Dataset Information

A family AA5_2 carbohydrate oxidase from Penicillium rubens displays functional overlap across the AA5 family.

ABSTRACT: Copper radical alcohol oxidases belonging to auxiliary activity family 5, subfamily 2 (AA5_2) catalyze the oxidation of galactose and galactosides, as well as aliphatic alcohols. Despite their broad applied potential, so far very few AA5_2 members have been biochemically characterized. We report the recombinant production and biochemical characterization of an AA5_2 oxidase from Penicillium rubens Wisconsin 54-1255 (PruAA5_2A), which groups within an unmapped clade phylogenetically distant from those comprising AA5_2 members characterized to date. PruAA5_2 preferentially oxidized raffinose over galactose; however, its catalytic efficiency was 6.5 times higher on glycolaldehyde dimer compared to raffinose. Deep sequence analysis of characterized AA5_2 members highlighted amino acid pairs correlated to substrate range and conserved within the family. Moreover, PruAA5_2 activity spans substrate preferences previously reported for AA5 subfamily 1 and 2 members, identifying possible functional overlap across the AA5 family.

SUBMITTER: Mollerup F

PROVIDER: S-EPMC6519835 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A family AA5_2 carbohydrate oxidase from Penicillium rubens displays functional overlap across the AA5 family.

Mollerup Filip F Aumala Ville V Parikka Kirsti K Mathieu Yann Y Brumer Harry H Tenkanen Maija M Master Emma E

PloS one 20190515 5

Copper radical alcohol oxidases belonging to auxiliary activity family 5, subfamily 2 (AA5_2) catalyze the oxidation of galactose and galactosides, as well as aliphatic alcohols. Despite their broad applied potential, so far very few AA5_2 members have been biochemically characterized. We report the recombinant production and biochemical characterization of an AA5_2 oxidase from Penicillium rubens Wisconsin 54-1255 (PruAA5_2A), which groups within an unmapped clade phylogenetically distant from ...[more]

PMID: 31091286

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Project description:In recent years, genome sequencing of filamentous fungi has revealed a high proportion of specialised metabolites with growing pharmaceutical interest. However, detecting such metabolites through in silico genome analysis does not necessarily guarantee their expression under laboratory conditions. However, one plausible strategy for enabling their production lies in modifying the growth conditions. Devising a comprehensive experimental design testing in different culture environments is time-consuming and expensive. Therefore, using in silico modelling as a preliminary step, such as Genome-Scale Metabolic Network (GSMN), represents a promising approach to predicting and understanding the observed specialised metabolite production in a given organism. To address these questions, we reconstructed a new high-quality GSMN for the Penicillium rubens Wisconsin 54-1255 strain, a commonly used model organism. Our reconstruction, iPrub22, adheres to current convention standards and quality criteria, incorporating updated functional annotations, orthology searches with different GSMN templates, data from previous reconstructions, and manual curation steps targeting primary and specialised metabolites. With a MEMOTE score of 74% and a metabolic coverage of 45%, iPrub22 includes 5,192 unique metabolites interconnected by 5,919 reactions, of which 5,033 are supported by at least one genomic sequence. Of the metabolites present in iPrub22, 13% are categorised as belonging to specialised metabolism. While our high-quality GSMN provides a valuable resource for investigating known phenotypes expressed in P. rubens, our analysis identifies bottlenecks related, in particular, to the definition of what is a specialised metabolite, which requires consensus within the scientific community. It also points out the necessity of accessible, standardised and exhaustive databases of specialised metabolites. These questions must be addressed to fully unlock the potential of natural product production in P. rubens and other filamentous fungi. Our work represents a foundational step towards the objective of rationalising the production of natural products through GSMN modelling.

Project description:To produce high levels of ?-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for ?-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production.IMPORTANCE Penicillium rubens is an important industrial producer of ?-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

Dataset Information

A family AA5_2 carbohydrate oxidase from Penicillium rubens displays functional overlap across the AA5 family.

Publications

A family AA5_2 carbohydrate oxidase from Penicillium rubens displays functional overlap across the AA5 family.

Similar Datasets

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