By Danny A. Flores1, 1440 Barberry Drive, Port Coquitlam, B. C. Canada V3B 1G3

The overall approach of functional amino acids delivery on low-quality fed diets is to balance or optimize the microbial cell protein (MCP) fraction with the escape dietary protein using other axenic approaches as with: (1) providing plant amino acids by boosting their free levels in green plant tissue (e. g. seed, leaves, flowers), and (2) that 'deliver' essential amino acids to rumen microbial metabolism including phenylalanine and methionine through non-GMO boosting including in host spp. that potentially respond significantly as has been observed as with Ruminococcus flavefaciens and R. albus (for the latter see: D. A. Flores, 1989).

Both approaches use the proposed non-GMO technology of Peptide Nucleic Acid (PNA) gene silencing using auxins as carriers which can enter and circulate through the plant's system with transcription factors (TFs) and their mRNAs tht feedback inhibit amino acid synthesis including histidine.  This has yet to be tested with an appropriate linker between the PNA and carrier as conjugate (see: M. Rownicki et al., 2017). 

We will deal soley with the former approach here with the use of PNA technology with plant crops due to manipulation of the biosynthetic regulatory elements of each controlling step for each amino acid involving their TFs downstream from the operator/promoter (O/P) region of the "cassette" with the mRNA in question.

It was reported in A. Stepanksy and T. Leustek (2006) that progress in studying histidine (HIS) biosynthesis in plants recently was made possible by findings that in some plants  many of the enzymatic steps are accounted for by single genes rather than redundancy and thus auxotrophic ustants in higher plants systmes could be useful for its elucidation; HIS is synthesized in 11 steps by the following enzymes: (1) N'-5'-phosphoribosyl-ATP transferase, (2) phosphribosyl ATP pyrophosphohydrolase, (3) phosphoribosyl-AMP cyclohydrolase, (4) BBMII or what is also known as N'-[(5'phosphoribosyl)-formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide, [5'-PRFAR] isomerase, (5-6) imidazoleglycerol-phosphate synthase, (7) imidazoleglycerol-phosphate dehydratase, (8) imidazoleacetolphosphate (IAP) aminotransferase, (9) histidinol phosphate phosphatase and finally (10-11) histidinol drhydrogenase.

Ingle et al. (2005) demonstrated that in Arabidopsis and 3 Alyssum spp. that free HIS in the steady-state pool correlated with the total HISN1 transcript levels in itssue using the AtHISN1B cDNA in roots to be in the order: A. lesbiacum >> A. serpylliforium > A. montanum > A. thaliana correlating with the size of the steady-state pool of root free HIS; further in Arabidopsis boosting free HIS up to 15-fold tightly correlated high levels of HIS1N1 gene expression and HIS pool size.  This suggests a genetic possibility in controlling mechanisms at the transcription level for endogenous free HIS in plant experimentation and eventually in crops.

Guyer et al. (1995) stated that regulatory mechanisms resembling the general control response in S. cerevisiae are also true with plants with respect to various amino acids as with lysine, tyrosine, phenylalanine, alanine, aspartate, glutamate, proline, threonine, tryptophan and valine.

In Stepansky and Leustek (2006) it was reveal that in yeat GCN4 TF, a B-21P TF up regulates, in addition to HIS biosynthesis, 539 genes in all amino acid pathways (except cysteine), genes encoding amino acid precursors, vitamin pathways, for peroxosomal components, mitochondrial carrier proteins and autophagy proteins. Genomics analysis in the Arabidopsis genome sequencing revealed that B-zip-type TFs exist but with no close sequence homology to yeast GCN4.  Also they stated that opaque 2 protein in maize was able to bind the promoter of GCN4-activated yeast genes resulting complementation of yeast GCN4 mutants.  Indeed GCN4 TF can be implicated as a regulatory element for amino acid metabolism or biosynthesis with HIS (and those unrelated to HIS) and storage proteins as well, being able to bind the promoters for these genes.  Complementarily, there is evidence of AACA-core sequences associated with the GCN4 motif and thus the GCN4 promoter element in all Arabidopsis HIS biosynthetic genes possibly suggesting a coordinated regulation of the pathway by GCN4 (and also other unrelated amino acid pathways).

It is presumed that elevated free amino acid concentration can also be effectively protected (cf. Browning reaction due to exposure to UV/light irradiation) with heat damage upon drying in the field after harvest, conditioning their availability and for MCP synthesis in rumen digestion with consequent uptake into MCP or its biomass.

 

 

References: 

1. D. A. Flores. 1989. Applications of recombinant DNA to rumen microbes for the improvement of low-quality feed utilization. J. Biotech. 10: 95-112.

2. D. Guyer, D. P. Patton and E. Ward. 1995. Evidence for cross-pathway regulation of metabolic gene expression in plants. Proc. Natl. Acad. Sci. USA  92: 4997-5000.

3. R. A. Ingle, S. J. Mugford, J. D. Reed, M.M. Campbell and A. C. Smith. 2005. Constitutively high expression of the histidine biosynthetic pathway contributes to nickel tolerance in hyperaccumulator plants. Plant Cell 17: 1-18.

4. M. Rownicki, M. Wojcechowska, A. J. Wierzba, J. Czarnecki, D. Bartosik, D. Gryko and J. Trylska. 2017. Vitamin B12 as a carrier of peptide nucleic acid (PNA) into bacterial cells. Sci. Rep. 7: 7644-7655.