Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi.

Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of cellulosomes, multienzyme complexes that synergistically colocalize enzymes to extract sugars from recalcitrant plant matter. However, a functional understanding of how fungal cellulosomes are deployed in vivo to orchestrate plant matter degradation is lacking, as is knowledge of how cellulosome production and function vary throughout the morphologically diverse life cycle of anaerobic fungi. In this work, we generated antibodies against three major fungal cellulosome protein domains, a dockerin, scaffoldin, and glycoside hydrolase (GH) 48 protein, and used them in conjunction with helium ion and immunofluorescence microscopy to characterize cellulosome localization patterns throughout the life cycle of Piromyces finnis when grown on simple sugars and complex cellulosic carbon sources. Our analyses reveal that fungal cellulosomes are cell-localized entities specifically targeted to the rhizoids of mature fungal cells and bodies of zoospores. Examination of cellulosome localization patterns across life stages also revealed that cellulosome production is independent of growth substrate in zoospores but repressed by simple sugars in mature cells. This suggests that further exploration of gene regulation patterns in zoospores is needed and can inform potential strategies for derepressing cellulosome expression and boosting hydrolytic enzyme yields from fungal cultures. Collectively, these findings underscore how life cycle-dependent cell morphology and regulation of cellulosome production impact biomass degradation by anaerobic fungi, insights that will benefit ongoing efforts to develop these organisms and their cellulosomes into platforms for converting waste biomass into valuable bioproducts. IMPORTANCE Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses. We developed and used imaging tools to observe cellulosome spatial localization patterns across life stages of the anaerobic fungus Piromyces finnis under different environmental conditions. The resulting spatial details of how anaerobic fungi orchestrate biomass degradation and uncovered relationships between life cycle progression and regulation of cellulosome production will benefit ongoing efforts to develop anaerobic fungi and their cellulosomes into useful biomass-upgrading platforms.

Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae.

Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-AL(CS) displayed significantly increased anaerobic growth rate (0.203±0.006 h⁻¹) and xylose consumption rate (1.866 g g⁻¹ h⁻¹) along with high ethanol conversion yield (0.41 g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain.

Effects of aromatic amino acids, phenylacetate and phenylpropionate on fermentation of xylan by the rumen anaerobic fungi, Neocallimastix frontalis and Piromyces communis.

AIMS: Anaerobic fungi are important members of the fibrolytic community of the rumen. The aim of this study was to study their requirement for aromatic amino acids (AA) and related phenyl acids (phenylpropionic and phenylacetic acids) for optimal xylan fermentation. METHODS AND RESULTS: Neocallimastix frontalis RE1 and Piromyces communis P were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia; ammonia plus a complete mixture of 20 AA commonly found in protein; ammonia plus complete AA mixture minus aromatic AA; ammonia plus phenyl acids; ammonia plus complete AA mixture without aromatic AA plus phenyl acids. Both species grew in all the media, indicating no absolute requirement for AA. The complete AA mixture increased (P<0.05) acetate concentration by 18% and 15%, sugar utilization by 33% and 22% and microbial yield by about 22% and 15% in N. frontalis and P. communis, respectively, in comparison with the treatments that had ammonia as the only N source. Neither the supply of aromatic AA or phenol acids, nor their deletion from the complete AA mixture, affected the fermentation rate, products or yield of either species. CONCLUSIONS: AA were not essential for N. frontalis and P. communis, but their growth on xylan was stimulated. The effects could not be explained in terms of aromatic AA alone. SIGNIFICANCE AND IMPACT OF THE STUDY: Ruminant diets should contain sufficient protein to sustain optimal fibre digestion by ruminal fungi. Aromatic AA or phenyl acids alone cannot replace the complete AA mixture.