09/28/2025
Here is a preliminary draft of a letter to your editor regarding recent updates or comments to the effect of encouraging further research in rumen microbiology, titled: "Per Os RNA-Based Regulation (PORR) of a Rumen Microbial Ecosystem Model Consisting of Boosted Commensal Fibrolytics, Proteolytics and Ligninolytics: an update," By D. A. Flores, Skye Blue Internet, Barberry House, 1440 Barberry Drive, Port Coquitlam, British Columbia, Canada V3B 1G3. Herein presented is my letter to the Editor, at this time:
Dear Editor,
Here is the the new concept introduced to our readers of Per Os RNA-based Regulation (or PORR), amongst other RNA-based regulatory gene-silencing techniques such as the other direct-applied ribonucleic acid-based regulation (DARR).
This paper uses plasmid-based genetics to modify enzyme expression and that conjugate with their cellular hosts producing a populated distribution in the rumen microbial ecosystem. Recent advances in fibrolytic enzymes from the rumen for microbial cellular energy (viz. ATP pool) from rumen fermentation are discussed in the following.
Boosting of all three major (3) fibroin types, that is, lignin, cellulose and hemicellulose (note, pectin is not included from forage in the experiment) is used in order to effectively create a boosted ATP pool for fibrolytics, proteolytics to utilize, and including fibrolysis via ligninolysis.
In regards microbial amino acid (AA) metabolism, there are methods that can be used to approach and to investigate which proteases to boost that are most critical for producing amino acids given their peptide, pre-formed amino acid (PFAA) profile in terms of their active site specificity and the frequency of most limiting acids in microbial AA cell metabolism that are most limiting in microbial metabolism as analyzed by proteomics techniques and with more new data needed about species microbial host AA metabolism and its enzymatic pathways and stipulating those AA that are most limiting to their metabolism.
The latter, can be further investigated in a biofermentor or chamber simulating the rumen environment as in a Rusitec(R) seeded in graded amounts of peptides previously showing an "enrichment" in the selected and most limiting AA in their metabolism.
Furthermore, those three (3) most major or predominant proteolytics as surveyed from the rumen that supply pre-formed amino acids (PFAA) as peptides in a common pool to the subpopulations of predominant cellulolytics, hemicellulolytics, and also pectinolytics, which are the characteristic digestors of grass and legume fodders can be used to generate such a pool of PFAA for a mixed rumen environment that has been boosted.
Furthermore, those ligninolytics are isolated and screened for their avidity to digest lignin substrate and their ligninolytic enzymes typed as to their active site mechanisms, viz. their classes as to being esterases (e. g. Faes), etherases or so-called lyases using systematic genomics/bioinformatics study, and additionally their potential boosted pool size optimally needed for the mixed commensal rumen ecosystem in sugars and PFAA.
Finally, there is a need to assess the microbial pool size that is optimally boosted for the fibrolytics, proteolytics and even other carbohydrolytics.
It is the desire to develop an energy pool size from ATP sufficient for the microbial biomass in the rumen to be maximized for production of a protein source amongst its major and secondary commensal players for this ecosystem environment.
In order to manipulate via Gene Editing plasmid-based genetics as an approach to manipulating rumen fermentation for boosting microbial cell protein (MCP) synthesis the author will employ a protocol to determine the relative contributions of plasmid versus chromosomal fibrolytic enzymes utilizing the equation (below):
Total Fibrolytic Activity = x (%) + y(%), x=plasmid-based
and Y=chromosomal-based activity
It will have to be proven that plasmid-based fibrolysis is quantitavely significant to make a difference when gene edited into the plasmid and the chromosomal DNA and subsequently boosted using the PORR approach (see above). The experiment involves effectively using pure culture measuring the clearance of lignocellulose on a plate before and after curing the specified fibrolytic plasmids. Cultures investigated or used from a collection will presumably have been typed for fibrolyticaly coded enzymes in their DNA via modern genomics/bioinformatics with DNA sequencing and analysis.
There was an original hypothesis stating from the literature that the microbial cell or model that is represented are "weakened" by rec-DNA manipulations and including our method of modifying gene editing, also termed a form of rec-DNA manipulation. In that case they could "wash out" in cases where reproduction is not fast enough in a highly competitive and continuously fermenting biofermentor. In fact, the author would like to mention in a personal communication here that in a pioneering study with K. Gregg, who the author conferred with back in 1992, at the University of New England, NSW in Australia, an attempt to introduce a culture into the rumen to detoxify ingested plant forage with secondary plant alkaloids, in this case, fluoroacetate, a common toxin from forage found in Australia and of great interest, apparently "washed out" when microbial numbers were sampled and counted by enumeration.
It should be pointed out here that the concern is whether or not just gene editing the plasmid fibrolytic enzymes genes leads to enough DNA "damage" to affect energy utilization with ATP, the energy currency and reserve of the microbe, in both its generation and use for metabolic anabolism in MCP synthesis.
It is the hope of this author to send best wishes to colleagues and friends in Pakistan and the rest of the Indian sub-continent where it is believed that animal species that predominate there, namely, the water buffalo species characteristic of the region, and their preference and adaptability towards roughage such as those from fibrous agricultural residues (FAR) would benefit the most and indeed present the most critical need for this PORR manipulation with fibre digestion in the rumen to improve MCP biomass formation and nutrition there.
Key words: Rumen RNA Gene Regulation Plasmid Microbes Enzymes Fibrolysis Proteolysis Ligninolysis
Warm Regards,
D. A. Flores
Skye Blue Internet
Port Coquitlam, BC
Canada V3B 1G3
+1 (236) 983 6419
[email protected]
