Abstract
Identification of missing genes or proteins participating in the metabolic pathways as enzymes are of great interest. One such class of pathway is involved in the eugenol to vanillin bioconversion. Our goal is to develop an integral approach for identifying the topology of a reference or known pathway in other organism. We successfully identify the missing enzymes and then reconstruct the vanillin biosynthetic pathway in Aspergillus niger. The procedure combines enzyme sequence similarity searched through BLAST homology search and orthologs detection through COG & KEGG databases. Conservation of protein domains and motifs was searched through CDD, PFAM & PROSITE databases. Predictions regarding how proteins act in pathway were validated experimentally and also compared with reported data. The bioconversion of vanillin was screened on UV-TLC plates and later confirmed through GC and GC-MS techniques. We applied a procedure for identifying missing enzymes on the basis of conserved functional motifs and later reconstruct the metabolic pathway in target organism. Using the vanillin biosynthetic pathway of Pseudomonas fluorescens as a case study, we indicate how this approach can be used to reconstruct the reference pathway in A. niger and later results were experimentally validated through chromatography and spectroscopy techniques.
Highlights
The identification of metabolic pathway’s genes and proteins is an emerging area of great interest
Two types of datasets were used for pathway construction: (1) Known or Reference data set - which includes protein sequences of five enzymes such as Eugenol hydroxylase cytochrome C subunit (EhyA)/Eugenol hydroxylase flavoprotein subunit (EhyB), Coniferyl alcohol dehydrogenase (CalA), CalB, Feruloyl CoA synthase (Fcs) and Enoyl CoA hydratase/aldolase (Ech), participating in the vanillin biosynthetic pathway of model organism i.e., P. fluorescens with conserved protein domains & motifs, (2) Predictive or Target data set - which includes potential homologous protein sequences such as Cytochrome-c CYC_ASPNG or hypothetical protein, hypothetical protein
The eugenol to vanillin bioconversion pathway of P. fluorescens was reconstructed by using a flexible computational methodology that combines sequence analysis, literature search and bioinformatics applications
Summary
The identification of metabolic pathway’s genes and proteins is an emerging area of great interest. It is a challenge to identify the correct bioconversion pathway that allows for the creation of sequence synteny based pathway network whose mechanism can be analyzed and tested against experimental observations To achieve such a goal, strategies that combine the different theoretical and wet lab confirmations to identify proteins and generate a set of plausible pathway network for the process of interest are needed. Such a process integrates homologous data and provides testable predictions and information about unexplored pathway mechanism in other organisms. The establishment of the bioconversion pathway in fungal systems would lead to hypothesize the metabolic fate of eugenol in eukaryotic systems
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