Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme

Abstract

The yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) secretes a xylose-induced biodegradable plastic-degrading enzyme (PaE). To suppress degradation of PaE during production and storage, we targeted the inhibition of proteolytic enzyme activity in P. antarctica. Proteases A and B act as upper regulators in the proteolytic network of the model yeast, Saccharomyces cerevisiae. We searched for orthologous genes encoding proteases A and B in the genome of P. antarctica GB-4(0) based on the predicted amino acid sequences. We found two gene candidates, PaPRO1 and PaPRO2, with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPaPRO1 and ΔPaPRO2, and evaluated PaE stability in culture by immunoblotting analysis. Both mutants exhibited sufficient production of PaE without degradation fragments, while the parent strain exhibited the degradation fragments. Therefore, we concluded that the protease A and B orthologous genes are related to the degradation of PaE. To produce a large quantity of PaE, we made a PaPRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPaPRO2 mutant of XG8 was able to produce PaE without the degradation fragments during large-scale cultivation in a 3-L jar fermenter for 3 days at 30°C. After terminating the agitation, the PaE activity in the XG8 ΔPaPRO2 mutant culture was maintained for the subsequent 48 h incubation at 25°C regardless of remaining cells, while activity in the XG8 control was reduced to 55.1%. The gene-deleted mutants will be useful for the development of industrial processes of PaE production and storage.

Diagnostic ability of Peptidase S8 gene in the Arthrodermataceae causing dermatophytoses: A metadata analysis

“…Studies report that proteases and subtilisin like genes are present in fungal saprophytes, phyto-pathogens, insect pathogens and human pathogens. Changes in the amounts and type of secreted proteases could be due to ecological switching related to a differential expression rather than genetic divergences of the genes encoding orthologous proteins [ 74 , 75 ].…”

Accelerated degradation of plastic products via yeast enzyme treatment

“…The PBAT production capacity is expected to increase by approximately 40% by 2025 34 . In addition, we had previously developed methods for efficient production 35 and stabilisation 36 , 37 of PaE, which would enable the use of PaE on a large scale and at a low cost. Therefore, PaE may prevent the accumulation and inadvertent dispersal of BP, thereby making it easier to use in both open and closed environments.…”

“…The BP-degrading enzyme PaE was purified using the affinity method, as described previously 29 to determine whether the PaE degrades the cast PBAT film. A 72-h xylose-fed batch culture filtrate using a jar fermenter of P. antarctica PaE-overexpressing strain XG8 35 , 36 was used for the submerged treatment of BP sheets and films in PaE, and that of P. antarctica GB-4 (0) -HPM7 [MAFF 3070010] 39 , 40 was used as an enzyme solution for film treatment in the field. The activity of PaE was evaluated based on the decrease in turbidity of emulsified PBSA (Bionolle EM-301, Showa Denko K. K.) 22 .…”