Synthesis of an antiviral drug precursor from chitin using a saprophyte as a whole-cell catalyst

Matthias G Steiger,Erwin E Rosenberg,Marko D Mihovilovic,Robert L Mach,Astrid R Mach-Aigner,Rita Gorsche

doi:10.1186/1475-2859-10-102

Matthias G Steiger, Erwin E Rosenberg + Show 4 more

Open Access

https://doi.org/10.1186/1475-2859-10-102

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Abstract

BackgroundRecent incidents, such as the SARS and influenza epidemics, have highlighted the need for readily available antiviral drugs. One important precursor currently used for the production of Relenza, an antiviral product from GlaxoSmithKline, is N-acetylneuraminic acid (NeuNAc). This substance has a considerably high market price despite efforts to develop cost-reducing (biotechnological) production processes. Hypocrea jecorina (Trichoderma reesei) is a saprophyte noted for its abundant secretion of hydrolytic enzymes and its potential to degrade chitin to its monomer N-acetylglucosamine (GlcNAc). Chitin is considered the second most abundant biomass available on earth and therefore an attractive raw material.ResultsIn this study, we introduced two enzymes from bacterial origin into Hypocrea, which convert GlcNAc into NeuNAc via N-acetylmannosamine. This enabled the fungus to produce NeuNAc from the cheap starting material chitin in liquid culture. Furthermore, we expressed the two recombinant enzymes as GST-fusion proteins and developed an enzyme assay for monitoring their enzymatic functionality. Finally, we demonstrated that Hypocrea does not metabolize NeuNAc and that no NeuNAc-uptake by the fungus occurs, which are important prerequisites for a potential production strategy.ConclusionsThis study is a proof of concept for the possibility to engineer in a filamentous fungus a bacterial enzyme cascade, which is fully functional. Furthermore, it provides the basis for the development of a process for NeuNAc production as well as a general prospective design for production processes that use saprophytes as whole-cell catalysts.

Highlights

Recent incidents, such as the SARS and influenza epidemics, have highlighted the need for readily available antiviral drugs
N-acetylneuraminic acid (NeuNAc) is prepared through extraction from natural sources, such as bird nests, milk, or eggs [5], through the hydrolysis of colominic acid in a culture broth of Escherichia coli K1 [6], or through chemical synthesis [7]
Engineering a NeuNAc synthesis pathway into Hypocrea The biosynthesis of NeuNAc begins with the formation of N-acetylmannosamine (ManNAc) from GlcNAc or UDP-N-acetylglucosamine (UDP-GlcNAc)

Summary

Introduction

Recent incidents, such as the SARS and influenza epidemics, have highlighted the need for readily available antiviral drugs. One important precursor currently used for the production of Relenza, an antiviral product from GlaxoSmithKline, is N-acetylneuraminic acid (NeuNAc) This substance has a considerably high market price despite efforts to develop cost-reducing (biotechnological) production processes. As a result of their location and their negative carboxylate functionality, sialic acids play important roles in mediating cellular recognition and adhesion processes [2] and in the infection cycles of severe viral diseases, such as influenza viruses A and B [3]. In these cases, de novo-synthesized viral particles attach to their respective sialic acids at the cell surface. The requirement for ATP or an excess of pyruvate and the subsequent expensive downstream processing has kept the costs of NeuNAc production considerably high (current market price is $100/g)

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