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15INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
METIONIN AMINOKISLOTASINING MIKROBIOLOGIK SINTEZI VA UNING REGULYATSIYASI
1Abdurahmonov A.G'., 2Davranov Q.D.
1O'zMU Biologiya fakulteti stajyor-tadqiqotchisi 2O'zRFA Mikrobiologiya instituti direktori b.f.d. akademik https://doi.org/10.5281/zenodo.13883847
KIRISH
Metioninning tarixi o'tgan asrning yigirmanchi yillarining boshlarida boshlanadi. Nyu-Yorkdagi Kolumbiya universiteti tadqiqotchisi JH Myuller 1922-yilda "oltingugurt saqlagan aminokislota"ni ajratgan bo'lsada, uning molekulyar formulasini noto'g'ri yozgan. Oradan uch yil o'tib, uning yaponiyalik hamkasbi Odake bu formulani tuzatgan va unga "metionin" deb nom bergan. Olti yil o'tgach, G. Barger va FP Koyne bu aminokislota tuzilishini aniqlaganlar. Metionin inson va chorvachilik ratsionida zarur bo'lgan muhim aminokislotalardan biri bo'lib hisoblanadi. Metionin yetishmovchiligi turli kasalliklar jumladan, toksemiya, bolalikni revmatik isitmasi, mushaklar falaji, soch to'kilishi, depressiya, shizofreniya, Parkinson jigarni shikastlanishi va o'sishning buzilishi bilan bog'liq (44) holatlarni yuzaga chiqarishga olib kelishi mumkin ekanligi aniqlangan. Metionin parrandachilik va xom ashyo sanoatida keng qo'llaniladi (52; 38; 15). Fermentatsiya jarayonlari ko'plab aminokislotalarni arzon narxlarda ta'minlay olganligi sababli, metioninni tijorat ishlab chiqarish uchun mikrobial jarayonni ishlab chiqishga katta qiziqish mavjud (43; 57; 39). Hozirgi vaqtda metionin kimyoviy sintez yoki oqsillarni gidrolizlash orqali olinadi. Bu jarayonlar iqtisodiy jihatdan birmuncha qimmatga tushadi. Kimyoviy sintez natijasida D- va L metioninning aralashmasi hosil bo'lsa, (31; 30) oqsillarning gidrolizi esa metioninni ajratish kerak bo'lgan murakkab aralashma bilan yakunlanadi. Metionin izomerlarining kimyoviy ishlab chiqarilgan aralashmasi immobilizatsiyalangan ferment bioreaktorlari yordamida hal qilinishi mumkinligiga qaramasdan (55); kimyoviy sintez keng yo'lga qo'yilmagan. Chunki u akrolein, metil merkaptan, ammiak va sianid kabi xavfli kimyoviy moddalardan foydalanishni talab qiladi (14).
Biologik faol L-metionin fermentativ sintez yoki mikroorganizmlar yordamida fermentatsiya yo'li bilan ishlab chiqarilishi mumkin. L-metioninning sintezi aminoatsilazalar yordamida N-atsil-DL-metioninning stereospesifik bo'linishi orqali fermentativ yo'l bilan sodir bo'ladi (9). Mavjud fermentatsiya jarayonlari yaxshi rentabellikka ega bo'lsada, ular qimmat substratlardan foydalanishni talab qiladi. Bu, mikrobiologik sintez yo'li bilan metionin ishlab chiqarish bo'yicha tadqiqotlarni yanada takomillashtirishga bo'lgan qiziqishlarni orttiradi.
Glutamin kislota sintez qiluvchi bakteriyalarni o'rganish natijasida: aksariyat aminokislotalarni ishlab chiqarishni samaradorligi, ko'proq fermentatsiya jarayonlariga bog'liq bo'lishini isbotladi. Shu bilan birga aminokislotalarni sintez qilish xususiyatiga ega bo'lgan mikroorganizmlar shtammlarini topilishi, bu soha sanoat mikrobiologiyasining muhim yo'nalishi ekanligini ko'rsatdi. Lizin, treonin, izoleysin va gistidin kabi aminokislotalar mikrobiologik sintez yo'li bilan muvaffaqiyatli ishlab chiqarilgan (12; 30; 40; 41; 26). Fermentatsiya yordamida biologik faol L-metioninni sintez qilish unumini oshirishga qaratilgan ko'plab tadqiqotlar olib borilishiga qaramasdan (22; 37; 46; 34, 28). , hanuzgacha metionin fermentatsiyasi tijoratlashtirilmagan.
L-metionin sintezi uchun mikroorganizmlarni tanlash
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Metioninni mikrobiologik sintezi uchun tijorat jihatdan foydali jarayonni muvaffaqiyatli o'rnatish uchun sintezlash faolligi yuqori bo'lgan mikroorganizmlarni topish yoki mutatsiya yo'li bilan hosil qilish kerak. Boshqa tipdagi shtammlar odatda sezilarli miqdorda metionin ishlab chiqarishga qodir emas, chunki uning biosintezi yuqori darajada tartibga solinadi (45; 18). Metioninni mikrobiologik sintezi etanol, sut kislotasi va limon kislotasi kabi boshqa an'anaviy fermentatsiyalardan sezilarli darajada farq qiladi, masalan,. Adenozin tri-fosfat (ATF) hosil qilish uchun glikoliz jarayonida ishtirok etuvchi etil spirti va sut kislotasidan farqli o'laroq, metionin sintezi energiya (ATF) talab qiladi(1-rasm).
1-rasm. Corynebacterium va Brevibacteriumda metionin biosintezini chizmasi. Ko'k chiziqlar ingibitorni, nuqtali chiziqlar esa repressiyani anglatadi.
Corynebacterium va Brevibacterium shtammlarida barcha fermentlar yakuniy mahsulotlar tomonidan ingirbirlanadi yoki E. coli ga qaraganda metionin ishlab chiqarish uchun ancha sodda tartibga solish mexanizmlariga ega ekanligi kuzatilgan (1-rasm). (56). Buning sababi, ko'rsatilgan mikroorganizmlarni aminokislotalarga kam bo'lgan muhitda rivojlanishi bilan tushuntiriladi. Agar organizm aminokislotalar tanqisligi sharoitida yashasa, aminokislotalar biosintezini tartibga solishning asosiy vazifasi, organizmning o'sish tezligiga javoban, uning kerakli metobolitni sintezi tezligini mehanizmlarini toppish bilan bog'liq bo'ladi. Shunday qilib, hujayrada sintez qilinadigan turli xil aminokislotalarning nisbatlarini to'g'rilashga hojat qolmaydi, chunki ortiqcha metionin ishlab chiqarilishi, ortiqcha treonin, lizin va izoleysin kabi aminokislotalarning ham ishlab chiqarilishiga olib keladi. Buning asoosiy sababi rasmda ko'rsatilganidek ular ham bir xil metobolik jarayonda sintez bo'lishi bilan izohlangan. Shunday qilib, metionin ishlab chiqarish uchun nisbatan o'z-o'zini tartibga soluvchi mexanizmlariga ega bo'lgan mikroorganizmlarni tanlash asosiy yechim hisoblanadi. Corynebacterium va Brevibacterium larga qo'shimcha ravishda, ba'zi laktobakteriyalar va xamirturushlar kabi mikroorganizmlar metionin va lizin sintez qilishi aniqlangan (39).
Mikroorganizmlarda metionin biosintezi
Aminokislotalarning aspartat oilasiga metionin, lizin, treonin va izoleysin kiradi (3-rasm). Ushbu oilaga kiruvchi aminokislotalarning biosintez yo'llari Stadtman va boshqalar tomonidan
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
izohlab berilgan va metionin biosintezi tafsilotlari o'rganib chiqilgan. Bundan tashqari E. coli, Salmonella typhimurium da metionin biosintezining to'liq metabolik yo'li va uning regulyatsiyasi o'rganilgan. Rukert va boshqalar. (2003) metionin biosintezi va uning Bacillus subtilisda regulyatsiyasini hamda genom ketma-ketligi yordamida Corynebacterium glutamicumda L-metionin biosintezi yo'lini aniqlaganlar (2-rasm).
To'g'ridan-to'g'ri sulfidlanish
2-rasm. Corynebacterium glutamicumda metionin biosintezi [1] Aspartat kinaza, [2] aspartaldegidegidrogenaza, [3] gomoserin degidrogenaza, [4] gomoserin O-atsetiltransferaza, [5] O-atsetilgomoserin (tiol)-liaza (sistationin g-sintaza), [6] sistationin g-liaza, [7] gomoserin S-metiltransferaza (vitamin B12 mustaqil yoki metH-kodlangan, vitamin B12 bog'liq), [8] gomoserin kinaza, [9] treonin sintaza, [10] O-asetilgomoserin sulfidrilaza, [11] serin gidrooksimetil transferaza.
Turli mikroorganizmlarda metionin biosintezi yo'llari ko'plab umumiy xususiyatlarga ega. Mikroorganizmlar turli xil biosintetik yo'llardan foydalansa ham, ko'pchilik bakteriyalar va zamburug'lar metioninni sintez qila oladi. Odatda, aspartat aspartat-kinaza ta'sirida 4-fosfoaspartatga aylanadi va so'ngra aspartaldegid degidrogenaza bilan oksidlanadi va aspartat yarim aldegid hosil qiladi. Ikkinchisi gomoserin degidrogenaza bilan oksidlanib, gomoserin hosil qiladi. Shu bilan bir qatorda, aspartat yarim aldegid digidropikolinat-sintaza ta'sirida digidropikolinatga aylanadi va lizin hosil bo'lishiga olib keladi. Gomoserindan bir metabolik yo'l metioninga, ikkinchisi esa treoninga, keyin esa izoleysinga olib keladi. Gomoserin suksinil KoA bilan kondensatsiyalanib, O-suksinil gomoserin hosil qiladi. E. colidagi bu reaksiya uchun mas'ul bo'lgan ferment gomoserin osuksiniltransferazadir. Aksariyat bakteriyalar oraliq sifatida O-suksinilgomoserin ishlab chiqaradi. Aksincha, ko'pchilik zamburug'lar va ba'zi bakteriyalar (masalan, Bacillus va Corynebacterium) O-suksinilgomoserin o'rniga O-asetilgomoserin sintez qiladi (13).
O-asetil gomoserindan metionin hosil bo'lishi ikkita metabolik yo'l orqali amalga oshadi. Bular gomosistein sistationin orqali (masalan, Neurospora crassa kabi ba'zi ichak bakteriyalari va zamburug'larida) yoki atsetil gomoserin O-asetil gomoserin (tiol)-liaza tomonidan bevosita
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
gomosisteinga aylanadi (24). Morinaga va boshqalar. (60) fakultativ metilotrof Pseudomonas FM-518 bilan shunga o'xshash kuzatuvlarni o'tkazgan. Bu mikroorganizm h-sistationinaza va O-atsetil gomoserin sulfidrilaza faolligiga ega. Sistationin O-suksinil gomoserin va sisteindan sistationin g-sintaza fermenti yordamida sintezlanadi (13). Bu bosqich teskari va kofaktor sifatida piridoksal fosfatni talab qiladi. Keyin sistationin piridoksal fosfatga bog'liq bo'lgan sistationin-h-liaza tomonidan gomosistein, piruvat va ammiakga bo'linadi (45). B. flavumdagi metioninning biosintetik yo'li gomoserin O-asetiltransferaza ta'sirida gomoserindan O-asetilgomoserin hosil bo'lishini va O-asetilgomoserin sulfidrilaza (AHS) reaksiyasi (42) orqali O-asetilgomoserindan bevosita gomosistein hosil bo'lishini o'z ichiga oladi.
Hwang va boshqalar. (20) C.glutamicum , C. lactofermentum va B. flavum kabi tegishli korineform bakteriyalarda metionin biosintezi uchun ikkita parallel yo'l, transsulfuratsiya yo'li va to'g'ridan-to'g'ri sulfidrillanish yo'li mavjudligi haqida xabar berganlar. Trans-sulfatlanish holatida, sistein E. coli (50) uchun tavsiflanganidek, L-sistationinga olib keladigan oasetil-L-gomoserin bilan reaktsiya uchun oltingugurt donori bo'lib xizmat qiladi. To'g'ridan-to'g'ri sulfidrillanish holatida noorganik sulfid Leptospira meyeri (4) uchun tavsiflanganidek, O-asetil-gomoserin hosil qilish uchun ishlatiladi.
Sistein va gomosistein to'g'ridan-to'g'ri oltingugurtdan yoki bu ikki metabolitning o'zaro konversiyasi bilan sintezlanishi mumkin. Tiolatsiya yo'llari mos ravishda sistein yoki gomosistein ishlab chiqarish uchun sulfidni O-asetilserin yoki O-asetilgomoseringa to'g'ridan-to'g'ri kiritadi. Ushbu reaksiyalar O-atsetilserin tioliaza (27) yoki O-asetilgomoserin tioliaza (61) tomonidan katalizlanadi. Saccharomyces cerevisiae (54) va B. flavum (42) va L. meyeri (4) kabi bakteriyalar gomosisteinni tiollash orqali sintez qilishi mumkin. Transsulfatlanish yo'llari sistationinning vositachi shakllanishi orqali gomosistein va sisteinning o'zaro konversiyasiga imkon beradi. Sisteindan gomosistein sintezi ichak bakteriyalarida trans-sulfatlanishning yagona vositasidir (16). E. coli da buning uchun met B gen mahsuloti sistationin g-sintaza va met C gen mahsuloti sistationin h-liazaning ketma-ket ta'siri talab qilinadi (11). Gomosistein S-metiltransferaza bilan metilatsiyalangan gomosistein metionin hosil bo'lishiga olib keladi (24; 5; 54). Gomosistein S-metiltransferaza bilan gomosisteinning metillanishi B-12 vitaminiga bog'liq yoki mustaqil bo'lishi mumkin, ammo 5-metiltetragidrofolat (poliglutamat hosilasi) ikkala holatda ham metil donori sifatida ishlaydi (29).
Metionin biosintezining metabolik regulyatsiyasi
Barcha mikroorganizmlarda faqat kerakli miqdordagi aminokislotalarni sintez qilish uchun fermentlarning miqdori va turlarini tartibga solish mexanizmlari mavjud. Ushbu tartibga solish mexanizmi ko'p miqdorda maqsadli aminokislota ishlab chiqarishni ta'minlash uchun faolsizlantirilishi yoki boshqa tarzda modifikatsiya qilinishi kerak. Aspartatlar oilasiga kiruvchi aminokislotalarning biosintezini metabolik regulyatsiyasi o'rganib chiqilgan (33; 21; 18). Aspartat kinaza (EC 2.7.2.4) metabolik yo'lning birinchi fermenti bo'lib, asparta oilasiga kiruvchi aminokislotalarning sintezida qatnashadi. Bu ferment aspartatning fosforlanishini katalizlaydi. E. colida bu metabolik yo'lning har bir mahsuloti yo'lning birinchi fermentini ingibirlaydi yoki repressiya qiladi (2-rasm), bu esa tartibga solish mexanizmi bir mahsulot boshqa mahsulotlarni sintezini to'xtatmasligi bilan izohlanadi. Buning sababi shundaki, E. coli tarkibida bir xil reaksiyani katalizlash uchun uchta alohida aspartat kinaza mavjud va ular turli xil aminokislotalarni regulyatsiya qiladi. Aspartat kinaza-I treonin tomonidan ingibirlanadi va uning sintezi treonin va izoleysin tomonidan pasaytiriladi. Aspartat kinaza II metionin tomonidan,
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
aspartat kinaz III esa lizin tomonidan ingibirlanadi. Ushbu murakkab metabolik yo'lni tartibga solish juda murakkab, chunki u o'z faoliyatini muntazam davomi uchun bir mahsulotning ko'p miqdorda sintez qilinishi zamirida butun metabolizmni to'xtatmasligini talab qiladi (18).
Aspartat-yarimaldegidning gomoseringa qaytarilishi ikki xil gomoserin degidrogenazalar tomonidan katalizlanadi. Gomoserin degidrogenaza I sintezi treonin va izoleysin tomonidan ingibirlanadi va uning faolligi treonin tomonidan, gomoserin degidrogenaza II esa metionin tomonidan ingibirlanadi (1-rasm). Agar metionin sintez qilishi ortsa-da, lekin lizin, treonin va izoleysinning sintezi past darajasi bo'lsa, unda aspartat kinazani tartibga solishning o'zi, metionin sintezining qolgan uchta aminokislotalar to'g'ri nisbatiga olib kelmaydi. Shunday qilib, har bir aminokislota odatda o'zining birinchi fermentini boshqaradi. Aksincha, Brevibacterium va Corynebacterium sp. aspartat oilasiga kiruvchi aminokislotalar sintezi ancha sodda tartibga solishga ega. Ushbu mikroorganizmlarda lizin va treonin tomonidan birgalikda ingibirlangan faqat bitta aspartat kinaza mavjud. Shuning uchun aspartat kinazaning samarali qayta ingibirlanishi uchun ham lizin, ham treonin mavjud bo'lishi kerak (1-rasm). Lizin va treonin bir vaqtning o'zida 1 mM da mavjud bo'lganda, ular aspartat kinazani 94% ga ingibirlaydi, har bir aminokislota esa 1 mMda mavjud bo'lsa, aspartat kinazani faqat 12-20% ingibirlaydi xolos (56). C. glutamicumda metionin biosintezining tartibga soluvchi jihatlari bo'yicha olib borilgan tadqiqotlar, ekzogen metionin yo'lining ba'zi fermentlari sintezning kuchli repressori ekanligini ko'rsatdi (21, 22). Gomoserin esterin sintezini katalizlovchi ferment E. coli va B. subtilisda metionin va S-adenosilmetionin tomonidan qayta aloqani ingibirlashi kuzatilgan (6; 16). Biroq, boshqa tadqiqotlarda, gomoserin degidrogenaza E. coli, B. subtilis va C. glutamicumda L-treonin tomonidan allosterik ravishda ingibirlanishi kuzatilgan.
Metionin biosintezi ham transkripsiya jarayonida tartibga solinadi. C. glutamicumda L-metionin biosintezi fermentlarini kodlovchi genlarning aksariyati yaxshi tavsiflangan (17; 25; 20; 47). ). E. coli, MetJ repressor va MetR aktivatorida bu nazoratda ikkita regulyator ishtirok etishi aniqlangan. MetJ repressori S-adenosilmetionin bilan o'zaro ta'sirlanib, Met qutisi ketma-ketligiga bog'lanadi va Met genlarining ko'pchiligining transkripsiyasini bostiradi. MetR geni metionin sintezini kodlaydigan metE va metH genlarining ifodalanishini faollashuviga ko'mak beradi. Gomosistein MetE ifodasining MetR faollashuvini sezilarli darajada oshiradi (58; 16).
B. subtilisdagi metionin yoki sistein biosintezida ishtirok etishi taxmin qilingan bir qator genlar va operonlar kodlash ketma-ketligidan yuqori darajada saqlanib qolgan ketma-ketlikni o'z ichiga oladi, bu esa tartibga solish transkripsiyaning muddatidan oldin tugashini nazorat qilinishini ko'rsatadi.. Olimlarning takidlashicha taxminiy transkripsion repressor McbR C. glutamicumda L-metionin sintezini boshqaradigan metabolik tarmoqni tartibga solishda ishtirok etadi.
Rouberi (57) mikroorganizmlarda norleysinga (metioninning analogi) qarshilik metioninning biosintetik fermentlarning birortasini repressiya qila olmasligi va bu esa qarshilik mutatsiyasining bitta regulyator joyini o'zgartirishini ko'rsatadi.
Repressiv shtammlar butun biosintetik ketma-ketlikni, diffuziyali repressor sintezi orqali boshqaradilar. Ba'zi organizmlarda metioninning o'zi emas, balki S-adenosilmetionin (SAM) yoki uning hosilalari metionin biosintezining korepressorlari bo'lishi mumkin. SAM metionin biosintezida ishtirok etadigan fermentlarni ingibirlaydi. SAM ning repressiya va ingibirlashda ishtirok etishi Corynebacteriumdagi metionin biosintezidagi yakuniy mahsulot bo'lishi mumkinligini ko'rsatadi (21). Bacillusda metionin biosintezining birinchi fermentining sinergik ingibirlash ham mavjud. Metionin va SAM Bacillus polymyxa ning gomoserin
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
atsetiltransferazasini ingibir qilishi va har bir ingibitor faol joydan farq qiluvchi boshqa o'ziga xos saytga bog'lanishi mumkinligi e'tirof etilgan (59).
S. cerevisiae da SAM metionin bilan birgalikda emas, balki yakka o'zi metionin biosintezi metabolik yo'lining birinchi fermenti bo'lgan gomoserin atsetiltransferazasini ingibir qila oladi. E. coli da metionin regulon genlari butun xromosoma bo'ylab tarqalgan. S-adenosilmetioninning korepressor sifatidagi va MetJ gen mahsulotining aporepressor sifatidagi tartibga solish funktsiyalari E. coli metionin biosintezi genlarining ba'zilarining "in vitro" ifodasini o'rganish bilan tasdiqlangan (49, 50). Biroq, MetJ va MetK izolyatsiya qilingan tartibga soluvchi mutantlarning hech biri metionin sintezi uchun to'liq nazoratdan chiqarilmagan (51; 16). E. coli da aspartaldegiddegidrogenaza fermenti faqat bir xil bo'lib, lizin, treonin va metionin tomonidan ko'p valentli repressiyaga uchraydi.
Olimlar MetE va MetH genlarini ifodalash uchun zarur bo'lgan tartibga soluvchi lokus, MetRni aniqlaganlar. Biroq, MetE geni in vitro tadqiqotlarida o'z sintezini avtomatik tartibga solishi ko'rsatilgan. (7). MetR geni MetE geni ifodasining trans-aktivatoridir va MetR geni avtogen regulyatsiya ostida va MetJ oqsili tomonidan repressiya qilinadi (32). Metionin biosintezining oxirgi bosqichlarida ishtirok etadigan ikkita ferment E. coli da B12 vitamini metionin tomonidan muvofiqlashtirilmagan tarzda repressiya qilinadi (29). Nakamori va boshqalar (36) (1999) 910 mg/l metionin hosil qiluvchi E. coli ning L-metioninga chidamli mutantlarini olishga muvaffaq bo'lgan (mutantni L-metioninga chidamliligi metobolizmda ko'proq L-metionin sintez qilinishiga sabab bo'ladi) va L-metionin sintez qiluvchi mutantlarda MetJ genida faqat bitta nuqta mutatsiyasi sodir bo'lganligini aniqlaganlar. Ular E. coli MetJ repressor proteinini kodlovchi MetJ genini klonlashgan va to'rtta mustaqil L-metionin sintez qiluvchi mutantlarda 54-pozitsiyada bitta aminokislota o'rnini bosuvchi ( Ser-Asn) topildi. Begona tip MetJ geni mutant MetJ geniga ega mutantlarga kiritilganda, transformantlarda ferment sintezi va L-metionin sintez qilish darajasi begona tip darajasiga qaytishi aniqlangan (36).
Ozuqa tarkibi va kultura sharoitlarining metionin sinteziga ta'siri
Sanoat fermentatsiyasining muvaffaqiyati, ko'p jihatdan ozuqaviy muhitni tanlash va optimallashtirishga bog'liq (10). Produkt uchun uchun ozuqa muhiti tarkibida barcha kerakli komponentlar tegishli konsentratsiyalarda bo'lishi kerak. Masalan Corynebacterium sp. o'sish uchun, ozuqa muhiti tarkibida biotin qo'shimchasi bo'lishini talab qiladi. Ozuqa tarkibi mikroorganizmlar fiziologiyasiga katta ta'sir ko'rsatadi va mahsulotni maksimal darajada ishlab chiqarish qobiliyati ko'pincha ma'lum fiziologik xususiyatlar asosida optimallashtiradi (31). Mikroorganizmlar o'sish muhitiga javoban fermentlar konsortsiumini o'zgartiradi va shuning uchun ozuqa muhitlari mahsulot shakllanishiga yordam beradigan tarzda tanlanishi kerak. Bir qator olimlar metionin sintezida mikroorganizmlarni qulay ozuqa muhitda o'stirish va optimallashtirishning empirik hamda statistik usullaridan foydalanganlar.
Uglerod, azot manbalari va ularning fermentatsiya muhitidagi nisbati alohida metabolitlarni sintezlanishida muhim rol o'ynaydi (10). Turli xil shtammlar tomonidan L-metionin sintezi uchun uglerod manbalari ham turlichadir. Pham va boshqalar.(43) metionin sintezi uchun uglerod manbalari sifatida shakarqamish sharbati, shinni, banan, kassava va kokos suvidan foydalanishgan. Glyukoza eng keng tarqalgan uglerod manbai hisoblanadi (21; 8), ammo Banik va Majumdar (3) ba'zi manbalarda maltoza metionin sintezi uchun eng yaxshi uglerod manbai sifatida qayd etilgan. Ozuqa muhitini mineral birikmalar bilan boyitishda karbamid, ammoniy nitrat, ammoniy sulfat, ammoniy digidrogen fosfat, ammoniy xlorid, natriy nitrat, ammoniy asetat,
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ammoniy tatarat, ammoniy sitrat va ammoniy oksalat kabi turli xil organik va noorganik azot manbalari ishlatilgan. , (60; 53).
Mondal va boshqalar.(34) Qayd qilinishicha turli azot manbalari va turli darajadagi biotinning metionin sintezida muhim rol o'ynashi, bunda 60 mM ammoniy nitrat va 5 Ag / l biotin bilan boyitilgan ozuqa muhitida mikroorganizmlar eng yaxshi metionin sintez qilishi haqida ma'lumot berganlar. Garchan, bir qancha tadqiqotchilar xamirturush ekstraktidan metionin ishlab chiqarish uchun N-manbai sifatida foydalangan bo'lsalar ham, organik azot manbalaridan foydalanishni tavsiya etilmaganlar, chunki ular odatda ko'p aminokislotalar (shu jumladan metioninni) o'z tarkibiy qismi hisoblanadi va mikroorganizm ozuqa muhitida metionin mavjudligi sabab uning sintez qilinmasligiga olib keladi.
Fermentatsiyada uglerod va azot manbalariga qo'shimcha ravishda minerallar va metall ionlari muhim rol o'ynaydi, chunki metall ionlari turli fermentlar uchun kofaktorlar hisoblanadilar. Turli mikroelementlarning metionin fermentatsiyasiga ta'siri o'rganib chiqilganda, ular oltingugurt manbai sifatida natriy sulfat, metall ionlari sifatida Fe+2, Mn+2 va vitamin sifatida siyanokobalamindan foydalangan holda maksimal metionin sintez bo'lishini kuzatganlar. Metilotrof xamirturush Candida boidinii ICCF26 ning L-metioninga boy mutantlari oltingugurtni yuqori talab qilishi haqida ega va bir hujayrali oqsil sifatida ishlatilishi mumkin (Avram va boshq., 1991).
Kase va Nakayama (21) turli xil aminokislotalarning metionin biosintezi metabolik yo'lining oraliq birikmasi bo'lgan O-asetil-l-gomoserin ishlab chiqarishga ta'sirini o'rganishgan. Kase va Nakayama Metionin sintezida (21, 22, 23) 10% glyukoza, 2% (NH4)2SO4, 0,05% K2HPO4, 0,05% KH2PO4, 0,1% MgSO4-7H2O, 0,001%FeSO4-7H2O, 0.001% MnSO4-4H2O, 100 Hg/l biotin va 2% CaCO3 kabi kompanentlarni o'z ichiga olgan ozuqa muhiti optimal hisoblanishi bayon qilingan. Banik va Majumdar (3) 5% maltoza, 0,8% ammoniy nitrat, 0,1% K2HPO4, 0,03% MgSO4-7H2O, 1mg/l Na2MoO4d, FeSO4, 5H2O va 1m/g biotin pH 7 bo'lgan ozuqa muhitida 4,5 g/l metionin hosil qilganligini ma'lum qilgan. Ghosh va Banerjee (8) hujayralarning o'tkazuvchanligiga ta'sir qiluvchi moddalarning ta'sirini o'rganishgan (masalan, penitsillin, Tween 80, EDTA) va bu birikmalar metionin sintezi samaradorlgiga hech qanday ta'sir ko'rsatmasligini ma'lum qilganlar. Kislorodning aminokislota fermentatsiyasiga ta'siri o'rganilganda, shu ma'lum bo'ldiki, (1, 1979; 19). Sharma va Gomes (48) C.lilium NTE 99 dan foydalanib olib borilgan fermentatsiya jarayonlari natijasi metionin sintezi uchun 40% kislorod bilan to'yintirish eng
maqbul variant deb etirof etildi. O'stirishda qo'yiladigan talablar 10% D-glyukoza
1% karbamid, xamirturush ekstrakti. 30°C, pH 7,0. Mineral tuzlar. Aeratsiya - 40-45 mg O2 / l min Fermentatsiya davomiyligi - 50 soat L-Met unumi 50 g/l (0,6 mol/mol)
Xulosa
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Metioninni mikrobiologik sintezi boshqa bakteriyalarga qaraganda, Corynebacterium va Brevibacterium larni maxsus shtammlarida ancha soddaroq kechishi kuzatilgan va uni mexanizmi o'rganib chiqilgan.
Turli mikroorganizmlarda metionin biosintezi umumiylikga ega ekanligi ko'rsatib berilgan bo'lsada, turli shtammlarda L-metionin sintezi uchun talab qilinadigan uglerod va azot manbalari har xil bo'lishi aniqlangan.
Metionin sinteziga kislorodni, hujayrani o'tkazuvchanligiga ta'sir ko'rsatuvchi moddalarni ta'siri o'rganilgan va turli produsentlarda turli natijalar olingan.
Umumiy mutagenez orqali yuqori mahsuldor shtammlarni yaratish va ularni ajratib olishning klassik usullari metioninning ko'proq sintez qilishni ta'minlashda muvaffaqiyatli bo'lib chiqmagan. Buning mumkin bo'lgan sababi, metionin biosintezida oltingugurtning assimilyatsiyasi yetarli darajada ko'rib chiqilmaganligi bilan bog'liq bo'lsa ajab emas. To'g'ridan-to'g'ri sulfillanish, transsulfatlanish va teskari transsulfatlanish jarayonlari metionin biosintezi va uning hujayradan chiqarilishida (sekretsiyasida) muhim rol o'ynaydi. Molekulyar biologiya va genetika sohasidagi yutuqlarni hisobga olgan holda, endi qayta aloqa ingibirlanishini bartaraf etish uchun metioninning biosintetik yo'lini o'zgartirish uchun ma'lumotlar mavjud. Potensiali yuqori metionin sintezlanishiga lizin va treonin uchun ikki tomonlama auksotrofiyaga ega mutant yordamida erishish mumkin, Yani, agar u mutant metionin va sistein bilan metobolizm jarayonida qayta aloqani ingibir qilishga muvaffaq bo'lsa. Metionin muhim aminokislotalardan biri bo'lib turli sohalarda foydalaniladi. Masalan, hayvonlarning ozuqa ratsionida, ayniqsa parrandachilik chorvachilik cho'chqachilik va akvakulturada.
FOYDALANILGAN ADABIYOTLAR
1. Akashui K, Shibai H, Hirose Y. Effect of oxygen supply on l-lysine, l-threonine and l-isoleucine fermentations. Agric Biol Chem 1979;43:2087- 92.
2. Avram D, Stan R, Vassu T, Vamanu A, Dan F. Isolation of l-methionine enriched mutants from methylotrophic yeast Candida boidinii ICCF26 in a sulfur deficient medium. Recent Adv Biotechnol 1991;495- 6.
3. Banik AK, Majumdar SK. Studies on methionine fermentation: Part I. Selection of mutants of Micrococcus glutamicus and optimum conditions for methionine production. Indian J Exp Biol 1974;12:363- 5.
4. Belfaiza J, Martel A, Margarita D, Saint-Girons I. Direct thiolation for methionine biosynthesis in Leptospira meyeri. J Bacteriol 1998;180:250- 5.
5. Bourhy P, Martel A, Margarita D, Saint-Girons I, Belfaiza J. Homoserine O-acetyltransferase, involved in the Leptospira meyeri methionine biosynthetic pathway, is not feedback inhibited. J Bacteriol 1997;179:4396- 8.
6. Brush A, Paulus H. The enzymic formation of O-acetylhomoserine in Bacillus subtilis and its regulation by methionine and S-adenosylmethionine. Biochem Biophys Res Commun 1971;45:735- 41.
7. Cai XY, Maxon ME, Redfield B, Glass R, Brot N, Weissbach H. Methionine synthesis in Escherichia coli: effect of the metR protein on metE and metH expression. Proc Natl Acad Sci U S A 1989;86:4407- 11.
8. Chattopadhyay MK, Ghosh AK, Sengupta S, Sengupta D. Threonine analogue resistant mutants of Escherichia coli K-12. Biotechnol Lett 1995a;17:567-70.
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9. Chibata I, Ishikawa T, Yamada S. Studies on amino acids and studies on the enzymatic resolution and specificity of mold acylases. Bull Agric Chem Soc Jpn 1957;21:304- 7.
10. Chisti Y, Moo-Young M. Fermentation technology, bioprocessing, scale-up and manufacture. In: Moses V, Cape RE, Springham DG, editors. Biotechnology: the science and the business, 2nd ed. New York7 Harwood Academic Publishers; 1999. p. 177- 222.
11. Duchange N, Zakin MM, Ferrara P, Saint-Girons I, Park I, Tran SV, et al. Structure of the metJBLF cluster in Escherichia coli K12. Sequence of the metB structural gene and of the 5V- and 3V-flanking regions of the metBL operon. J Biol Chem 1983;258:14868-71.
12. Fan C, Chen L, Zheng S. Characteristics of l-lysine high yielding strain FMC8611. Weishengwuxue Zazhi 1988;8:11 -7.
13. Flavin M. Metabolic sulphur compounds. In: Greenberg DM, editor. Metabolic pathways. New York7 Academic Press; 1975. p. 457- 503.
14. Fong CV, Goldgraben GR, Konz J, Walker P, Zank NS. Condensation process for dl-methionine production. In: Goldfrab AS, editor. Organic chemicals manufacturing hazards. Ann Arbor7 Ann Arbor Science Publishers; 1981. p. 115-94.
15. Funfstuck R, Straube E, Schildbach O, Tietz U. Prevention of reinfection by l-methionine in patients with recurrent urinary tract infection. Med Klin 1997;92:574-81.
16. Greene RC. Biosynthesis of methionine. In: Neidhardt FC, Curtis III R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE, editors. Escherichia coli and Salmonella: Cellular and molecular biology, 2nd ed. Washington, DC7 American Society for Microbiology; 1996. p. 542- 60.
17. Grossmann K, Herbster K, Mack M. Rapid cloning of metK encoding methionine adenosyltransferase from Corynebacterium glutamicum by screening a genomic library on a high density colony-array. FEMS Microbiol Lett 2000;193:99-103.
18. Herrmann KM, Somerville RL. Amino acids biosynthesis and genetic regulation. New York7 Addison-Wesley; 1983. p. 147- 244.
19. Hillinger M, Hanel F. Process analysis of l-lysine fermentation under different oxygen supply. Biotechnol Lett 1981;3:219 - 24.
20. Hwang BJ, Yeom HJ, Kim Y, Lee HS. Corynebacterium glutamicum utilizes both trans-sulfuration and directsulfhydrylation pathways for methionine biosynthesis. J Bacteriol 2002;184:1277- 86.
21. Kase H, Nakayama K. Isolation and characterization of S-adenosylmethionine requiring mutants and role of Sadenosylmethionine in the regulation of methionine biosynthesis in Corynebacterium glutamicum. Agric Biol Chem 1975b;39:161-8.
22. Kase H, Nakayama K. l-methionine production by methionine analog-resistant mutants of Corynebacterium glutamicum. Agric Biol Chem 1975a;39:153- 60.
23. Kase H, Nakayama K. O-acetylhomoserine as an intermediate in methionine biosynthesis in Arthrobacter paraffineus, Corynebacterium glutamicum and Bacillus species. Agric Biol Chem 1975c;39:687-93.
24. Kerr DS, Flavin M. The regulation of methionine synthesis and the nature of cystathionine g-synthase in Neurospora. J Biol Chem 1970;245:1842- 55.
25. Kim JW, Kim HJ, Kim Y, Lee MS, Lee HS. Properties of Corynebacterium glutamicum metC gene encoding cystathionine-h-lyase. Mol Cell 2001;11:220- 5.
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_25-26 SEPTEMBER, 2024_
26. Kircher M, Pfefferle W. The fermentative production of l-lysine as an animal feed additive. Chemosphere 2001;43:27 -31.
27. Kredich NM. Biosynthesis of cysteine. In: Neidhardt, et al., editors. Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. Washington, DC7 American Society for Microbiology; 1987. p. 514- 27.
28. Kumar D, Bisaria VS, Sreekrishan TR, Gomes J. Production of methionine by a multi-analogues resistant mutant of Corynebacterium lilium. Process Biochem 2003;38:1165-71.
29. Kung HF, Spears C, Greene RC, Weissbach H. Regulation of the terminal reactions in the methionine biosynthesis by vitamin B12 and methionine. Arch Biochem Biophys 1972;150:23-31.
30. Leuchtenberger W. Amino acids—technical production and uses. In: Rehm HJ, Reed G, Phuler A, Stadler P, editors. Products of primary metabolism. Biotechnology, vol. 6. Weinheim7 VCH; 1996. p. 492.
31. Mannsfeld SP, Pfeiffer A, Tanner H, Liebertanz E. Continuous process for the manufacture of methionine. US Patent 04069251; 1978.
32. Maxon ME, Redfield B, Cai XY, Shoeman R, Fujita K, Fisher W, et al. Regulation of Methionine synthesis in Escherichia coli: effect of the metR protein on metE and metH expression. Proc Natl Acad Sci U S A 1989;86:85 - 9.
33. Miyajima R, Shiio I. Regulation of aspartate family amino acid biosynthesis in Brevibacterium flavum: VII properties of homoserine O-transacetylase. J Biochem 1973;73:1061- 8.
34. Mondal S, Chatterjee SP. Enhancement of methionine production by methionine analogue resistant mutants of Brevibacterium heali. Acta Biotechnol 1994;14:199- 204.
35. Mondal S, Das YB, Chatterjee SP. l-methionine production by double auxotrophic mutants of an ethionine resistant strain of Brevibacterium heali. Acta Biotechnol 1994;14:61-6.
36. Nakamori S, Kobayashi T, Nishimura H, Takagi H. Mechanism of l-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of l-methionine biosynthetic enzymes. Appl Microbiol Biotechnol 1999;52:179- 85.
37. Nakayama K, Araki K, Kase H. Microbial production of essential amino acid with Corynebacterium glutamicum mutants. Adv Exp Med Biol 1978;105:649- 61.
38. Neuvonen PJ, Tokola O, Toivonen ML, Simell O. Methionine in paracetamol tablets, a tool to reduce paracetamol toxicity. Int J Clin Pharmacol Ther Toxicol 1985;23:497- 500.
39. Odunfa SA, Adeniran SA, Teniola OD, Nordstorm J. Evaluation of lysine and methionine production in some lactobacilli and yeasts from Ogi. Int J Food Microbiol 2001;63:159-63.
40. Okamoto K, Ikeda M. Development of industrially stable process for l-threonine fermentation by an lmethionine auxotrophic mutant of Escherichia coli. J Biosci Bioeng 2000a;89:87- 9.
41. Okamoto K, Kino K, Ikeda M. Hyper production of l-threonine by an Escherichia coli mutant with impaired lthreonine uptake. Biosci Biotechnol Biochem 2000b;61:1877- 82.
42. Ozaki H, Shiio I. Methionine biosynthesis in Brevibacterium flavum: properties and essential role of Oacetylhomoserine thiolase. J Biochem 1982;91:1163-71.
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43. Pham CB, Galvez CF, Padolina WG. Methionine fermentation by batch fermentation from various carbohydrates. ASEAN Food J 1992;7:34-7.
44. Rose WC. The nutritive significance of the amino acids. Physiol Rev 1938;18:109-36.
45. Rowbury RJ, Woods DD. Further studies of the repression of methionine synthesis in Escherichia coli. J Gen Microbiol 1961;24:129-44.
46. Roy SK, Mishra AK, Nanda G. Extracellular production of l-methionine. Curr Sci 1984;53:1296- 7.
47. Rqckert C, Pqhler A, Kalinowski J. Genome-wide analysis of the l-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation. J Biotechnol 2003;104:213- 28.
48. Sharma S, Gomes J. Effect of dissolve oxygen on continuous production of methionine. Chem Eng Technol 2001;1:69-73.
49. Shoeman R, Redfield B, Coleman T, Greene RC, Smith AA, Brot N, et al. Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the expression of the metF gene. Proc Natl Acad Sci US A 1985a;82:3601 -5.
50. Smith DA. S-amino acid metabolism and its regulation in Escherichia coli and Salmonella typhimurium. Adv Genet 1971;16:141- 65.
51. Su CH, Greene RC, Holloway CT. Regulation of S-adenosylmethionine synthetase in Escherichia coli K-12. Bacteriol Proc 1970;70:136- 9.
52. Tabor H, Rosenthal SM, Tabor CW. Biosynthesis of spermidine and spermine from putrescine and methionine. J Biol Chem 1958;233:907- 17.
53. Tani Y, Lim WJ, Yang HC. Isolation of l-methionine-enriched mutants of a methylotrophic yeast, Candida boidinii No 2201. J Ferment Technol 1988;66:153-8.
54. Thomas D, Surdin-Kerjan Y. Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 1997;61:503-32.
55. Tosa T, Mori T, Fuse N, Chibata I. Studies on continuous enzyme reactions for preparation of a DEAE-sephadexaminoacylase column and continuous optical resolution of acyl-dl-amino acids. Biotechnol Bioeng 1967;9:603 -8.
56. Tosaka O, Takinami K. Biotechnology of amino acid production. In: Aida K, Chibata I, Nakayama K, editors. Progress in industrial microbiology, vol. 24. Tokyo7 Elsevier; 1986. p. 152-72.
57. Umerie SC, Ekwealor IA, Nawabo IO. Lysine production from various carbohydrates and seed meals. Bioresour Technol 2000;75:249- 52.
58. Weissbach H, Brot N. Regulation of methionine synthesis in Escherichia coli. Mol Microbiol 1991;5:1593-7.
59. Wyman A, Paulus H. Purification and properties of homoserine transacetylase from Bacillus polymyxa. J Biol Chem 1975;250:3897- 903.
60. Yamada H, Morinaga Y, Tani Y. l-methionine over-production by ethionine resistant mutants of obligate methylotroph strain Om 33. Agric Biol Chem 1982;46:47-55.
61. Yamagata S. Roles of O-acetyl-l-homoserine thiolases in micro-organisms. Biochimie 1989;71:1125-43.
