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Sujitha D.,Kumar H. G. Jalendra,Thapliayal Garima,Pal Garima,Vanitha P. A.,Uttarkar Akshay,Patil Mahesh,Reddy B. H. Rajashekar,Niranjan Vidya,Rayalcheruvu Usha,Govind Geetha,Udayakumar M.,Vemanna Ramu 한국식물생명공학회 2023 Plant biotechnology reports Vol.17 No.6
Reactive oxygen species (ROS) increases under stress and damages cellular processes leading to decrease in productivity. Many genes have been known to be involved in scavenging ROS. We report the identification of master regulators of oxidative stress responsive genes from contrasting rice genotypes. Using microarray analysis, we identified 52 differentially expressed transcription factors (TFs) from the contrasting rice genotypes under oxidative stress. Upregulation of these TFs induces the expression of many genes in resistant or sensitive genotypes. The promoters of these TFs are enriched with reactive oxygen species binding elements (ROSE). The promoter analysis of genes that respond to oxidative stress also revealed that these TF binding sites were present and that these genes expressed differently in contrasting rice genotypes. The transcript levels of TFs correlate with expression level of stress responsive genes coding for various pathways such as polyol, ABA, JA biosynthesis and signaling. Functional validation of HSF-C1a using virus-induced gene silencing (VIGS), showed reduced expression of its target genes. Our study demonstrates that identified TFs could act as major transcriptional regulators of oxidative stress tolerance. These TFs can be used as markers and are potential candidates to improve stress tolerance in plants.
Recent Advances in Enzyme Engineering through Incorporation of Unnatural Amino Acids
원유미,Amol D. Pagar,Mahesh D. Patil,Philip E. Dawson,윤형돈 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.4
The development of new enzyme engineering technologies has been actively pursued as the industrial use of biocatalysts is rapidly increasing. Traditional enzyme engineering has been limited to changing the functional properties of enzymes by replacing one amino acid with the other 19 natural amino acids. However, the incorporation of unnatural amino acids (UAAs) has been exploited to manipulate efficient enzymes for biocatalysis. This has been an effective enzyme engineering technique by complementing and extending the limits of traditional enzymatic functional changes. This review paper describes the basic functions of the new functional groups of UAAs used in enzyme engineering and the utilization of UAAs in the formation of chemical bonds in the proteins. The recent developments of UAA-mediated enzymology and its applicability in industry, pharmaceutical and other research areas to overcome the limitations of existing enzymes is also emphasized.
Yoo, Hee-Wang,Kim, Joonwon,Patil, Mahesh D.,Park, Beom Gi,Joo, Sung-yeon,Yun, Hyungdon,Kim, Byung-Gee Elsevier 2019 Bioresource technology Vol.291 No.-
<P><B>Abstract</B></P> <P>In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the <I>E. coli</I>. Consequently, AlkL with signal peptide FadL (AlkL<SUB>f</SUB>) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkL<SUB>f</SUB> and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from <I>Pseudomonas pelagia</I> (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 ± 7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 ± 1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Bioconversion ofdodecanoic acid methyl esterintovalue-addedchemicals. </LI> <LI> Introduce host-based signal peptide to enhance expression level of transporter. </LI> <LI> Maximize product production by controlling the expression level of multi enzymes. </LI> <LI> Found novel enzymes with good activity to dodecanoic acid methyl ester. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
Enzymatic synthesis of sitagliptin intermediate using a novel ω-transaminase
Kim, Geon-Hee,Jeon, Hyunwoo,Khobragade, Taresh P.,Patil, Mahesh D.,Sung, Sihyong,Yoon, Sanghan,Won, Yumi,Choi, In Suk,Yun, Hyungdon Elsevier 2019 Enzyme and microbial technology Vol.120 No.-
<P><B>Abstract</B></P> <P>Enantiopure β-amino acids are essential precursors of various pharmaceuticals, agrochemicals and other industrially important chemicals. In this study, we selected sixteen potential ω-Transaminases (ω-TAs) by BLAST and phylogenetic tree analysis. These ω-TAs were cloned, purified and tested for their reactivity for the synthesis of model β-amino acid (<I>R</I>)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid [3-ATfBA], a key precursor for sitagliptin. In an enzymatic cascade, lipase converted β-ketoester substrate to β-keto acid, which was subsequently aminated by the selected ω-TA to its corresponding β-amino acid. A potent enzyme from <I>Ilumatobacter coccineus</I> (ω-TAIC) was identified for the production of 3-ATfBA. The pH dependency of the product inhibition suggested that lowering the reaction pH to 7.0 can circumvent the inhibition of ω-TAIC by 3-ATfBA and about 92.3% conversion of 100 mM β-keto ester substrate could be achieved. The applicability of this enzymatic system was further evaluated at the scale of 140 mM, wherein 3-ATfBA was generated with excellent conversion (81.9%) and enantioselectivity (99% <I>ee</I>). Furthermore, ω-TAIC was successfully used for the synthesis of various β-amino acids from their corresponding β-keto ester substrates.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A potent ω-TA was identified using bioinformatic tools from 16 potential enzymes. </LI> <LI> 3-ATfBA was produced with good conversion (81.9%) and 99% <I>ee.</I> </LI> <LI> ω -TAIC was successfully used for the synthesis of various β-amino acids. </LI> <LI> Lowering the reaction pH to 7.0 can circumvent the inhibition of ω -TAIC by 3-ATfBA. </LI> </UL> </P>
Sung, Sihyong,Jeon, Hyunwoo,Sarak, Sharad,Ahsan, Md Murshidul,Patil, Mahesh D.,Kroutil, Wolfgang,Kim, Byung-Gee,Yun, Hyungdon The Royal Society of Chemistry 2018 Green Chemistry Vol.20 No.20
<P>Running two two-step cascades in parallel anti-sense to transform an alcohol to an amine allowed the conversion of ω-hydroxy fatty acids (ω-HFAs) and α,ω-diols to the corresponding ω-amino fatty acids (ω-AmFAs) and α,ω-diamines, respectively. The network required only two enzymes namely an aldehyde reductase (AHR) and a transaminase (TA). Benzylamine served on the one hand as amine donor and on the other hand after deamination to benzaldehyde also as oxidant. All ω-HFAs tested were efficiently transformed to their corresponding ω-AmFAs using purified enzymes as well as a whole-cell system, separately expressing both the enzymes, with conversions ranging from 80-95%. Additionally, a single-cell co-expressing all enzymes successfully produced the ω-AmFAs as well as the α,ω-diamines with >90% yield. This system was extended by employing a lactonase, enabling the transformation of ε-caprolactone to its corresponding ω-AmFA with >80% conversion.</P>