Improvement in the production of L-Lysne by ENU treated...

Current Trends in Biotechnology and PharmacyVol. 13 (4) 406-423, October 2019, ISSN 0973-8916 (Print), 2230-7303 (Online)

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Improvement in the production of L-Lysne by ENUtreated Chemical mutagenesis of ddh gene recombinant

strain of Corynebacterium glutamicum MTCC25069

Bhushan Vanasi1, Ramesh Malothu*1

1Department of Biotechnology, Institute of Science Technology, Jawaharlal Nehru TechnologicalUniversity:Kakinada, Andhra Pradesh, India.

*Corresponding Author : [email protected]

Improvement in the production of L-Lysne by ENU treated

ABSTRACT:Auxotrophic mutant formed from ddh generecombinant MTCC25069with blocked homoserinedehydrogenase showed an increased yield of LLysine of 24.89 g/l from normal ddh generecombinant MTCC25069strain which had a yieldof 20.66 g/l of L Lysine. The maximum yield of L-lysine for the auxotrophic mutant is attained at7.5 pH, 300C of temperature and an incubationtime of 96 hrs. The Auxotrophic mutant of ddhrecombinant C. glutamicum showed nearly 6.52g/l more amount of l lysine than Auxotrophic mutantof wild type with 18.57 g/l of L Lysine. TheChemical mutagen ENU caused mutation in theHomoserine serine dehydrogenase enzymediverted the Aspartyl â semialdehyde to bind with2,3Dihydrodipicolinate synthase to participate inthe L Lysine synthesis through 2,3 meso-Diaminopimelate (Meso-Dap). Being arecombinant for diaminopimelate dehydrogenase(ddh) the auxotrophic mutant for the homoserinedehydrogenase follows the ddh pathway byoverexpression of ddh by deviating theAcetyltransferase and Succinyl transferase is thereason for the high yield of L Lysine production.

Keywords: Aspartyl â semialdehyde, 2,3Dihydrodipi-colinate synthase, Homoserinedehydrogenase, Diaminopimelate dehydrogenase(ddh). L, L-diaminopimelate (2, 3-meso DAP).

INTRODUCTION:L Lysine is one of the most important

essential amino acids which could be used in many

biophysical mechanisms in the living organisms.Corynebacterium glutamicum is used to produceL Lysine commercially1. L-Lysine is an essentialamino acid which is utilized in many biochemicalreactions like phosphorylation and also used asan additive for fodder crops2. Annually around 80,00, 00 tones were produced which made L Lysinesecond among global amino acid synthesis atindustrial scale3,4. Chemical synthesis, enzymaticmethod, fermentation, extraction from proteinHydrolysate, genetic engineering and protoplastfusions were several kinds of technologiesemployed in L Lysine synthesis fromCorynebacterium glutamicum3,26. L-lysine is oneof the most deficient components found in the foodof both human and animals. Animal feed generallycontains a less quantity of L-lysine and is notsynthesized by cattle, poultry or other livestock,so L-lysine will be added as a food supplement foranimals to meet feed requirements6. L-Lysine, oneof the eight essential amino acids for animals andhumans which is used as feed additives, dietarysupplements and also as an ingredient ofpharmaceuticals and cosmetics7.

Corynebacterium glutamicum is a non-lethaland non-emulsifying gram-positive bacterium. Itexhibits a low protease activity in the culture andcan secrete protease-sensitive proteins into theculture supernatant14. C.glutamicum is a gram-negative bacteria with the absence oflipopolysaccharide removed in the production oftherapeutic proteins15 increases the yield byreducing the purification steps. C. glutamicum is


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Bhushan Vanasi and Ramesh Malothu

generally recognized as safe (GRAS) for theindustrial biochemical production of L Lysine andL glutamate16.

Corynebacterium glutamicum is one of themajor microorganisms used in amino acidsynthesis. The Corynebacterium glutamicum isa rod-shaped bacteria, aerobic and gram-positivebacteria grows in the soil, on the surfaces ofvegetables and fruits17. C.glutamicum has thecapability to metabolize glucose, fructose, andsucrose18, 29. C. glutamicum utilizes many differentkinds of carbohydrates, organic acids, and alcoholas a carbon and energy source for rapid microbialgrowth and for many amino acids synthesis24, 25.The glucose, or sucrose or any carbon source isutilized by the Corynebacterium glutamicum for llysine synthesis by fermentation28. The time ofincubation is reported for maximum L Lysine isbetween 48 hrs to 72 hrs30, 31. The ddh recombinantCorynebacterium glutamicum MTCC25069produces more amounts of L Lysine compared toWild type. This is because of the expression ofmore amount of ddh which acts as an enzyme forthe substrate 2,6 dicarboxylic acid with theparticipation of less number of enzymes. Chemicalmutagenesis with ENU increased the yield of LLysine in the mutant than the Wild type strain27.The ENU causes insertion or deletion mutationand shows its effect on protein synthesis. TheENU causes mutation in Homoserinedehydrogenase gene to cause the HomoserineAuxotrophsof C. glutamicum21.

Generally, the Aspartyl â semialdehyde isproduced in two ways. In the Krebs cycle ofCorynebacterium glutamicum, the Oxaloaceticacid (OAA)19,20 undergoes transamination reactionwith the presence of glutamate: oxaloacetate:transaminase enzymes produce aspartyl â semialdehyde which further produces homoserine andL L diaminopimelate (2,3 meso- DAP) by twodifferent pathway1. The Aspartyl â semialdehydeis also formed from Aspartate dehydrogenase fromAspartyl phosphate which was formed fromAspartate by Aspartate kinase2,19. The aspartylâ-semi aldehyde acts as a common substrate toproduce L Lysine through L L diaminopimelate

(2,3-DAP) and Methionine or threonine throughhomoserine1,28. The aspartylâ-semi aldehydeconverts to Homoserine by reacting withhomoserine dehydrogenase4 which participates inthe Homoserine pathway in the production ofThreonine and Methionine22,23. Homoserine reactswith MetA6 and produces O-Acetylhomoserinewhich reacts with Met B synthesize Cystathioninefurther reacts with C7 to produce Homocysteinefinally reacts with Met E or Met H to producemethionine or Homoserine reacts with homoserinekinase produces L homoserine phosphate andconverts to threonine11 by Threonine synthase inHomoserine pathway. Aspartyl â semi aldehydereacts with 2,3 Dihydrodipicolinate synthaseproduces 2,3 Dihydropicolinate which furtherreduces to 2,6 Dicarboxylic acids by 2,3Dihydrodipicolinate reductase. Corynebacteriumglutamicum chose three kinds of enzymes namelyAcetyltransferase or Succinyl Transferase ordiaminopimelate dehydrogenase (ddh) to produceL L diaminopimelate (2,3 meso DAP). The LLdiaminopimelate (2,3 meso DAP) converts to LLysine by Lysine synthase. By Recombination withddh gene with a constitutive promoter enhancesthe productivity of L Lysine by diverting theacetyltransferase and succinic transferasepathway to ddh pathway. The Chemical MutagenN-nitroso-N-ethyl urea (ENU)3,9 has the capabilityto cause deletion or insertion mutation in theHomoserine dehydrogenase18 enzyme and blocksthe Homoserine Pathway which generally leadsto the production of threonine and Methionine. Thisblock in the homoserine pathway diverts theaspartic â-semialdehyde to react with 2,3Dihydrodipicolinate synthase the enzyme toproduce more amounts of “-diaminopimelate (“DAP) through ddh pathway.2,3 Dihydrodipicolinatesynthase 35 converts aspartic â-semialdehyde to2,3 Dihydropicolinate. In the presence of reductase2,3 Dihydropicolinate reduces to Piperidine 2,6,dicarboxylic acid. The formation of DAP will bedone by binding of Piperidine 2,6, dicarboxylic acidwith three different enzymes acetyltransferase orSuccinyl transferase or diaminopimelatedehydrogenase enzymes leads to three differentpathways for the L Lysine production through DAP.


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2,6 dicarboxylic acid reacts with acetyl transferaseproduces the N – acetyl 2 –amino 6-keto L-pimelate which reacts with the enzymeaminotransferase produces N- Acetyl- L- L -diaminopimelate produces L- L- DAP bydeacetylase in the acetyltransferase pathwaywhich is a three-step pathway. 2,6 dicarboxylatereacts with Succinyl transferase to produce NSuccinyl 2- amino 6 keto L pimelate which reactswith dap C Produces N Succinyl L-Ldiaminopimelate which again reacts with dap Egives L L diaminopimelate is also a three steppathway. The ddh recombinant strain producesmore L L diaminopimelate (2,3 meso DAP) byoverexpression of ddh enzyme which follows ddhpathway for the production of L L diaminopimelate(2,3 meso-DAP) by reacting with 2,6 Dicarboxylicacid as substrate by eliminating the more numberof reactions that were in the remainingacetyltransferase and Succinyltransferasepathways by overexpression of ddh by the ddhrecombinant strain of C. glutamicumMTCC25069strain with constitutive promoter. Thegeneral events that took place in the Lysineproduction are illustrated in fig 1.

MATERIALS AND METHODS:Bacterial cell cultures: The bacterial strain usedin this research is a recombinant of ddh gene witha constitutive promoter of C. glutamicum(MTCC25069) developed by cloning32 in the lab ofRamesh Malothu, school of biotechnology, JNTUKwhich had an increased yield of 20.66 g/L of l-lysine.

Recombinant strain developed in ourlaboratory from the MTCC25069wild type C.glutamicum, by recombining ddh gene with aconstitutive promoter was utilized in the processof chemical mutagenesis with-nitroso-N-ethyl urea(ENU). The mutagenic power of N-ethyl-N-nitrosourea (ENU) stems from the generation ofdiazomethane33, 34.

Chemical mutagenesis method: Seed culturemedium (D-Glucose 10 g/l, Peptone 5 g/l, Yeastextract 3.75 g/l, NaCl 5 g/l, (NH4)2SO4 17.5 g/l,K2HPO4 25 g/l, KH2PO4 25 g/l, Threonine 20 g/l,

Methionine 20 g/l, ZnSO40.5 g/l, MgSO4.7H2O25 g/l, FeSO4.7H2O 1 g/l and MnSO4.5H2O0.5g/l), LB medium (Tryptone 10 g/l, NaCl 10 g/l andYeast 5 g/l). The ddh recombinant MTCC25069strain were grown on LB medium and collectedinto test tubes with 3 ml each into 5 test tubeswhich were used for inducing the chemicalmutation. Then the cell cultures were incubatedfor 24 hrs. at 370C in an orbital shaker. Centrifugethe tubes at 10000xg for 5mins and collect thepellets. These collected pellets were suspendedin 3 ml sodium citrate buffer and again centrifugethese pellets at 10000xg for 5 mins. The pellet iscollected and resuspended into 3 ml buffercontaining sodium citrate buffer with a PH of 4.1and 1.2 ml of N-nitroso-N-ethyl urea (ENU) with100 nM concentration. The cultures were incubatedseparately for 0,5,10,15,20,25 and 30 min andcentrifugation has done at 10000xg for 5 mins afterthe stipulated time. The pellets were collected andsuspended in 3 ml sodium citrate buffer to washthe mutagen ENU and the resulted pellets wereagain suspended in the 3ml sodium citrate bufferto remove the traces of ENU. These pellets areincubated at 300c for 3 days in the threonine andMethionine enriched seed culture media.

Isolation of Auxotrophs: The growth obtainedafter 3 days of incubation was inoculated in 1 mlseed culture media without Threonine orMethionine and in 300c in an orbital shaker. 50units of penicillinase were added to each tube andleft for 10 minutes. 100μl of this growth inoculatedon to seed culture media with threonine andmethionine and also onto the seed culture mediawithout threonine or methionine and incubated for3 days at 300C in the orbital shaker. After the timeof incubation is completed the samples werescreened for Lysine production. The samples werecentrifuged at 15000xg for 10 min. The supernatantis collected for lysine analysis. Quantitativeanalysis of L Lysine was done by SDS PAGE.

Optimization of fermentation parameters:Different parameters like Concentration of ENU,Time of exposure of ENU, PH of the culture media,Temperature and time were tested to find the bettergrowing conditions of the recombinant strain when



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Fig 1: The Homoserine pathway for Threonine and Methionine synthesis and Acetyltransferase, SuccinylSynthase and diaminopimelate dehydrogenase pathways in the synthesis of L Lysine through L Ldiaminopimelate (2,3 meso- DAP).



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treated with ENU for the high amount of productionof L Lysine was tested.

In the chemical mutagenesis, we tested fordifferent concentrations of 25, 35, 50, 75, 100 and120 nm of the concentration of ENU and differenttimes of exposure of ENU of 5, 10, 15, 20, 25 and30 minutes to the Bacterial strain were tested atConstant temperature, Time, and pH. Which areoptimized conditions of wild type C. glutamicumfor L-Lysine production (13). The recombinant strainafter treating with ENU, temperature and time ofincubation was kept constant and tested fordifferent pH values of 6, 6.5, 7.0, 7.5, 8 and 8.5.After checked for different PH the ENU treatedddh recombinant cultures were tested for differenttemperatures of 10, 25, 29, 30,31 and 35 bykeeping the Volume, pH and time of incubationconstant. The 100 nm ENU treated recombinantC. glutamicum was tested for different time periodsof incubation 24, 48, 72, and 96 hrs by keepingthe pH at 7.5 and temperature 300C constant.

By checking for all the parameters we chosethe best-adapted values of chemically mutatedrecombinant strain which had given a high amountof yield of L Lysine to culture the recombinant strainand compared with Wild Type C. Glutamicum forproductivity of L Lysine.

Culturing the Chemical Mutagen ENU (100 NM)Treated ddh Recombinant strain of C.glutamicum MTCC25069under OptimizedConditions: After optimizing the differentconditions of Temperature, PH and time ofincubation 20 min chemical mutagen ENU (100NM) exposed ddh gene recombinant strainMTCC25069was cultured in under theseconditions. The ENU treated ddh recombinant wascultured at 300C of temperature, 7.5 PH and withan incubation time of 96 hrs. was tested for theproductivity of L Lysine from mutant ddhrecombinant strain compared with normal wild typeMTCC25069C. glutamicum mutant.

Molecular Docking analysis of Aspartyl â semialdehyde with Homoserine dehydrogenaseand 2,3 Dihydrodipicolinate Synthase:Molecular docking was performed for Aspartyl â

semi aldehyde with Homoserine dehydrogenaseand 2,3 Dihydrodipicolinate synthase to find thebonding interactions between the Protein andligand. The amino acid sequence of Homoserinedehydrogenase enzyme of C. glutamicumMTCC25069with accession number NP_600409.1and 2,3Dihydrodipicolinate synthase enzyme ofC. glutamicum MTCC25069with accessionnumber NP_601846.1 was collected and fromNCBI and checked verified in the UniProt and thesequences from UniProt is used to build a proteinmodel by Homology modeling in the SWISS-MODEL Server belongs to Swiss Institute ofBioinformatics (SIB). Protein model quality builtby Swiss model server analyzed through thePDBsum database. After checking theRamachandran plot and RMSD values we choosethe protein models of Homoserine dehydrogenaseand 2,3 Dihydrodipicolinate synthase to dock withligand Aspartyl â semialdehyde in the PyREXsoftware. Finally, the image analysis and aminoacid interactions in the Protein ligand are generatedin the discovery studio.

RESULTS AND DISCUSSION:Chemical mutagenesis:Effect of Concentration of ENU on therecombinant strain of C. glutamicum: Theamount of ENU used to treat plays a pivotal role incausing Mutagenesis in the bacterial species. Therecombinant C. glutamicum showed highproductivity of L Lysine of 23.28 g/l of yield bykeeping the temperature, time of incubation, andPH constant at 100 Nm concentration of ENU.Optimized conditions to grow the wild type C.glutamicum MTCC25069strain for maximum yieldof L Lysine of 96 hrs. time of incubation, 30 C oftemperature and PH 7.5. Kept constant bychecking the yield for 25,35,50,75, 100 and 120nm of concentration produced. 20.66 g/l, 20.11g/l,21.05 g/l, 22.10 g/l, 23.28 g/l and 18.17 g/l of LLysine respectively. We got high productivity of LLysine at 100 nm concentration of ENU 23.28 g/lfor Auxotrophic ddh recombinant mutant and a highyield of 16.23 g/l for Auxotrophic mutant of wildtype C. glutamicum when compared to 35 nm, 50nm,75nm concentrations of ENU.



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Table.1: Table for L Lysine production at different ENU concentrations.

S.No Time of Temperature Concentration pH Lysine LysineIncubation (00 c) of ENU treated concentration concentration

(hours) Auxotrophic AuxotrophicWild Type Mutant

1 96 30 25 7.5 13.26 20.112 96 30 35 7.5 13.50 20.663 96 30 50 7.5 14.40 21.054 96 30 75 7.5 15.80 22.105 96 30 100 7.5 16.23 23.286 96 30 120 7.5 14.99 18.17

Table.2: Table for L Lysine production at different times of exposure of ENU to ddh recombinantstrain.

S.No Time of Tempe- Time of Concentration pH Lysine LysineIncubation rature Exposure of of ENU in concentration concentration

(hours) (00 c) ENU in Mins. Nano Auxotrophic Auxotrophicmolars Wild Type Mutant

1 96 30 5 100 7.5 13.05 20.222 96 30 10 100 7.5 14.78 22.673 96 30 15 100 7.5 15.23 23.174 96 30 20 100 7.5 16.48 24.025 96 30 25 100 7.5 15.54 22.236 96 30 30 100 7.5 14.23 21.56

Table 3: Table for L Lysine production at different times of exposure of ENU to ddh recombinant strain.

S.No Time of Temperature pH Lysine L-LysineIncubation concentration concentration

Auxotrophic AuxotrophicWild Type Mutant

1 96 30 6.0 14.78 16.502 96 30 6.5 15.02 18.173 96 30 7.0 16.35 22.164 96 30 7.5 17.02 24.325 96 30 8.0 16.21 19.176 96 30 8.5 13.23 17.29



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Graph 1: A plot between Concentration of ENUexposed vs Concentration of L Lysine produced.The L-Lysine concentration showed an increasedpattern with an increase in the concentration from25 nm till 100 nm with a maximum yield of 23.28g/l and decreased at 125 nm. concentration atconstant values, time of Incubation 96 hrs.,Temperature 300C and pH 7.5.

The amounts of L Lysine produced shownincreased trend from 25 nm concentration up to amaximum amount of L Lysine achieved at 100 nmconcentration of ENU 23.28 g/l which is increasedfrom 20.66g/l represented in table 1 and plotted inthe graph 1 by taking L Lysine concentration onY-axis and Concentration of ENU on X-axis. Thepattern of increase of L Lysine production shownillustrated in graph 1.

The effect of time of exposure of ENU onRecombinant C. glutamicum ATCC 13032: TheENU treated recombinant C. glutamicum showedmaximum yield of L Lysine of 24.02 g/l for 20 minsof exposure of chemical mutagen at 100 nm ofconcentration were compared to different times ofexposure chemical mutagen at 100 nmconcentration for 5, 10, 15, 25 and 30 mins. Theeffect of ENU on the L Lysine is recorded maximumat 20 min of exposure before washing with citratebuffer in the process of creating the Auxotrophicmutant4 from the recombinant ATCC 13032. Theincreased productivity of L Lysine Yield by takingthe time of exposure of ENU is represented in thegraph 2 by plotting the graph between Time ofexposure of ENU on X-axis and Concentration ofL Lysine on Y axis from the values of table 2 got

for different times of exposure of ENU by keepingTime of incubation 96 hrs., Temperature 30 C and100 NM Concentration of ENU and pH of 7.5Constant. The trend of the graph increased from 5mins of exposure till 20 mins of exposure recorded20.22 g/l, 22.67 g/l, 23.17 g/l and 24.02 g/l for 5mins, 10mins, 15mins and 20 mins of exposureto ENU respectively. The maximum yield of LLysine achieved by the Auxotrophic mutant of ddhrecombinant is 24.02 g/l and for Auxotrophicmutant of the wild type strain is 16.48 g/l at 20mins of exposure of ENU. After 25 mins of exposuredecreased the amount of L Lysine yield to 22.23g/l and at 30 min of exposure, it was 21.56 g/l.

Graph 2: A Plot between Time of Exposure of 100n M ENU and L. LysineYield. The L Lysineconcentration showed an increased pattern withan increase in the Time of exposure of ENU from5 mins. To 20mins. with a maximum yield of 24.02g/l and 16.48g/l for Recombinant and wild typemutants respectively and decreased at 25 mins.of ENU exposure at a constant time of Incubation96 hrs., Temperature 300C and PH 7.5.

Isolation of Auxotrophic Mutant: After threedays of incubation, the strains that show growthin the presence of Methionine or Threonine or thepresence of both the amino acids were consideredas auxotrophic mutants and were isolated andcultured. Due to lack of homoserinedehydrogenase, the mutant strains utilize theThreonine and methionine supplemented in themedia. The auxotrophic mutants require morequantity of threonine and methionine in their mediasignifies the lack of Homoserine pathway that could



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occur in the normal strains of both Wild type aswell as ddh recombinant varieties of C. glutamicumATCC 13032. These auxotrophic mutants wereused further to know the optimized conditions ofPH, Temperature and time of incubation.

The Chemical mutagen had shown itsmaximum activity at 100 nm by blocking theHomoserine dehydrogenase a key enzyme in theproduction of Threonine and methionine. The ENUcaused mutation in Homoserine dehydrogenasegene which generally combines with Aspartyl BSemi aldehyde to produce the homoserine. Thisblockage in the Homoserine pathway favors theDAP pathway by combining with 2,3, Dihydrodi-picolinate synthase produced 2,3, Dihydrodi-picolinate which reduces to 2,6, a dicarboxylicacid.

Optimization of PH, Temperature and Timeof Incubation for Chemical mutagen ENUtreated ddh recombinant strain of C.glutamicum ATCC 13032:Effect of pH on the Recombinant strain of C.glutamicum: The ENU concentration used tomutate the recombinant strain is 100 nm whichwas an optimized condition for high productivity ofL Lysine was used which was treated onRecombinant strain for 20 mins. The recombinantstrain has shown maximum lysine productivity of22.16 g/l at 7.5 pHs when compared to ENUmutated wild type strain MTCC25069at the same7.5 pHs. The Productivity of l lysine is increasedconsiderably in the recombinant strain for all thePh values when compared with wild type strain.But both the strains had shown maximumproductivity at 7.5 pH.

The trend of production of L Lysine increasedfrom PH 6.0 to 7.5 and shown decreasedproductivity for PH 8 and PH 8.5. The L Lysineyield recorded maximum at PH 7.5 with an LLysine concentration of 24.32 g/l. 16.50 g/l, 18.17g/l, 22.16 g/l, 24.32 g/l, 19.17 g/l and 17.29 g/l ofL Lysine yield was reported for 6.0,6.5 7.0, 7.5,8.0 and 8.5 PH values respectively represented inthe table 3. The L Lysine yield recorded maximum

at 7.5 PH with 24.32 g/l for Auxotrophicrecombinant mutant and 17.02 g/l for anauxotrophic mutant of wild type C. glutamicum.The graph was plotted by taking PH values on theX-axis and Yield on the Y-axis and illustrated thetrend of increase in the productivity of L Lysine ingraph 3.

Graph 3: Plot between a PH of the Media and LLysine Yield. The L Lysine concentration showedincreased pattern with increase with the increasein value of PH, from 6.0 to 7.5 reached themaximum yield of 24.32 g/l L-Lysine anddecreased for further increase in the PH by keepingtime of Incubation for 96 hrs., Temperature 300Cconstant after treating with ENU (100 NM) with20mins of exposure before washing.

The PH showed its impact on mediautilization and glucose consumption by maximumuptake of glucose at 7.5 pH had supported theincreased participation of the C. glutamicum inthe Dap pathway for the L- Lysine synthesis. ThePH had shown its impact on the fluidity of bacterialcell wall and Plasma membrane of the C.glutamicum at 7.5 which helped in the glucoseconsumption.Effect of Temperature on the Recombinantstrain of C. glutamicum: Here the strain treatedwith 100 Nm concentration of ENU which wasexposed to ENU for 20 mins was tested. Theincubation was kept for different temperatures andPH of 7.5 and time of incubation 96 hrs. was keptconstant. The recombinant strain showedincreased productivity of L Lysine with themaximum amount of L Lysine was 24.20 g/l at 30C of temperature. All the recombinant strains



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Graph 5: A Plot between Time of Incubation forddh gene Recombinant C. glutamicum and L Lysineyield. The L Lysine concentration showed anincreased pattern with an increase with theincrease in time of incubation, from 12 hrs. to 96hrs. reached the maximum yield of 24.16 L and17.98 g/l Lysine for Auxotrophic recombinant

showed an increased pattern in the L Lysineproduction till 30 C and productivity decreased for31C and 32 C of temperature. L Lysine yield of17.11 g/l, 19.52 g/l, 22.78 g/l, 24.20 g/l, 22.16 g/land 18.17 g/l for temperatures 27.00C, 28.00C,29.00C, 30.00C, 31.00C and 32.00 C respectivelywas reported is represented in the table 4. TheAuxotrophic mutant of ddh recombinant strain hadshown the maximum L Lysine productivity of 24.20g/l and wild type auxotrophic mutant shown 18.13maximum L Lysine yield at 300C. The trend forthe increase in the productivity of L Lysine isillustrated in graph 4 by taking the PH on the X-axis and L Lysine Yield on Y axis.

3.2.3 effect of Time of incubation on ENUtreated recombinant strain ofC. glutamicum:The recombinant strain has shown maximumproductivity of 24.16 g/l after 72 hrs. and also shownincreased productivity of when compared with ENUmutated wild type strain. Time of incubation is 96hrs. for wild type strain which is also same forboth Recombinant and ENU treated Recombinantstrains of C. glutamicum MTCC25069. The Timeof incubation signifies the growth kinetics of C.glutamicum. Further incubating the strain shownthe decrease in the quantity of L lysine productiondue to the strain entering into death phase between96 to 120 hrs. of incubation.

The L Lysine productivity increased whilethe time of incubation is being increased. The LLysine productivity for 12 hrs.,24 hrs.,48 hrs., 72hrs., 96 hrs., and 120 hrs. was 10.23 g/l, 13.21 g/l, 17.16 g/l, 18.68, g/l, 24.16 g/l and 18.17 g/lrespectively represented in the Table 5 and plottedin graph 5 by taking Time of incubation on the X-axis and L Lysine yield on the Y-axis. The trend ofthe time of incubation increased from 12 hrs. to96 hrs. reached the maximum L Lysineconcentration of 24.16 g/l for Auxotrophicrecombinant mutant and 17.98 g/l for Auxotrophicmutant of L Lysine further decreased to 18.17 g/land 15.23 for Recombinant and Wild type mutantsrespectively at 120 hrs. of incubation.

Graph 4: A plot between Change in theTemperature VS L Lysine Yield. The L Lysineconcentration showed an increased pattern withan increase in temperature, from 270C to 300Creached the maximum yield of 24.20 g/l and 18.13g/l L- Lysine respectively for Auxotrophic mutantsof ddh recombinant and wild type and decreasedfor a further increase in the temp. by keeping thetime of Incubation for 96 hrs., PH 7.5 constantafter treating with ENU (100 NM) with 20 mins ofexposure before.

The temperature had shown its significanteffect on the rate of metabolism favoring theenzymes and substrates that participates in thesynthesis of Amino acids. The temperature of 300Chad favored the enzyme-substrate complexformations and showed its impact on the increasedrate of reactions by reducing the activation energiesof catalytic enzymes that were involved in the LLysine pathway by keeping the Time, Volume andPH are constant.



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TABLE 4: Table for L Lysine production at different temperatures of ENU treated ddh recombinantC.glutamicum.

S.No Time of Temperature pH Lysine LysineIncubation concentration concentration

Auxotrophic AuxotrophicWild Type Mutant

1 96 27.0 7.5 15.23 17.112 96 28.0 7.5 16.07 19.523 96 29.0 7.5 17.45 22.784 96 30.0 7.5 18.13 24.205 96 31.0 7.5 17.66 22.166 96 32.0 7.5 16.52 18.17

TABLE 5: Table for L Lysine production at different times of incubation of ENU treated ddhrecombinant C.glutamicum.

S.No Time of Temperature pH L-Lysine L-LysineIncubation (0o C) concentration concentration

(hours) Auxotrophic AuxotrophicWild Type Mutant

1 12 30 7.5 14.56 15.782 24 30 7.5 15.11 16.433 48 30 7.5 16.32 17.164 72 30 7.5 16.43 18.685 96 30.0 7.5 17.98 24.166 120 30.0 7.5 15.23 18.17

TABLE 6: Table for L Lysine production for ddh recombinant mutant and wild type mutant at theoptimized parameters of ddh recombinant mutant.

S.no Optimized Parameters L Lysine in L Lysine in ARWt.mutant (g/l) Mutant(g/l)

1. Conc. Of ENU (100 nM) 16.12 23.282. Time of exposure (20 min.) 16.48 24.023. PH (7.5 ) 17.02 24.324. Temperature (300C) 18.13 24.205. Time of incubation 96 hrs. 17.98 24.16



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mutant and auxotrophic wild type mutantrespectively and decreased for a further increasein the time by keeping temperature 300C., PH 7.5constant after treating with ENU(100NM) with 20mins of exposure before washing.

The Corynebacterium glutamicum hadentered the stationary phase after the 96 hrs. ofincubation is the reason for and production of LLysine increased substantially after the logarithmicphase and reached the maximum amount of LLysine production between 72 hrs. to 96 hrs. ofincubation.

Comparative analysis of maximum Yields ofL Lysine for Auxotrophic ddh recombinantmutant and Auxotrophic wild type mutant atOptimized Conditions: By optimizing the growthof ENU treated ddh RecombinantMTCC25069strain of C. glutamicum we got 23.28g/l of L Lysine and 16.2 g/l of L lysine for wild typeauxotrophic mutant for 100 NM concentration ofENU. By treating the ENU (100 NM) concentrationfor 20 minutes we got the better yield of 24.02 g/lfor ddh recombinant strain and 16.48 g/l for theWild type. After isolating the Auxotrophic mutantfor Threonine and Methionine after exposing to ENU(100 NM) for 20 mins of time of exposure of ENUbefore washing with Citrate buffer was checkedfor different PH, different Temperatures and fordifferent times of incubation for ddh generecombinant ATCC auxotrophic mutant of C.glutamicum and wild type auxotrophic mutant. wegot a better yield of L Lysine of 24.32 g/L for theddh Recombinant auxotrophic mutant and 17.02g/l for wild type auxotrophic mutant at 7.5 PH. Wegot a better yield of 24.20 g/l of. Lysine forAuxotrophic mutant of ddh recombinant C.glutamicum and 18.13 g/l for wild type auxotrophicmutant at 300C of temperature and highproductivity of 24.16 g/l and 17.98 g/l of L Lysinerespectively after 96 hrs. of time of Incubation.Hence The best optimizing conditions for ENUtreated ddh Recombinant mutant strain ofCorynebacterium glutamicum shown for high yieldof L Lysine at the 100 NM concentration of ENUwith an exposure time of 20 mins created anAuxotroph which show a better yield of L Lysine

at 7.5 PH, 300C of temperature and 96 hrs. ofIncubation. The Optimized Parameters and amountof L Lysine Produced for Recombinant mutant andquantity of L Lysine produced by wild typeauxotrophic mutant at the same conditions ofrecombinant strain was tabulated in table 6.

Graph 6: Maximum Yield of L Lysine forauxotrophic wild type mutant (WT) and auxotrophicddh recombinant mutant (AR Mutant) based onOptimized parameters of Recombinant strain.

Molecular Docking analysis of Aspartyl â semialdehyde with Homoserine dehydrogenaseand 2,3 Dihydrodipicolinate Synthase:Molecular docking analysis of Aspartyl â semialdehyde ligand with Homoserinedehydrogenase protein: Homology model isdeveloped in the SWISS-MODEL server for theamino acid sequence collected from UniProt [40].The Protein model built in the Swiss model [36].collected in the pdb format. The protein model isfurther analyzed in PDBsum[37]. database tocheck the protein quality for docking. TheRamachandran plot had shown nearly 91.5% ofresidues in the favorable regions and RMS distancefrom planarity is around 2 signified good proteinquality. The ligand of aspartyl semi aldehydecollected from PubChem is used to dock in thePyREX [39] software with the Modeled Homoserinedehydrogenase protein. The ligand Aspartyl â semialdehyde had shown its interaction with glycineGLY (151) with a hydrogen bond and also carbon-hydrogen bond with glycine GLY (288). Proline PRO(B: 287) Alanine ALA (B:289), tyrosine TYR (B:178,155), Glycine GLY (B:268, 151), AsparagineASN (B:270) and Leucine LEU (B: 154) are theinteracting amino acids with the ligand in



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Homoserine dehydrogenase. Homoserinedehydrogenase enzyme is with one conventionalhydrogen bond. The Homoserine dehydrogenaseshowed a good interaction with the ligand Aspartylâ semi aldehyde with a binding energy of -5.2 Kcal.Homoserine dehydrogenase had nine interactingamino acids is the reason for having high bindingenergy even though having only a hydrogen bondand a Carbon-hydrogen bond.

Molecular docking analysis of Aspartylâ semi aldehyde ligand with 2,3 Dihydrodipi-colinate Synthase: Homology model is developedin the SWISS-MODEL server for the amino acidsequence 2,3 Dihydrodipicolinate synthasecollected from UniProt. The Protein model built inthe Swiss model collected in the .pdb format. Theprotein model is further analyzed in the PDBsumdatabase to check the protein quality for docking.

Fig: a) Ligand Aspartyl â semialdehyd e) Protein Homoserine dehydrogenase c&d) Protein-ligand interaction(Docking in Pyrex)e) lid plot analysis (PDBsum) of Ligand Aspartyl â semi aldehyde with Protein Homoserinedehydrogenase: GLY (151), H-bond and C-H bond with GLY (288). PRO (B: 287), ALA (B:289), TYR (B:178,155), GLY (B:268, 151), ASN (B:270) and LEU (B: 154) are the interacting amino acids in proteinHomoserine dehydrogenase with the ligand Aspartyl â semi aldehyde.f). Ramachandran plot for HSD with91.5% favored regions, h) RMSD value of HSD between 1.5 to 2.0 for HSD signifying the good quality ofProtein to be docked with the ligand.



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The Ramachandran plot had shown nearly 90.05%of residues in the favorable regions and RMSdistance from planarity is between 1.5 to a signifyinggood quality of modeled protein. The ligand ofaspartyl semi aldehyde collected from PubChemis used to dock in the PyREX software with theModeled 2,3 Dihydrodipicolinate synthase protein.The ligand Aspartyl â semi aldehyde had shownits interaction with Leucine (B:206), Serine (B:261),Aspartate ASP (B:205) and Isoleucine (B:257) witha conventional hydrogen bond. 2,3,Dihydrodipicolinate synthase is with 4 conventionalhydrogen bonds. Leucine LEU (B:206), AspartateASP (B:205), Serine SER (B:261) and IsoleucineILE (B:257) were the interacting amino acids of2,3, Dihydrodipicolinate synthase. The 2,3Dihydrodipicolinate synthase showed a goodinteraction with the ligand Aspartyl semi aldehydewith a binding energy of -4.4 Kcal. Even though ithad less amino acid interactions but due to thepresence of 4 hydrogen bonds it can able to showconsiderable binding with the Ligand.

The aspartyl b semi aldehyde had shownhigher binding energy of -5.2 k. cal with homoserinedehydrogenase with 9 interacting amino acids andtwo hydrogen bonds than 2,3 Dihydrodipicolinatesynthase enzyme with - 4.5 k.cal of binding energy,4 interacting amino acids with 4 Hydrogen bonds.As the chemical mutagen, ENU caused mutation

in the homoserine dehydrogenase caused proteinnot to express or might have produced a proteinwhich could not bind properly with Aspartyl B semialdehyde diverted the Aspartyl semialdehyde tobind with 2,3 Dihydrodipicolinate synthase enzymeentered the dap pathway. As the binding energy ismore for homoserine dehydrogenase enzyme itshowed higher reactivity with the Aspartyl B semialdehyde but lack of it caused the Aspartylsemialdehyde to bind with2,3 Dihydrodipicolinatesynthase which directed to L -Lysine synthesisthrough producing L L diaminopimelate. Being addh recombinant strain the Corynebacteriumglutamicum produced more ddh which deviatedthe 2,6 dicarboxylic acid away from reacting withacetyltransferase or Succinyl transferase toproduce L Lysine.The L Lysine yield in the newly developedauxotrophic mutant of ddh recombinant strainof Corynebacterium glutamicum MTCC25069strain in the presence of Homoserine basedamino acids:

Auxotrophic ddh recombinant strain in thepresence of methionine and threonine hadproduced g/l of L Lysine, in the presence ofThreonine only had produced 22.34 g/l and 23.54g/l in methionine only and 22.92 g/l of L Lysine inthe absence of both methionine and Threonine fromthe auxotrophic recombinant mutant.



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Graph 7:L Lysine yield of Auxotrophic mutantof ddh Recombinant strain of C. glutamicum.In the presence of both Methionine and Threonine,the L Lysine Yield is 24.89 g/l, in the presence ofthreonine only the L Lysine yield is 23.54 g/l,methionine only is 22.34 g/l and in the absenceof Methionine and threonine is 21.92 g./l for theAuxotrophic mutant of ddh recombinantMTCC25069strain of C. glutamicum. Discussion:

The ddh Recombinant mutant producedmore amounts of L Lysine under because of effectchemical mutagenesis of ENU by blocking theHomoserine dehydrogenase of homoserinepathway diverting the aspartate â-semialdehydeto bind with 2,3 Dihydrodipicolinate synthase. Theauxotrophic recombinant mutant had shown a yield24.89 g/l L Lysine in the presence of threonineand Methionine.23.54, g/l in the presence ofthreonine only and 22.34 g/l in the methionine only

and 22.92 g/l without threonine and methionine inthe media signifies the mutation had occurred inthe Homoserine dehydrogenase which participatesin the Homoserine pathway of the auxotrophicmutant of ddh recombinant Corynebacteriumglutamicum ATCC 13032. Binding energy for theprotein homoserine dehydrogenase of – 5.2 K. Calwith one conventional hydrogen bond binds morestrongly with -0.7 k.cal greater than 2,3Dihydrodipicolinate synthase with -4.5 K.Cal with4 conventional hydrogen bonds with aspartate â-semialdehyde. Due to the lack of Homoserinedehydrogenase in the auxotrophic mutant of ddhrecombinant, the energy utilized in the synthesisof Threonine and Methionine by Homoserinepathway will be utilized in the L Lysine synthesisby binding with 2,3 Dihydrodipicolinate synthaseto produce L Lysine through L L diaminopimelate(2,3 meso-DAP). Even the number of hydrogenbonds and interacting amino acids of the proteinsplay an important role in the diverting theHomoserine pathway to DAP pathway can beanalyzed in the molecular docking analysis ofProtein and ligand.

Being a recombinant for ddh gene, therecombinant mutant expressed more amounts ofDDH enzyme which further diverts the strain fromentering 3 enzymes involved pathways ofacetyltransferase and Succinyl transferase to asingle enzyme involved ddh pathway in theproduction of L L diaminopimelate (2,3 meso-DAP)for the production of High yield of L Lysine.

Table 7: Ramachandran plot statistics representing the quality of proteins used for Modeling

S.No Plot statistics Homoserine 2,3 Dihydrodipi-dehydrogenase colinate Synthase:

1 Residues in most favored regions[A,B,L] 1124 91.5% 447 90.5%2 Residues in additional allowed regions[a,b,l,p] 92 7.5% 42 8.5%3 Residues in generously allowed regions [~a,~b,~l,~p] 8 0.7% 2 0.4%4 Residues in disallowed regions 4 0.3% 3 0.6%5 Number of non-glycine and non Proline residues 1228 100.0% 494 100%6 A number of end-residues (excl. Gly and Pro) 8 47 Number of glycine residues (shown as triangles) 92 248 Number of Proline residues 48 189 Total number of residues 1376 540



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Conclusion:By culturing the Chemically mutated ddh

recombinant MTCC25069strain the L Lysine yieldwas increased to 24.89 g/l at optimized parameters

of PH 7.5, temperature 30 C and 96 hrs. of time ofincubation compared to an auxotrophic mutant ofwild type strain with a yield of 18.57 g/l of L Lysine.



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Acknowledgement:I sincerely thank Science and Engineering

Research Board (SB/FTP/ETA-168/2012, Dt: .20-5-2013, DST, Govt, New Delhi-110070) for foundingthis entire project and also to JNTUK Kakinada forenabling me to continue my research project.

Conflict of Interest: The authors declare that theyhave no conflicts of interest.References:1. Min Ju Lee and Pil Kim,2018, Recombinant

Protein Expression System in Coryne-bacterium glutamicum and Its Application,Frontiers in Microbiology. doi.org/10.3389/fmicb.2018.02523

2. Wendisch VF, Bott M (2005) In Handbook ofCorynebacterium glutamicum, CRC Press.Taylor & Francis, Boca Raton, pp 377–396.doi.org/10.1201/9781420039696

3. Anastassiadis, S. 2007. L-lysine fermen-tation. Recent Patents Biotechnol., 1: 11-24. doi: 10.2174/187220807779813947

4. Shah, A.H., A. Hameed, and G.M. Khan.2002b. Fermentative production of L-lysine:bacterial fermentation. I. J. Med. Sci., 2: 52-157. Doi: 10.3923/jms.2002.152.157

5. Tosaka, O. 1983. The Production of L-lysineby Fermentation. Trends in Biotechnol., 1:70-74. doi: 10.1016/0167-7799(83)90055-0

6. Ikeda, M. 2003. Amino acid productionprocess. Adv. Biochem. Eng. Biotechnol.,79: 1– 35.doi:10.1016/0167-7799 (83) 90055-0

7. Xu JZ, Han M, Zhang JL, Guo YF, ZhangWG. Metabolic engineering Corynebacteriumglutamicum for the l lysine production byincreasing the flux into the l lysinebiosynthetic pathway. Amino Acids.2014;46:2165–75. doi: 10.1007/s00726-014-1768-1

8. Hilliger M, Haenel F, Menz J (1984) Inûuenceof temperature on growth and L-Lysineformation in Corynebacterium glutamicum.J Appl Microbiol 24:437–441. Doi: 10.1002/jobm.19840240704

Graph8: Yield of L Lysine of Auxotrophic mutantsof ddh recombinant and Wild Type C. glutamicumat Optimized conditions of ddh Recombinantmutant. R- L Lysine in an auxotrophic mutant ofddh Recombinant C. glutamicum MTCC25069is24.89(g/l). L -Lysine in an auxotrophic mutant ofwild type C. glutamicum MTCC2506918.57 (g/l).

The wild type mutant and ddh recombinantmutant had Chemical mutagen ENU inducedblockage in the homoserine pathway andoverexpression of ddh gene directed DAPsynthesis in ddh recombinant strain with theinvolvement of fewer enzymes compared withAcetyltransferase and Succinyl transferasepathways lead to increased production of L Lysinethan auxotrophic mutant Wild Type of C.glutamicum. Nearly 4.23 g/l amount of L Lysinewas increased in this newly developed Strain whencompared with normal ddh recombinant strainwhich had a yield of 20.66 g/l L Lysine. The LLysine Yield of Auxotrophic mutant of ddhrecombinant is 6.32 g/l L Lysine Yield more thanauxotrophic mutant wild type with a yield of 18.57g/l L Lysine. In the case of Auxotrophic mutant ofddh recombinant strain shown the maximum yieldin the presence of both threonine and methionineof 24.89 g/l L Lysine, 23.54 g/l L Lysine in thepresence of threonine only, 22.34 g/lL Lysine inthe presence of only methionine, and finally 22.92g/l of L Lysine in the absence of both methionineand threonine. From this study, the amount oflysine production enhanced is discussed on thebasis of molecular docking which further supportedour results. Hence we developed an auxotrophicmutant from the ddh gene recombinant ofCorynebacterium glutamicum MTCC25069whichwill be helpful in industrial L Lysine Productionthrough this new Auxotrophic mutant.



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9. Leuchtenberger W, Huthmacher K, Drauz K(2005) Biotechnological production of aminoacids and derivatives: current status andprospects. Appl Microbiol Biotechnol69(1):1–8. Doi: 10.1007/s00253-005-0155-y

10. Patek M (2007) Branched-chain amino acidsbiosynthesis pathways, regulation, andmetabolic engineering. Springer, Heidelberg778.

11. Hanson RS, Dillingham RL, Olson P, LeeGH, Cue D, Schendel FJ, Bremmon C,Flickinger MC (1996). Production of L-lysineand some other amino acids by mutants ofB. methanolicus. In M. E. Lidstrom and F.R. Tabita (ed.) Microbial growth on C1compounds. Kluwer Academic Publishers,Dordrecht, the Netherlands, pp. 227-234.Doi: 10.1007/978-94-009-0213-8_31

12. Tryfona T, Mak T. Fermentative productionof lysine by Corynebacterium glutamicum:transmembrane transport and metabolic fluxanalysis Process Biochem 2005; 40: 499-508. doi:10.1093/nar/gku989. PMC 4384041.PMID 25348405.

13. Hirono S, Sugimoto M, Nakan E, Izui M,HayakawaY. Method of producing L-lysine.United States Patent 6090597. 2000.

14. Liu, L., Yang, H., Shin, H. D., Li, J., Du, G.,and Chen, J. (2013). Recent advances inrecombinant protein expression byCorynebacterium, Brevibacterium, andStreptomyces: from transcription andtranslation regulation to secretion pathwayselection. Appl. Microbiol. Biotechnol. 97,9597–9608. doi: 10.1007/s00253-013-5250-x.

15. Liu, L., Yang, H., Shin, H. D., Li, J., Du, G.,and Chen, J. (2013). Recent advances inrecombinant protein expression byCorynebacterium, Brevibacterium, andStreptomyces: from transcription andtranslation regulation to secretion pathwayselection. Appl. Microbiol. Biotechnol. 97,9597-9608. doi: 10.1007/s00253-013-5250-x.

16. Lee, J. Y., Na, Y. A., Kim, E., Lee, H. S.,and Kim, P. (2016). Erratum to: The actino-

bacterium Corynebacterium glutamicum, anindustrial workhorse. J. Microbiol.Biotechnol. 26:1341. doi: 10.4014/jmb. 2016.2607.1341.

17. Hanson RS, Dillingham RL, Olson P, LeeGH, Cue D, Schendel FJ, Bremmon C,Flickinger MC (1996). Production of L-lysineand some other amino acids by mutants ofB. methanolicus. In M. E. Lidstrom and F.R. Tabita (ed.) Microbial growth on C1compounds. Kluwer Academic Publishers,Dordrecht, the Netherlands, pp. 227-234.Doi: 10.1007/978-94-009-0213-8_31

18. Tryfona T, Mak T. Fermentative productionof lysine by Corynebacterium glutamicum:transmembrane transport and metabolic fluxanalysis Process Biochem 2005; 40: 499-508. doi:10.1093/nar/gku989. PMC 4384041.PMID 25348405.

19. Xu JZ, Han M, Zhang JL, Guo YF, Qian H,Zhang WG. Improvement of l lysineproduction combines with minimization of byproducts synthesis in Corynebacteriumglutamicum. J Chem Technol Biot. 2014;89:1924–33.doi: 10.1007/s00726-014-1768-1

20. Van Ooyen J, Noack S, Bott M, Reth A,Eggeling L. Improved l lysine production withcorynebacterium glutamicum and systemicinsight into citrate synthase flux and activity.Biotechnol Bioeng. 2012; 109:2070–81. doi:10.1002/bit.24486

21. OZAKI, H. AND SHIIO, I., 1983. Productionlysine by pyruvate kinase mutants ofBrevibacterium flavum. Agri. Biol. Chem., 47:1569-1576. Doi: 10.1080/00021369. 1983.10865820

22. Coloìn, G. E.; Jetten, M. S.; Nguyen, T. T.;Gubler, M. E.; Follettie, M. T.; Sinskey, A.J.; Stephanopoulos, G. Effect of induciblethrB expression on amino acid production inCorynebacterium lactofermentum ATCC21799. Appl. Environ. Microbiol. 1995, 61,74"78.

23. Reinscheid, D. J.; Kronemeyer, W.; Eggeling,L.; Eikmanns, B. J.; Sahm, H. Stable



423

Expression of hom-1-thrB in Corynebacte-rium glutamicum and Its Effect on the CarbonFlux to Threonine and Related Amino Acids.Appl. Environ. Microbiol. 1994, 60, 126"132.

24. Kircher, M. and W. Pfeerle. 2001. The fermen-tative production of L-lysine as a annimalfeed additive. Chemosphere, 43: 27-31.

25. Seibold, G., M., Auchter, S. Berens, J.Kalinowski and B.J. Eikmanns. 2006.Utilizationn of soluble starch by arecombinant Corynebacterium glutamicumstrain: growth and lysine production. J.Biotechnol., 124: 381-391. Doi:10.1016/j.jbiotec.2005.12.027

26. Nelofer, R., Q. Syed and M. Nadeem. 2008.Statistical Optimization of Process Variablesfor L-lysine production by Corynebacteriumglutamicum. Turk. J. Biochem., 33 (2): 50- 57.

27. Shah, A. H., A. Hameed and G.M. Khan.2002a. Improved microbial Lysine productionby developing a new auxotrophic mutantofCorynebacteriumm glutamicum. Pak. J.Biol. Sci., 5: 80-83. DOI: 10.3923/pjbs.2002.80.83

28. Shah, A.H., A. Hameed and G.M. Khan.2002b. Fermentative production of L-lysine:bacterial fermentation. I. J. Med. Sci., 2: 52-157. DOI: 10.3923/jms.2002.152.157

29. Shah, A.H., A. Hameed, S.U. Rehman andA.H. Jan. 2012. Nutritional and mutationalaspects of Lysine production by Coryne-bacterium glutamicum auxotrophs. Pak. J.Zool., 44(1): 141-149.

30. PHAM, C. B., KATAOKA, H. AND MATSU-MURA, M., 1993. Optimization of batch fer-mentation conditions for L-lysine production.Annl. Rep. IC Biotech., 16: 506509.

31. MATOS, M. V. AND COELLO, N., 1999. L-Lysinee production by Corynebacteriumglutamicum non-growing cells.Acta CientVenez., 50: 233-239. PMID:10974714

32. Hanahan, D. 1985. Techniques fortransformation of E. coli. In D. M. Glover(ed.), DNA cloning, vol. 1. IRL Press, Oxford.

33. Ohnishi J, MitsuhashiA S, Hayashi M, AndoS, Yokoi K, Ochiai k, Ikeda M (2002) A novelmethodology employing Corynebacteriumglutamicum genome information to generatea new l-lysine producing mutant. AppliedMicrobiology and Biotechnology 58: 217-223.DOI 10.1007/s00253-001-0883-6

34. WittmannC, Heinzle E (2002) MetabolicEngineering of the Valine pathway inCorynebaceriumglutamicum – Analysis andModelling. Applied and EnvironmentalMicrobiology 68: 5843-5849.

35. Nishida H, Nishiyama M, Nobuyuki K,Kosuge T, Hoshino T, Yamane H (1999) Aprokaryotic gene cluster involved inthesynthesis of lysine through theaminoadipatee pathway: A key to the evolution ofamino acid biosynthesis. Genome Research9: 175- 1183. doi:10.1101/gr.9.12.1175

36. Waterhouse, A., Bertoni, M., Bienert, S.,Studer, G., Tauriello, G., Gumienny, R., Heer,F.T., de Beer, T.A.P., Rempfer, C., Bordoli,L., Lepore, R., Schwede, (2018) T.SWISS-MODELL: homology modelingg of proteinstructures and complexes. Nucleic AcidsRes. 46(W1), W296-W303. https://doi.org/10.1093/nar/gky427

37. Laskowski R A, Jab³oñska J, Pravda L,VaøekováRSS, Thornton J M (2018).PDBsum: Structural summaries of PDBentries. Prot. Sci., 27, 129-134. https://doi.org/10.1002/pro.3289

38. Laskowski R A, MacArthur M W, MossDSS,Thornton J M (1993). PROCHECK - aprogram to check the stereochemical qualityof protein structures. J. App. Cryst., 26, 283-291. 10.1107/S0021889892009944

39. Designing quorum sensing inhibitors ofPseudomonas aeruginosa utilizing FabI: anenzymic drug target fromthe fatty acidsynthesis pathway. Kalia M, Yadav VK,Singh PK, Dohare S, Sharma D, Narvi SS,Agarwal V. 3 Biotech. 2019 Feb;9(2):40. Doi:10.1007/s13205-019-1567-1

40. UniProt, Consortium. (January 2015).“UniProt: a hub for protein information”.Nucleic Acids Research. 43 (Databaseissue): D204. doi: 10.1093/nar/gku989


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