Parascedosporium putredinis NO1 tailors its secretome for different lignocellulosic substrates

Abstract

Parascedosporium putredinis NO1 is a plant biomass-degrading ascomycete with a propensity to target the most recalcitrant components of lignocellulose. Here we applied proteomics and activity-based protein profiling (ABPP) to investigate the ability of P. putredinis NO1 to tailor its secretome for growth on different lignocellulosic substrates. Proteomic analysis of soluble and insoluble culture fractions following the growth of P. putredinis NO1 on six lignocellulosic substrates highlights the adaptability of the response of the P. putredinis NO1 secretome to different substrates. Differences in protein abundance profiles were maintained and observed across substrates after bioinformatic filtering of the data to remove intracellular protein contamination to identify the components of the secretome more accurately. These differences across substrates extended to carbohydrate-active enzymes (CAZymes) at both class and family levels. Investigation of abundant activities in the secretomes for each substrate revealed similar variation but also a high abundance of "unknown" proteins in all conditions investigated. Fluorescence-based and chemical proteomic ABPP of secreted cellulases, xylanases, and β-glucosidases applied to secretomes from multiple growth substrates for the first time confirmed highly adaptive time- and substrate-dependent glycoside hydrolase production by this fungus. P. putredinis NO1 is a promising new candidate for the identification of enzymes suited to the degradation of recalcitrant lignocellulosic feedstocks. The investigation of proteomes from the biomass bound and culture supernatant fractions provides a more complete picture of a fungal lignocellulose-degrading response. An in-depth understanding of this varied response will enhance efforts toward the development of tailored enzyme systems for use in biorefining.IMPORTANCEThe ability of the lignocellulose-degrading fungus Parascedosporium putredinis NO1 to tailor its secreted enzymes to different sources of plant biomass was revealed here. Through a combination of proteomic, bioinformatic, and fluorescent labeling techniques, remarkable variation was demonstrated in the secreted enzyme response for this ascomycete when grown on multiple lignocellulosic substrates. The maintenance of this variation over time when exploring hydrolytic polysaccharide-active enzymes through fluorescent labeling, suggests that this variation results from an actively tailored secretome response based on substrate. Understanding the tailored secretomes of wood-degrading fungi, especially from underexplored and poorly represented families, will be important for the development of effective substrate-tailored treatments for the conversion and valorization of lignocellulose.

Keywords: CAZymes; Parascedosporium putredinis NO1; activity-based protein profiling; lignocellulose; proteomics.

Fig 1

Investigating the distribution of proteins across bound and supernatant fractions in the P. putredinis NO1 secretome. The number of proteins identified in at least one replicate across all substrates for the bound fraction compared to the supernatant fraction within the predicted P. putredinis NO1 secretome from growth on six lignocellulosic substrates for 4 days.

Fig 2

Differences in the P. putredinis NO1 secretome across lignocellulosic substrates. Molar percentage values for proteins identified on at least one substrate in the bound (A) and supernatant (B) fractions of the P. putredinis NO1 secretome scaled to Z-scores across substrates. EF, empty fruit bunch; LI, kraft lignin; RS, rice straw; SC, sugar cane bagasse; WB, wheat bran; WS, wheat straw.

Fig 3

Differences in functional categories of the P. putredinis NO1 secretome across lignocellulosic substrates. The molar percentage abundance of proteins identified as CAZymes, that were not identified as CAZymes but assigned to GO categories, or that were not annotated as CAZymes or with GO categories (unknown) were calculated proportionally for each substrate of the bound (A) and supernatant fractions (B) of the P. putredinis NO1 secretome.

Fig 4

Proportional abundances of enzyme activities in the bound fraction of the P. putredinis NO1 secretome. The molar percentage abundance of proteins assigned to GO terms was calculated proportionally for the bound fraction of the P. putredinis NO1 secretome after 4 days of growth on six lignocellulosic substrates. EF, empty fruit bunch (A); LI, kraft lignin (B); RS, rice straw (C); SC, sugar cane bagasse (D); WB, wheat bran (E); WS, wheat straw (F). Average molar percentage ± standard error (n = 3, n = 2 for SC substrate).

Fig 5

Proportional abundances of enzyme activities in the supernatant fraction of the P. putredinis NO1 secretome. The molar percentage abundance of proteins assigned to GO terms was calculated proportionally for the supernatant fraction of the P. putredinis NO1 secretome after 4 days of growth on six lignocellulosic substrates. EF, empty fruit bunch (A); LI, kraft lignin (B); RS, rice straw (C); SC, sugar cane bagasse (D); WB, wheat bran (E); WS, wheat straw (F). Average molar percentage ± standard error (n = 3, n = 2 for SC, LI, and WB substrates).

Fig 6

Differences in proportional catalytic CAZyme class abundance of the P. putredinis NO1 secretome across lignocellulosic substrates. The molar percentage abundance of proteins belonging to each catalytic class of CAZyme was calculated proportionally to the total abundance of CAZymes for each substrate for the bound (A) and supernatant fractions (B) of the P. putredinis NO1 secretome.

Fig 7

Differences in CAZyme family abundance of the P. putredinis NO1 secretome across lignocellulosic substrates. Molar percentage values for proteins annotated as CAZymes and identified on at least one substrate of the P. putredinis NO1 secretome scaled to Z-scores across substrates for the bound (A) and supernatant (B) fractions separately.

Fig 8

Differences in P. putredinis NO1 glycoside hydrolase production over time visualized with activity-based probes. Fluorescence imaging following SDS-PAGE is shown for single replicates of samples of culture supernatants taken at days 3, 4, 5, 6, 7, and 10 of growth. They were treated with a triplex probe mixture targeting cellulases, xylanases, and retaining β-glucosidases. EF, RS, SC, WB, WS, alongside PageRuler (Thermo) prestained protein ladder. Secretomes were stained in triplicate, a single replicate is shown here.

Fig 9

ABPP-determined variation in relative active enzyme levels over time during P. putredinis NO1 growth on various substrates. Resolved bands running at different apparent MW values (left column in each block) were integrated into the Cy2 (β-glucosidase probe), Cy3 (cellulase probe), or Cy5 (xylanase probe) channels. Average band integration values (n = 3) are shown as color intensity varying from white (not detected) to full color (~1,000,000 counts) to black (saturation) on a logarithmic scale. Secretomes were prepared and stained in biological triplicate following different culture times (labels above columns).