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  August 12, 2020
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Medica Communications 9

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Description:

An Preview of Sample CHAP for the Book Publications, "A Compilation of Lignocellulose Feedstock and Related Research for Feed, Food and Energy - Vol II, 1st Edition," (c) D. A. Flores. Skye Blue Publications. Port Coquitlam, B. C. Canada V3B 1G3. 
 
 
CHAP Genetic Analyses of E. coli and Yeast Strains for the Overproduction of Histidine.



By D. A. Flores


The Problem and the Potential.

Three different strains are discussed that were earlier investigated to elucidate the biosynthesis of histidine (HIS) were: 1) E. coli HISGE271K mutant strai, 2) E. coli HISEA83, a constructed strain, and 3) of Kluyveromyces marxianus, a yeast, KMMIG1 mutant strain for MIG1 disruption, and which were used to suggest the mechanisms in cellular metabolism for boosting or urpregulating HIS levels.  We will close the discussion by suggesting what is called Peptide Nucleic Acid (PNA) technologies for gene silencing using transcription factors in such a way that the resulting biosynthetic operons overproduced phenotypically activities that result in greater HIS levels in the cell.


Aspects of Microbial Genetics in E. coli and the Yeast  Kluyveromyces marxianus.

The first mutant strain to be discussed is called HISGE271K where HisG, also called ATP-phosphoribosyltransferase or ATP-PRT, initially results in feedback inhibition (FBI) by HIS.  The AMP structural analogue 5-Aminoimidazole-4-carboxamide ribonucleotide, or AICAR, formed during HIS biosynthesis (and from de novo purine biosynthesis) can competitively inhibit, viz. via FBI, as do ADP/AMP analogues to substrate ATP-PRT interacting with the enzyme HisG at the active site (see: E. A. Malykh et al., 2018).
 
One suggestion to upregulate HIS biosynthesis in this case is to use trasncription factor engineering (TFE) on the operon that increases levels of HisG expression and effective against FBI of AICAR on HisG.

In another study, a constructed strain HISEA83 produced more HIS due to less AICAR around and its FBI of its HisG enzyme due  to: 1) over expression  of purH  and purA in the purine biosynthetic pathway. AICAR is converted to ATP via purH and purA overexpression with also use of Pi; 2) the pitA transport system was gene deleted apparently creating a larger availability of Pi; 3) and lastly, it was found that the alkaline phosphatase activity in the cell was not FBI'd as much at low Pi concentrations (see: E. A. Malykh et al., 2018).

For the yeast species Kluyveromyces marxianus, the biosynthetic pathway of HIS has been attributed to 10 different steps and 7 different genes, HIS1 -> HIS7, with HIS4 accounting for 4 biosynthetic steps leaving the rest implicated with each of 1 biosynthetic steps (see: M. Nurcholis et al., 2020).
 
TFE for the HIS1 -> HIS7 genes and MIG1, a global regulon already implicated with HIS4 metabolism can be used to upregulate HIS biosynthetic pathways.


The Non-GMO Option for Overproducing Strains.

In actual fact, a non-GMO supplement would call for techniques like peptide nucleci acid (PNA) cojugated with a "carrier" like B12 to transbound the microbial cell wall and membrane with the gene silencing element as with the mechnanism using a palindromic binding nucleic acid-based reagent with central and outward symmetry binding in the "reverse" symmetry and thus silencing the expressed mRNAs from target DNA sequence or gene.  Most likely, as was mentioned earlier, the operator/promoter (O/P) binding elements or products will be blocked from translation that "feedback" and inhibit HIS biosynthesis at the rate-limiting biosynthetic juncture points or steps.


A Current Industrial Process.

In fact, it has been described for the overproduction of HIS that Corynebacterium glutamicum involves recombinant strains through transformation with a stronger tac promoter involved in L-HIS biosynthesis leading to its overproduction in culture (Y. Cheng et al., 2013).


Conclusions.

Whether in the recent past of just lately, there is evidence in the biochemistry of HIS biosynthesis to suggest ways to bring about TFE together with more modern and acceptable gene silencing techniques referred to as PNA technology, also more being more acceptable by public perception, being non-GMO in nature compared to current recombinant strains through transformation with C. glutamicum.
 
 
References:
 
(1) Y. Cheng, Y. Zhou, L. Yang, C. Zhang, Q. Xu, X. Xie, M. Chen. 2013. Modification of histidine biosynthesis pathway genes and the impact on production of L-histidine in Corynebacterium glutamicum. Biotechnol. Lett. 35(5): 735.
 
(2) E. A. Malykh, I. A. Butov, A. B. Ravsheeva, A. A. Krylov, S. V. Mashko, N. V. Stoynova. 2018. Specific features of L-histidine production by Escherichia coli concerend with feedback control of AICAR formation and inorganic phosphate/metal transport. Microb. Cell Fact. 17(1): 42.
 
(3) M. Nurcholis, M. Murata, S. Limtong, T. Kusaka and M. Yamada. 2020. MIG1 as a positive regulator for the histidine biosynthesis pathway and as a global regulator in the thermotolerant yeast Kluyveromyces marxianus. Scientific Reports 10(1): 4382. 
 
 
 


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