Expanding salivary biomarker detection by creating a synthetic neuraminic acid sensor via chimeragenesis.

Accurate and timely diagnosis of oral squamous cell carcinoma (OSCC) is crucial in preventing its progression to advanced stages with a poor prognosis. As such, the construction of sensors capable of detecting previously established disease biomarkers for the early and non-invasive diagnosis of this and many other conditions has enormous therapeutic potential. In this work, we apply synthetic biology techniques for the development of a whole-cell biosensor (WCB) that leverages the physiology of engineered bacteria in vivo to promote the expression of an observable effector upon detection of a soluble molecule. To this end, we have constructed a bacterial strain expressing a novel chimeric transcription factor (Sphnx) for the detection of N-acetylneuraminic acid (Neu5Ac), a salivary biomolecule correlated with the onset of OSCC. This WCB serves as the proof-of-concept of a platform that can eventually be applied to clinical screening panels for a multitude of oral and systemic medical conditions whose biomarkers are present in saliva.

Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators.

The ability of bacteria to sense specific molecules within their environment and trigger metabolic responses in accordance is an invaluable biotechnological resource. While many transcription factors (TFs) mediating such processes have been studied, only a handful have been leveraged for molecular biology applications. To expand the repertoire of biotechnologically relevant sensors we present a strategy for the construction and testing of chimeric TF libraries, based on the fusion of highly soluble periplasmic binding proteins (PBPs) with DNA-binding domains (DBDs). We validate this concept by constructing and functionally testing two unique sense-and-respond regulators for benzoate, an environmentally and industrially relevant metabolite. This work will enable the development of tailored biosensors for novel synthetic regulatory circuits.

Unraveling the specific regulation of the central pathway for anaerobic degradation of 3-methylbenzoate.

The mbd cluster encodes the anaerobic degradation of 3-methylbenzoate in the ß-proteobacterium Azoarcus sp. CIB. The specific transcriptional regulation circuit that controls the expression of the mbd genes was investigated. The PO, PB 1, and P3 R promoters responsible for the expression of the mbd genes, their cognate MbdR transcriptional repressor, as well as the MbdR operator regions (ATACN10GTAT) have been characterized. The three-dimensional structure of MbdR has been solved revealing a conformation similar to that of other TetR family transcriptional regulators. The first intermediate of the catabolic pathway, i.e. 3-methylbenzoyl-CoA, was shown to act as the inducer molecule. An additional MbdR-dependent promoter, PA, which contributes to the expression of the CoA ligase that activates 3-methylbenzoate to 3-methylbenzoyl-CoA, was shown to be necessary for an efficient induction of the mbd genes. Our results suggest that the mbd cluster recruited a regulatory system based on the MbdR regulator and its target promoters to evolve a distinct central catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as the central metabolite. All these results highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.

Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.

The mbd cluster encoding genes of the 3-methylbenzoyl-CoA pathway involved in the anaerobic catabolism of 3-methylbenzoate and m-xylene was characterized for the first time in the denitrifying ß-Proteobacterium Azoarcus sp. CIB. The mbdA gene product was identified as a 3-methylbenzoate-CoA ligase required for 3-methylbenzoate activation; its substrate spectrum was unique in activating all three methylbenzoate isomers. An inducible 3-methylbenzoyl-CoA reductase (mbdONQP gene products), displaying significant amino acid sequence similarities to known class I benzoyl-CoA reductases catalysed the ATP-dependent reduction of 3-methylbenzoyl-CoA to a methyldienoyl-CoA. The mbdW gene encodes a methyldienoyl-CoA hydratase that hydrated the methyldienoyl-CoA to a methyl-6-hydroxymonoenoyl-CoA compound. The mbd cluster also contains the genes predicted to be involved in the subsequent steps of the 3-methylbenzoyl-CoA pathway as well as the electron donor system for the reductase activity. Whereas the catabolic mbd genes are organized in two divergent inducible operons, the putative mbdR regulatory gene was transcribed separately and showed constitutive expression. The efficient expression of the mbd genes required the oxygen-dependent AcpR activator, and it was subject of carbon catabolite repression by some organic acids and amino acids. Sequence analyses suggest that the mbd gene cluster was recruited by Azoarcus sp. CIB through horizontal gene transfer.

A finely tuned regulatory circuit of the nicotinic acid degradation pathway in Pseudomonas putida.

The biochemistry of nicotinic acid (NA) degradation is known but the transcriptional control of the genes involved is still poorly studied. We report here the transcriptional regulatory circuit of the nic genes responsible for the aerobic degradation of NA in Pseudomonas putida KT2440. The three NA-inducible catabolic operons, i.e. nicAB, encoding the upper pathway that converts NA into 6-hydroxynicotinic acid (6HNA), and the nicCDEFTP and nicXR operons, responsible for channelling 6HNA to the central metabolism, are driven by the Pa, Pc and Px promoters respectively. The nicR regulatory gene encodes a MarR-like protein that represses the activity of the divergent Pc and Px promoters being 6HNA the inducer molecule. A new gene, nicS, that is associated to the nicAB genes in the genomes of different γ- and ß-Proteobacteria, encodes a TetR-like regulator that represses the activity of Pa in the absence of the NA/6HNA inducers. The NA regulatory circuit in P. putida has evolved an additional repression loop based on the NicR-dependent cross regulation of the nicS gene, thus assuring a tight transcriptional control of the catabolic genes that may prevent depletion of this vitamin B3 when needed for the synthesis of essential cofactors.

Identification of the Geobacter metallireducens bamVW two-component system, involved in transcriptional regulation of aromatic degradation.

Regulation of aromatic degradation in obligate anaerobes was studied in the Fe(III)-respiring model organism Geobacter metallireducens GS-15. A two-component system and a sigma54-dependent promoter were identified that are both involved in the regulation of the gene coding for benzoate-coenzyme A ligase, catalyzing the initial step of benzoate degradation.

Anaerobic catabolism of aromatic compounds: a genetic and genomic view.

Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.