Huarong Tan

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

SUMMARY Streptomycetes are the most abundant source of antibiotics. Typically, each species produces several antibiotics, with the profile being species specific. Streptomyces coelicolor, the model species, produces at least five different antibiotics. We review the regulation of antibiotic biosynthesis in S. coelicolor and other, nonmodel streptomycetes in the light of recent studies. The biosynthesis of each antibiotic is specified by a large gene cluster, usually including regulatory genes (cluster-situated regulators [CSRs]). These are the main point of connection with a plethora of generally conserved regulatory systems that monitor the organisms physiology, developmental state, population density, and environment to determine the onset and level of production of each antibiotic. Some CSRs may also be sensitive to the levels of different kinds of ligands, including products of the pathway itself, products of other antibiotic pathways in the same organism, and specialized regulatory small molecules such as gamma-butyrolactones. These interactions can result in self-reinforcing feed-forward circuitry and complex cross talk between pathways. The physiological signals and regulatory mechanisms may be of practical importance for the activation of the many cryptic secondary metabolic gene cluster pathways revealed by recent sequencing of numerous Streptomyces genomes.

Autoregulation of antibiotic biosynthesis by binding of the end product to an atypical response regulator

In bacteria, many “atypical” response regulators (ARRs) lack the conserved residues important for phosphorylation by which typical response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. However, such mechanisms have not been established. JadR1, an OmpR-type ARR of Streptomyces venezuelae, appears to activate the transcription of jadomycin B (JdB) biosynthetic genes while repressing its own gene. JadR1 activities were inhibited in cells induced to produce JdB, which was found to bind directly to the N-terminal receiver domain of JadR1, causing JadR1 to dissociate from target promoters. The activity of a NarL-type ARR, RedZ, that regulates production of another antibiotic was likewise modulated by the end product (undecylprodigisines), implying that end-product-mediated control of antibiotic pathway-specific ARRs may be widespread. These results could prove relevant to knowledge-based improvements in yield of commercially important antibiotics.

A pathway‐specific transcriptional regulatory gene for nikkomycin biosynthesis in Streptomyces ansochromogenes that also influences colony development

DNA sequence analysis of a 7.5 kb XhoI DNA fragment from the region flanking the nikkomycin biosynthesis gene cluster in Streptomyces ansochromogenes revealed one 3.3 kb open reading frame (ORF), designated sanG. The deduced product of sanG (1061 amino acids), which is similar to PimR of Streptomyces natalensis, contains an OmpR‐like DNA binding domain in its N‐terminal portion and A‐ and B‐type nucleotide binding motifs in the middle of the protein. Disruption of sanG abolished nikkomycin biosynthesis, reduced sporulation and led to brown pigment accumulation. All aspects of this complex phenotype were complemented by a single copy sanG which was integrated into the chromosome. The introduction of multiple copies of sanG resulted in increased nikkomycin production. S1 mapping results indicated that sanG is transcribed from at least three promoters (P1, P2 and P3), P1 being strongly upregulated when production of nikkomycins starts. Two putative transcription units for nikkomycin biosynthesis, starting from sanN and sanO, are dependent on the expression of sanG, whereas a putative transcription unit starting from sanF was not regulated by sanG. These results suggested that sanG encodes a transcriptional activator important for nikkomycin biosynthesis that, unusually, also has pleiotropic effects on secondary metabolism and development.

“Pseudo” γ-Butyrolactone Receptors Respond to Antibiotic Signals to Coordinate Antibiotic Biosynthesis

In actinomycetes, the onset of secondary metabolite biosynthesis is often triggered by the quorum-sensing signal γ-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated “pseudo” GBL receptor. Intriguingly, it could bind the endogenous antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic antibiotic jadomycin. It is shown to coordinate their biosynthesis via direct repression of JadR1, which activates jadomycin biosynthesis while repressing chloramphenicol biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate antibiotics, and the interactions lead to the derepression of jadR1. The antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate antibiotic biosynthesis by binding and responding to antibiotics signals. Such an antibiotic-mediated regulatory mechanism could be a general strategy to coordinate antibiotic biosynthesis in the producing bacteria.

Characterization of the Polyoxin Biosynthetic Gene Cluster from Streptomyces cacaoi and Engineered Production of Polyoxin H

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics.

The pleiotropic regulator AdpA-L directly controls the pathway-specific activator of nikkomycin biosynthesis in Streptomyces ansochromogenes

The nikkomycin‐producing strain Streptomyces ansochromogenes has a homologue (adpA‐L) of the key pleiotropic Streptomyces regulatory gene adpA. Gene disruption and genetic complementation revealed that adpA‐L was required for both nikkomycin biosynthesis and morphological differentiation. Transcriptional analysis suggested that the transcription of sanG, the specific activator gene for nikkomycin biosynthesis, was dependent on AdpA‐L. In gel‐shift and DNase 1 footprinting assays, the purified His6‐tagged recombinant AdpA‐L protein bound the upstream region of sanG at five sites, which are spread over more than one kilobase of DNA and most of which is inside the transcribed region. A consensus AdpA‐L‐binding sequence, 5′‐TGGCNNVWHN‐3′ (V: C, A or G; W: A or T; H: A, T or C; N: any nucleotide) was found in these binding sites. Transcriptional analysis of sanG carrying mutated AdpA‐L binding sites showed that transcription of sanG was eliminated when site I was mutated and its trascription was decreased when site V was mutated, whereas it was increased when the binding sites II, III or IV were mutated. Meanwhile, nikkomycin production of the mutated site III strain was enhanced comparing with the wild‐type strain as control. This work highlights a new level of complexity in the regulation of nikkomycin biosynthesis.

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.

Differential regulation of antibiotic biosynthesis by DraR‐K, a novel two‐component system in Streptomyces coelicolor

A novel two‐component system (TCS) designated as DraR‐K (sco3063/sco3062) was identified to be involved in differential regulation of antibiotic biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow‐pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR‐K activates ACT but represses yCPK production directly through the pathway‐specific activator genes actII‐ORF4 and kasO, respectively, while its role on RED biosynthesis was independent of pathway‐specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at −124 to −98 nt and −24 to −1 nt relative to the respective transcription start point of actII‐ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR‐binding sequence, 5′‐AMAAWYMAKCA‐3′ (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel‐retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR‐K in primary metabolism. DraR‐K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR‐Ksav (sav_3481/sav_3480, homologue of draR‐K) in the industrial model strain S. avermitilis NRRL‐8165 led to similar abnormal antibiotic biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR‐K‐mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of antibiotic biosynthesis in Streptomyces.

Nucleoside antibiotics: biosynthesis, regulation, and biotechnology

The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics.

PolY, a transcriptional regulator with ATPase activity, directly activates transcription of polR in polyoxin biosynthesis in Streptomyces cacaoi.

polY, a transcriptional regulatory gene in the polyoxin biosynthetic cluster of Streptomyces cacaoi, was analysed, and its deduced product (PolY) showed amino acid sequence homology to AfsR from Streptomyces coelicolor A3(2). PolY contains an OmpR‐like DNA binding domain at its N‐terminal and an ATPase domain in the middle of the protein. Disruption of polY abolished polyoxin biosynthesis, which could be restored by the integration of a single copy of polY into the chromosome of the disruption mutant. Transcription of polR, a pathway‐specific regulatory gene of polyoxin biosynthesis, was controlled by polY. Electrophoretic mobility shift assay and DNase I protection experiments indicated that PolY bound to the promoter region of polR, and the binding site contained a direct nucleotide repeat typical of Streptomyces antibiotic regulatory protein binding sites. PolY exhibited ATPase activity in vitro. Additionally, binding of ADP/ATPγS to ATPase domain triggered the oligomerization of PolY and enhanced its DNA binding activity. Consistently, further experiments in vivo demonstrated that changes of ADP/ATP concentrations significantly affected PolY activity in the cell. These results suggested that the ATPase domain might be a sensor of endogenous pool of ADP/ATP, whose change modulated PolY activity under the physiological conditions.