Javier Santos-AberturasJohn Innes Centre · Department of Molecular Microbiology
Javier Santos-Aberturas
PhD Molecular Biology and Microbiology
About
59
Publications
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1,115
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Introduction
My research is focused on the discovery of new specialized metabolites from Bacteria by bioactivity screening and genome mining, as well as on the elucidation of the biosynthetic pathways leading to the production of structurally unusual compounds, lately with a particular emphasis towards RiPPs. Originally trained as a Molecular Microbiologist, through my career I have acquired an important array of skills in analytical chemistry, biochemistry and bioinformatics.
Additional affiliations
March 2015 - present
November 2012 - February 2015
April 2011 - August 2012
Publications
Publications (59)
Stress memory is a phenomenon whereby exposure to initial stress event influences a response to subsequent stress exposures. Studying stress memory is important to understand the cellular behavior in dynamic environment, especially nowadays, in times with growing environmental instability. Stress memory has been characterized in vascular plants but...
Bacterial secondary metabolites represent an invaluable source of bioactive molecules for the pharmaceutical and agrochemical industries. Although screening campaigns for the discovery of new compounds have traditionally been strongly biased towards the study of soil-dwelling Actinobacteria, the current antibiotic resistance and discovery crisis ha...
Stress memory is a phenomenon whereby exposure to initial stress event influences a response to subsequent stress exposures. Studying stress memory is important to understand the cellular behaviour in dynamic environment, especially nowadays, in times with growing environmental instability. Stress memory has been characterized in vascular plants bu...
In recent years, genome mining has become a powerful strategy for the discovery of new specialized metabolites from microorganisms. However, the discovery of new groups of ribosomally synthesized and post-translationally modified peptides (RiPPs) by employing the currently available genome mining tools has proven challenging due to their inherent b...
d-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of l-amino acids into their d-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a d-aspartate (d-...
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
The rational discovery of new specialized metabo-lites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters ar...
Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces st...
The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are...
The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription...
The different expression level and solubility showed by each protein variant represents an important challenge during screening campaigns: Usually, the total activity measurement constitutes the only criterion for identifying improved variants. This hampers the chances of finding interesting mutants, especially if the aim is to improve activity: On...
Metalloproteinase inhibitors often feature hydroxamate moieties to facilitate the chelation of metal ions in the catalytic center of target enzymes. Actinonin and matlystatins are potent metalloproteinase inhibitors that comprise rare N-hydroxy-2-pentyl-succinamic acid warheads. Here we report the identification and characterization of their biosyn...
The YcaO superfamily of proteins catalyzes the phosphorylation of peptide backbone amide bonds, which leads to the formation of azolines and azoles in ribosomally synthesized and post-translationally modified peptides (RiPPs). Bottromycins are RiPPs with potent antimicrobial activity, and their biosynthetic pathway contains two divergent, stand-alo...
Bottromycin A2 is a structurally unique ribosomally synthesized and post-translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrug-resistant bacteria. The structural novelty of bottromycin stems from its unprecedented macrocyclic amidine and rare β-methylated amino acid residues. The N-terminus of a prec...
A fully automated robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiterplate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and mul...
Pimaricin (natamycin) is a small polyene macrolide antibiotic used worldwide. This efficient antimycotic and antiprotozoal agent, produced by several soil bacterial species of the genus Streptomyces, has found application in human therapy, in the food and beverage industries and as pesticide. It displays a broad spectrum of activity, targeting ergo...
Here, we report a widely and generally applicable strategy to obtain reliable information in high-throughput protein screenings of enzyme mutant libraries. The method is based on the usage of the split-GFP technology for the normalization of the expression level of each individual protein variant combined with activity measurements, thus resolving...
Streptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of non-glycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in biological membranes and as a tool for the diagnosis of Niemann-Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have prove...
PAS-LuxR regulators are highly conserved proteins devoted to the control of antifungal production by binding to operators located in given promoters of polyene biosynthetic genes. The canonical operator of PimM, archetype of this class of regulators, has been used here to search for putative targets of orthologous protein PteF in the genome of Stre...
Even though there is an urgent need for new antifungals with improved clinical properties, the substrate promiscuity of tailoring enzymes has been poorly studied as a source of new structural diversity for polyene macrolides. We explore the acceptance of different polyene macrolides by the glutamine amidotransferase PscA and the catalytic effect of...
The DNA region encoding the filipin gene cluster in Streptomyces avermitilis (pte) contains a PAS-LuxR regulatory gene, pteF, orthologue to pimM, the final pathway-specific positive regulatory protein of pimaricin biosynthesis in Streptomyces natalensis. Gene replacement of the gene from S. avermitilis chromosome resulted in a severe loss of filipi...
Background
FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influenc...
Primers used for RT-PCR analysis (This file presents a list of primers and their corresponding sequences, that have been used for RT-PCR experiments).
Table containing primers for PCR amplifications of the target putative regulatory genes (The file presents primers and their corresponding sequences, that have been used for PCR amplification of whole genes or homologous regions and promoter regions).
Schematic representation of FkbR and FkbN protein domains and deleted regions (This file illustrates FkbR and FkbN proteins and their organization before and after inactivation).
The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely
by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacr...
Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the Streptomyces antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding r...
Purification of GST-fusion protein in E.coli BL21. Purification of GST-PimRSARP by affinity chromatography on Glutathione Sepharose. Lane T, total E. coli cell extract; lane P, purified proteins after affinity chromatography. Left lane, molecular size markers (in kDa).
(TIF)
Primer efficiency. The efficiency of each set of primers was calculated according to the equation E = 10[−1/slope]−1. Using 5-fold dilutions of genomic DNA, the resulting Ct values were plotted against the logarithm of the DNA quantity as shown in A (primers for pimM), B (primers for pimR) and C (primers for rrnA1). Data are from three replicates a...
LAL regulators (Large ATP-binding regulators of the LuxR family) constitute a poorly studied family of transcriptional regulators. Several regulators of this class have been identified in antibiotic and other secondary metabolite gene clusters from actinomycetes, thus they have been considered pathway-specific regulators. In this study we have obta...
Differentially expressed genes showing increased transcript levels in the LAL mutants when compared to the parental strain.
Mc and p-values for the contrasts between the indicated conditions (A: S. coelicolor A3(2) M145. B: S. coelicolor Δ0877. and C: S. coelicolor Δ7173 strains). Among the 322 genes with statistically significant results the table...
Determination of the quality flag for array spots. The Feature Extraction software quantifies spot fluorescence and provides a set of Boolean values to assess the quality of the results. These quality indicators evaluate both red and green channel data of each spot (indicators are listed in the first row). Based on previous observations of the sign...
Complete list of
Mc
and
p-
values for all genes passing the statistical tests are provided in an Excel spreadsheet.
(XLS)
Validation of microarray results using qRT-PCR. Correlation between qRT-PCR and microarray results for 11 different genes (see text). Samples for S. coelicolor Δ0877 are shown by triangles while those for S. coelicolor Δ7173 are indicated by squares. A least square straight line fit is also shown.
(TIF)
Assigned weights to each spot flags. To obtain spot quality weights that could be entered into the data analysis, we followed the idea behind the array weight estimation of Ritchie et al. [51]. In our case, the data of the 15 array hybridizations (3 conditions×5 biological replicates) were normalized, the array weights calculated, and a linear mode...
Sequence of primers employed for RT-PCR.
(DOC)
Differentially expressed genes showing decreased transcript levels in the LAL mutants when compared to the parental strain.
Mc and p-values for the contrasts between the indicated conditions (A: S. coelicolor A3(2) M145. B: S. coelicolor Δ0877. and C: S. coelicolor Δ7173 strains). The p-values BvA and CvA are indicated in bold type when found stati...
Sequence of primers used for qPCR.
(DOC)
Control of polyene macrolide production in Streptomyces natalensis is mediated by the PAS-LuxR transcriptional activator PimM. Expression of target genes in this strain is positively regulated by binding of the regulator to 14-nucleotide sites showing dyad symmetry, and overlapping the -35 element of each promoter. These sequences have been found i...
Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM. This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. PimM, truncated forms of the protein without the PAS domain (PimM(ΔPAS))...
We present the X-ray structure of PimD, both substrate-free and in complex with 4,5-desepoxypimaricin. PimD is a cytochrome P450 monooxygenase with native epoxidase activity that is critical in the biosynthesis of the polyene macrolide antibiotic pimaricin. Intervention in this secondary metabolic pathway could advance the development of drugs with...
Polyenes represent a major class of antifungal agents characterised by the presence of a series of conjugated double bonds in their planar hydroxylated macrolide ring structure. Despite their general interest, very little is known about the factors that modulate their biosynthesis. Among these factors, we have recently discovered a new inducing com...
Sequencing of the DNA region on the left fringe of the pimaricin gene cluster revealed the presence of a 579 bp gene, pimM, whose deduced product (192 aa) was found to have amino acid sequence homology with bacterial regulatory proteins. Database comparisons revealed that PimM combines an N-terminal PAS domain with a C-terminal helix-turn-helix (HT...
The gene cluster responsible for pimaricin biosynthesis in Streptomyces natalensis contains a cholesterol oxidase-encoding gene (pimE) surrounded by genes involved in pimaricin production. Gene-inactivation and -complementation experiments revealed that pimE encodes a functional cholesterol oxidase and, surprisingly, that it is also involved in pim...
Questions
Questions (2)
I am looking for DNA binding sites for transcrptional regulators in bacterial promoters.
I have been trying to perform Marfey assys lately for the determination of the stereochemistry in peptides, but starting from pure standards and samples I do not get repetitive results and background is generally higher than one would expect. Any tricks about the HCl hydrolisis, the glassware or the derivatization steps? It seems quite trivial when described in papers and protocols, but I am not a chemist and something is obviously going wrong.Thanks a lot!
