotic production by exogenous antibiotic exemplifies an important survival strategy more sophisticated than the induction of antibiotic resistance.

An engineered strong promoter for streptomycetes.

Well-characterized promoters are essential tools for metabolic engineering and synthetic biology. In Streptomyces coelicolor, the native kasOp is a temporally expressed promoter strictly controlled by two regulators, ScbR and ScbR2. In this work, first, kasOp was engineered to remove a common binding site of ScbR and ScbR2 upstream of its core region, thus generating a stronger promoter, kasOp3. Second, another ScbR binding site internal to the kasOp3 core promoter region was abolished by random mutation and screening of the mutant library to obtain the strongest promoter, kasOp* (where the asterisk is used to distinguish the engineered promoter from the native promoter). The activities of kasOp* were compared with those of two known strong promoters, ermEp* and SF14p, in three Streptomyces species. kasOp* showed the highest activity at the transcription and protein levels in all three hosts. Furthermore, relative to ermEp* and SF14p, kasOp* was shown to confer the highest actinorhodin production level when used to drive the expression of actII-ORF4 in S. coelicolor. Therefore, kasOp* is a simple and well-defined strong promoter useful for gene overexpression in streptomycetes.

A novel role of ‘pseudo’γ‐butyrolactone receptors in controlling γ‐butyrolactone biosynthesis in Streptomyces

In streptomycetes, a quorum‐sensing mechanism mediated by γ‐butyrolactones (GBLs) and their cognate receptors was known to trigger secondary metabolism and morphological differentiation. However, many aspects on the control of GBL signal production are not understood. In this work, we report that ScbR2, the pseudo GBL receptor in Streptomyces coelicolor, negatively controls the biosynthesis of γ‐butyrolactone (SCB1) by directly repressing the transcription of scbA, which encodes the key enzyme for SCB1 biosynthesis. Similarly, the pseudo GBL receptor JadR2 in Streptomyces venezuelae was shown to repress the expression of jadW1, which also encodes the putative GBL synthase. These regulatory relationships were verified in Escherichia coli using lux‐based reporter constructs. Additionally, the temporal expression profiles of scbA, scbR2 and scbR (receptor gene for SCB1) were examined in Streptomyces coelicolor, which showed the sequential expression of ScbR/R2 regulators in the control of SCB1 production. Overall, our results clearly demonstrated that pseudo GBL receptors play a novel role in controlling GBL biosynthesis in streptomycetes. As ScbR/R2 homologues and their binding sites upstream of GBL synthase genes are commonly found in Streptomyces species, and ScbR2 homologues cross‐recognize each others target promoters, the ScbA/R/R2 quorum‐sensing regulatory system appears to represent an evolutionarily conserved signal control mechanism.

Identification of JadG as the B ring opening oxygenase catalyzing the oxidative C-C bond cleavage reaction in jadomycin biosynthesis.

Jadomycin B is a member of atypical angucycline antibiotics whose biosynthesis involves a unique ring opening C-C bond cleavage reaction. Here, we firmly identified JadG as the enzyme responsible for the B ring opening reaction in jadomycin biosynthesis. In vitro analysis of the JadG catalyzed reaction revealed that it requires FMNH(2) or FADH(2) as cofactors in the conversion of dehydrorabelomycin to jadomycin A. The cofactors could be supplied by either a cluster-situated flavin reductase JadY or the Escherichia coli Fre. JadY was characterized as a NAD(P)H-dependent FMN/FAD reductase, with FMN as the preferred substrate. Disruption mutant of jadY still produced jadomycin, indicating that the function of JadY could be substituted by other enzymes in the host. JadG represents the biochemically verified member of an enzyme class catalyzing an unprecedented C-C bond cleavage reaction.

Genome-wide identification and evaluation of constitutive promoters in streptomycetes

BackgroundStreptomycetes attract a lot of attention in metabolic engineering and synthetic biology because of their well-known ability to produce secondary metabolites. However, the available constitutive promoters are rather limited in this genus.ResultsIn this work, constitutive promoters were selected from a pool of promoters whose downstream genes maintained constant expression profiles in various conditions. A total of 941 qualified genes were selected based on systematic analysis of five sets of time-series transcriptome microarray data of Streptomyces coelicolor M145 cultivated under different conditions. Then, 166 putative constitutive promoters were selected by following a rational selection workflow containing disturbance analysis, function analysis, genetic loci analysis, and transcript abundance analysis. Further, eight promoters with different strengths were chosen and subjected to experimental validation by green fluorescent protein reporter and real-time reverse-transcription quantitative polymerase chain reaction in S. coelicolor, Streptomyces venezuelae and Streptomyces albus. The eight promoters drove the stable expression of downstream genes in different conditions, implying that the 166 promoters that we identified might be constitutive under the genus Streptomyces. Four promoters were used in a plug-and-play platform to control the expression of the cryptic cluster of jadomycin B in S. venezuelae ISP5230 and resulted in different levels of the production of jadomycin B that corresponded to promoter strength.ConclusionsThis work identified and evaluated a set of constitutive promoters with different strengths in streptomycetes, and it enriched the presently available promoter toolkit in this genus. These promoters should be valuable in current platforms of metabolic engineering and synthetic biology for the activation of cryptic biosynthetic clusters and the optimization of pathways for the biosynthesis of important natural products in Streptomyces species.

Development of a Synthetic Oxytetracycline-Inducible Expression System for Streptomycetes Using de Novo Characterized Genetic Parts

Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (KD = 1.7 × 10(-10) M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01-4 μM of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchmarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Potr* presents a new versatile tool for fine-tuning gene expression in streptomycetes.

ScbR- and ScbR2-mediated signal transduction networks coordinate complex physiological responses in Streptomyces coelicolor.

In model organism Streptomyces coelicolor, γ-butyrolactones (GBLs) and antibiotics were recognized as signalling molecules playing fundamental roles in intra- and interspecies communications. To dissect the GBL and antibiotic signalling networks systematically, the in vivo targets of their respective receptors ScbR and ScbR2 were identified on a genome scale by ChIP-seq. These identified targets encompass many that are known to play important roles in diverse cellular processes (e.g. gap1, pyk2, afsK, nagE2, cdaR, cprA, cprB, absA1, actII-orf4, redZ, atrA, rpsL and sigR), and they formed regulatory cascades, sub-networks and feedforward loops to elaborately control key metabolite processes, including primary and secondary metabolism, morphological differentiation and stress response. Moreover, interplay among ScbR, ScbR2 and other regulators revealed intricate cross talks between signalling pathways triggered by GBLs, antibiotics, nutrient availability and stress. Our work provides a global view on the specific responses that could be triggered by GBL and antibiotic signals in S. coelicolor, among which the main echo was the change of production profile of endogenous antibiotics and antibiotic signals manifested a role to enhance bacterial stress tolerance as well, shedding new light on GBL and antibiotic signalling networks widespread among streptomycetes.

Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus

BackgroundOxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus. Despite its importance, little is known about the regulation of OTC biosynthesis, which hampered any effort to improve OTC production via engineering regulatory genes.ResultsA gene encoding a Streptomyces antibiotic regulatory protein (SARP) was discovered immediately adjacent to the otrB gene of oxy cluster in S. rimosus and designated otcR. Deletion and complementation of otcR abolished or restored OTC production, respectively, indicating that otcR encodes an essential activator of OTC biosynthesis. Then, the predicted consensus SARP-binding sequences were extracted from the promoter regions of oxy cluster. Transcriptional analysis in a heterologous GFP reporter system demonstrated that OtcR directly activated the transcription of five oxy promoters in E. coli, further mutational analysis of a SARP-binding sequence of oxyI promoter proved that OtcR directly interacted with the consensus repeats. Therefore, otcR was chosen as an engineering target, OTC production was significantly increased by overexpression of otcR as tandem copies each under the control of strong SF14 promoter.ConclusionsA SARP activator, OtcR, was identified in oxy cluster of S. rimosus; it was shown to directly activate five promoters from oxy cluster. Overexpression of otcR at an appropriate level dramatically increased OTC production by 6.49 times compared to the parental strain, thus demonstrating the great potential of manipulating OtcR to improve the yield of OTC production.

A platform for the development of novel biosensors by configuring allosteric transcription factor recognition with amplified luminescent proximity homogeneous assays

A wide range of chemicals can be sensed by allosteric transcription factors (aTFs) in bacteria. Herein, we report a biosensing platform by using isolated aTFs as recognition elements in vitro. Moreover, a general strategy to increase the sensitivity of the aTF-based biosensors is provided. As a proof-of-concept, we obtained by far the most sensitive uric acid and oxytetracycline biosensors by using aTF HucR and OtrR as recognition elements, respectively. As a large number of aTFs are present in bacteria, our work opens a novel route to develop sensitive aTF-based biosensors.

A genetic biosensor for identification of transcriptional repressors of target promoters

Transcriptional repressors provide widespread biological significance in the regulation of gene expression. However, in prokaryotes, it is particularly difficult to find transcriptional repressors that recognize specific target promoters on genome-scale. To address this need, a genetic biosensor for identifying repressors of target promoters was developed in Escherichia coli from a de novo designed genetic circuit. This circuit can convert the negative input of repressors into positive output of reporters, thereby facilitating the selection and identification of repressors. After evaluating the sensitivity and bias, the biosensor was used to identify the repressors of scbA and aco promoters (PscbA and Paco), which control the transcription of signalling molecule synthase genes in Streptomyces coelicolor and Streptomyces avermitilis, respectively. Two previously unknown repressors of PscbA were identified from a library of TetR family regulators in S. coelicolor, and three novel repressors of Paco were identified from a genomic library of S. avermitilis. Further in vivo and in vitro experiments confirmed that these newly identified repressors attenuated the transcription of their target promoters by direct binding. Overall, the genetic biosensor developed here presents an innovative and powerful strategy that could be applied for identifying genome-wide unknown repressors of promoters in bacteria.