Closely related fungi employ diverse enzymatic strategies to degrade plant biomass

Background Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails. Results It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition. Conclusions These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.Peer reviewe

The Genome of Anopheles darlingi, the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vectorhuman and vectorparasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles- darlingi. © 2013 The Author(s)

Overexpression, purification and characterisation of homologous α-L-arabinofuranosidase and endo-1,4-β-D-glucanase in Aspergillus vadensis

In the recent past, much research has been applied to the development of Aspergillus, most notably A. niger and A. oryzae, as hosts for recombinant protein production. In this study, the potential of another species, Aspergillus vadensis, was examined. The full length gDNA encoding two plant biomass degrading enzymes, i.e. α-L-arabinofuranosidase (abfB) (GH54) and endo-1,4-β-D-glucanase (eglA) (GH12) from A. vadensis were successfully expressed using the gpdA promoter from A. vadensis. Both enzymes were produced extracellularly in A. vadensis as soluble proteins and successfully purified by affinity chromatography. The effect of culture conditions on the expression of abfB in A. vadensis was examined and optimised to give a yield of 30 mg/L when grown on a complex carbon source such as wheat bran. Characterization of the purified α-L-arabinofuranosidase from A. vadensis showed an optimum pH and temperature of pH 3.5 and 60 °C which concur with those previously reported for A. niger AbfB. Comparative analysis to A. niger AbfA demonstrated interesting differences in temperate optima, pH stability and substrate specificities. The endo-1,4-β-D-glucanase from A. vadensis exhibited a pH and temperature optimum of pH 4.5 and 50 °C, respectively. Comparative biochemical analysis to the orthologous EglA from A. niger presented similar pH and substrate specificity profiles. However, significant differences in temperature optima and stability were noted

Analysis of the molecular basis for the non-amylolytic and non-proteolytic nature of Aspergillus vadensis CBS 113365

Aspergillus vadensis CBS 113365, a close relative of A. niger, has been suggested as a more favourable alternative for recombinant protein production as it does not acidify the culture medium and produces very low levels of extracellular proteases. The aim of this study was to investigate the underlying cause of the non-amylolytic and non-proteolytic phenotype of A. vadensis CBS 113365. Our results demonstrate that the non-functionality of the amylolytic transcription factor AmyR in A. vadensis CBS 113365 is primarily attributed to the lack of functionality of its gene's promoter sequence. In contrast, a different mechanism is likely causing the lack of PrtT activity, which is the main transcriptional regulator of protease production. The findings presented here not only expand our understanding of the genetic basis behind the distinct characteristics of A. vadensis CBS 113365, but also underscore its potential as a favourable alternative for recombinant protein production.This research was supported by the China Scholarship Council for financial support (grant no. 202107720032). SG. was supported by a grant of the Applied Science Division (TTW) of NWO and the Biotechnology and Safety Program of the Ministry of Infrastructure and Water Management 15807 to RPdV. SG currently holds a Juan de la Cierva Incorporación grant (IJC 2020–042749-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union Next Generation EU/PRTR.Peer reviewe

Additional file 1: of Closely related fungi employ diverse enzymatic strategies to degrade plant biomass

Combines Additional File Figures S1–S4 and Table S1. Figure S1: Hydrolytic enzyme activity profiles of the eight species. Figure S2: Laccase activity of the eight species. Figure S3: Differences in feruloyl esterase production. Figure S4: Conserved SDS-PAGE profiles for isolates of the same species. Table S1. Strains used in this study

Additional file 2: of Closely related fungi employ diverse enzymatic strategies to degrade plant biomass

Combines Additional file Tables S2–S4 (excel format). Table S2A. Numbers of putative genes per CAZy family for the 10 genomes addressed in this study. Table S2B. Numbers of putative genes per Plant Polysaccharide Degradation-related CAZy family for the 10 genomes addressed in this study. Table S3A. Orthology clusters of feruloyl esterase (SF), glycoside hydrolase (GH), carbohydrate esterase and polysaccharide lyase (PL) families. Table S3B. Orthology clusters of auxiliary activities (AA). Table S4A. Detection of proteins in cultures grown on wheat bran sorted by CAZy family. Table S4B. Detection of proteins in cultures grown on sugar beet pulp sorted by CAZy family. Table S4C. Detected proteins in cultures grown on wheat bran sorted by number of species that contain an orthologue. Table S4D. Detected proteins in cultures grown on sugar beet pulp sorted by number of species that contain an orthologue