Breaking Down Substrate

The process by which fungus convert sawdust into mushroom fruiting bodies never ceases to amaze me. How the fungus converts what looks like dusty powder into beautiful and colorful mushrooms seems like magic. How does this process work behind the scenes?

For some background knowledge, hardwood sawdust is composed primarily of three major groups: lignin, cellulose, and hemicellulose (composing of ~80% of the dry weight). The approximate ratios (per GPT) are as follows:

·      Cellulose: 38 – 49% (the main structural component)

·      Hemicellulose: 19 – 26% (branched mix of sugars like xylose and mannose)

·      Lignin: 23 – 30% (complex rigid polymer that binds cellulose and hemicellulose together)

The following is what Chat GPT spits out when prompting it regarding the mushroom metabolic pathways. It seems more focused on the process of metabolizing sugar used for the building of the mushroom biomass, so we can focus on that on this first article. In the next article, we can focus on the biochemical reactions related to the actual fruiting of the mushrooms.

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Step 1: Initial Lignin Breakdown

Enzyme laccase and manganese peroxide (MnP) use oxygen and hydrogen peroxide (H2O2) to attack lignin’s surface and deeper parts, which turn it into reactive fragments and open up sawdust.

·      Reaction: Lignin + O2  + Manganese + H2O2  Smaller lignin pieces + Water

Further explanation:

·      Laccase is an enzyme which breaks down lignin, a complex polymer that most wood is made from. It belongs to a group of enzymes called oxidases and uses oxygen (O2) to oxidize (remove electrons from) molecules like lignin.

·      Manganese is an enzyme whose main job is to break down lignin as well. It uses hydrogen peroxide (H2O2) to oxidize manganese ions (Mn2+), which creates Mn3+, a highly reactive form of manganese. Mn3+ then acts as a chemical sword which breaks down lignin into smaller pieces

·      The reason why both laccase and manganese are necessary is that the former breaks down the phenolic parts of lignin (parts with hydroxyl groups attached to aromatic rings, which are easier to oxidize), while the latter breaks down phenolic parts (larger portion of lignin and harder to degrad)

Step 2: Complete Lignin Degradation

Versatile peroxidase (VP) and glyoxal oxidase finish breaking tough lignin with H2O2 (produced from lignin bits), splitting it into small compounds like aldehydes

·      Reaction: Lignin + H2O2  Small aromatic compounds + Acid + More H2O2

Further explanation:

·      Versatile peroxidase (VP) is a hybrid enzyme with functions similar to MnP – it fiishes breaking apart stubborn lignin fragments left behind by laccase and MnP. It is the best for large, non-phenolic structures – the hardest parts to degrade.

·      Glyoxal oxidase is an enzyme that helps generate hydrogen peroxide (H2O2) by oxidizing small aldehydes and alcohols (which are lignin byproducts). This provides H2O2 needed by VP (and also MnP) and keeps the breakdown reactions going by fueling peroxidases

·      VP and GLOX are like the cleanup crew and power supply. They finish off stubborn lignin pieces and generate H2O2 needed to keep lignin-degrading system running smoothly.

Step 3: Hemicellulose Breakdown & Release of Hemicellulose

Endo-xylanase enzymes cut the branches of hemicellulose (side fibers in sawdust) into shorter sugar chains, like xylans

·      Reaction: Hemicellulose + Water  Shorter xylan chains

Exo-xylanase and B-xylosidase enzymes trim the ends and split those chains, releasing individual sugars like xylose.

·      Reaction: Xylan chain + Water  Xylose sugars

Further explanation:

·      Endo-xylanase is a protein that acts like tiny scissors. It cuts up hemicellulose (a tough plant fiber into smaller sugar bits

·      Sawdust is composed of Cellulose, hemi-cellulose, and lignin. Cellulose is tough and straight and only made from glucose. Hemicellulose is softer and branched, and made of mixed sugars (xylose, arabinose, mannose, galactose, little bit of glucose). Oyster mushrooms target hemi-cellulose as easier to digest.

·      Exo-xylanase is an enzyme that cuts xylan from the ends of the chain, releasing smaller sugar units (like xylose) – like nibbling off the tips of a rope

·      B-Xylosidase is an enzyme that further breaks down the small sugar units into single xylose sugars – like chopping last bits into bite-sized pieces.

Step 4: Cutting Cellulose Chains:

Endoglucanase enzymes slice the main cellulose fibers in sawdust into shorter chains.

·      Reaction: Cellulose + H2O  Shorter sugar chains

Further explanation:

·      Endo-glucanase functions like endo-xylanase in the sense that it targets cellulose and breaks it into smaller pieces. They just target different substrates have different functions that xylan chain branched where is cellulose is like longer chain

·      Although cellulose is just one sugar, it is more difficult to break apart than hemi-cellulose as cellulse forms tight, crystalline fibers that are super strong and organized, like tightly packed cables.

Step 5: Releasing Sugar Pairs from Cellulose:

Exoglucanase enzymes trim the ends of cellulose chains, releasing cellobiose (two linked sugar molecules)

·      Reaction: Cellulose chain + water  Cellobiose

Further explanation:

·      Cellobiose is a disaccharide made of two glucose units linked together by a B-1, 4-glucosidic bond.

Step 6: Turning Cellobiose into Sugar

B-Glucosidase enzymes split cellobiose into individual glucose sugars, which the mushrooms can easily use

·      Reaction: Cellobiose + water  2 Glucose

Step 7: Building Mushroom Tissue

The mushroom uses glucose and xylose in its metabolism (like glycolysis) to produce energy and building blocks for growing its cell walls (chitin), proteins, and mushrooms. About 20% of the sawdust’s carbon becomes mushroom mass.

·      Reaction: Glucose + Xylose  Energy + Mushroom biomass

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Summary:

This GPT thread basically broke down the main processes that oyster mushrooms use to degrade sawdust, which is composed of cellulose, hemi-cellulose, and lignin. In the case of cellulose and hemi-cellulose, it uses enzymes such as endo-xylanase or endo-glucanase to break up the substrate and then a series of other enzymes to further process them down into the base sugars. In the case of lignin, it seems necessary to do a one-two punch to break down the matter using heavier hitters.

This does not include the metabolizing of nitrogen based supplements, which is necessary for large mushroom flushes or the metabolic processes occurring during pinning or the fruiting itself. Perhaps we can examine some of those processes at a later time.