Analysis and optimisation of plant biomass degrading enzyme production in Aspergillus

Abstract

Much research over the past 25 years has been applied to the development of filamentous fungi, most notably Aspergillus, as hosts for recombinant protein production. Their inherent abilities to grow at high rates and to high biomass densities and their exceptional capacity to secrete high levels of homologous product are well recognized. Despite there being many advances made in the hyper-production of heterologous proteins in filamentous fungi, their ability to produce and secrete homologous proteases along with different native protein glycosylation still requires further strain improvement to efficiently produce a wide range of heterologous proteins. The aim of this thesis was to develop an efficient fungal expression system for the production of recombinant proteins, in particular those involved in plant biomass degradation. Due to its potential in biomass degradation and with its favourable fermentation capabilities, Aspergillus was chosen for this study. In Chapter 2 eight different Aspergillus species were compared with respect to their genomic ability to degrade plant cell wall biomass. While all tested Aspergilli had a similar potential to degrade plant biomass, results showed that even in closely related species, their strategies differed markedly. Combining the approaches from different species is likely to result in better enzyme mixtures for industrial applications, such as the saccharification of plant biomass for biofuel production. In Chapter 3, the molecular and phenotypical differences between A. vadensis and six other species of black Aspergilli were examined. Growth on varying carbon sources and extracellular enzyme profiles when grown on maltose/starch indicated significant and unique differences between A. vadensis and the other black Aspergilli. Further analysis of the partial genome of A. vadensis genome, combined with gene expression data when grown on maltose indicate that its aberrant phenotype is likely caused by the low expression of prtT and amyR regulators or their associated genes and not a mutation or deletion as was originally concluded. In Chapter 4 six novel constitutive promoters from A. niger (pef1α, ptktA, pef1β, ptal1, pcetA and ppgkA) and a further five from A. vadensis (pef1α, prps31, pgpdA, pubi1 and poliC) were tested in A. vadensis using a gene encoding a secreted arabinofuranosidase from Fusarium oxysporum as a reporter for heterologous protein production. Of the promoters tested, 3 from A. niger (pef1α > ptal > ppgkA) and 3 from A. vadensis (pef1α > poliC > prps31) all resulted in higher ABF activity than for that of the commonly used gpdA promoter from A. nidulans. In Chapter 5 the potential of A. vadensis as an expression host was tested by successfully expressing an α-L-arabinofuranosidase (abfB) (GH54) and an endo-1,4-β-D-glucanase (eglA) (GH12) from A. vadensis under the control of the gpdA promoter from A. vadensis. In Chapter 6 an evolutionary screening method was used to improve the inulin degradation potential of Aspergillus oryzae through the upregulation of exo-inulinase. As an organism with no predicted endo-inulinase function, improved inulin degradation would be largely dependent on the overproduction of this enzyme. Subsequent generation growth of Aspergillus oryzae (Rib40) on inulin for 9 weeks successfully resulted in exo-inulinase overproducing mutants.