Publication: Metabolik mühendisliği yöntemlerini kullanarak Corynebacterium glutamicum microorganizmasında L- DOPA üretiminin geliştirilmesi
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Date
2019-01-01
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Abstract
Bu çalışmada bir bitki patojeni olan Ralstonia solanacearum tirozinaz geniCorynebacterium glutamicum toprak bakterisinde uygun pEKEx2 plazmidine aktarılarakyeni plazmide pEKVx2 adı verilmiştir. Genin kontrolü yapıldıktan sonra bu rekombinantplazmid C. glutamicum’a aktarılmış ve L-DOPA üretimi gerçekleştirilmiştir. CGXII tanılıbesi yerinde yapılan çalışmada hiç bir optimizasyon çalışması yapılmadan 1 mg/ml L-tirozin ilavesi ile 211 mg/L düzeyinde L-DOPA eldesi sağlanmıştır. Aynı plazmid ile C.glutamicum MB001 (DE3) hücrelerinde üretim %57 oranında artmıştır.
The increase in the number of elderly people suffering from the symptoms of Parkinson’sdisease is leading to an expansion in the market size of L-DOPA (3,4-dihydroxyphenyl-L-alanine) which is an amino acid used for the treatment of this disease. The need for betterqualityproductsthrougheconomicallyfeasibleprocessesmakesbiotechnologicalapproaches attractive. The current project uses metabolic engineering approaches tosynthesize L-DOPA in Corynebacterium glutamicum using the tyrosinase gene from the plantpathogen Ralstonia solanacearum.The gram-positive soil bacterium C. glutamicum is currently well known as an industrialworkhouse. Initially it was identified as a glutamate producer. Accelerated with the progressin metabolic engineering, which is based on the development and integration of geneticengineering tools, systems biology and omics-based global analysis techniques, it is nowpossible to expand its capabilities for the production of a huge number of bio-based valueadded chemicals and recombinant proteins. Based on the fact that L-DOPA is an amino acid,we have hypothesized that the metabolism of C. glutamicum can be expanded to produce L-DOPA.To this end, the codon optimized tyrosinase gene from R. solanacearum has been insertedwith the required ribosome binding site into the pEKEx2 plasmid, suitable for expression in C.glutamicum. The plasmid harboring the gene with the correct sequence has been named aspEKVx2. Then this plasmid has been transformed into C. glutamicum cells for L-DOPAsynthesis. In CGXII defined medium with 0.4 mM CuSO 4 in the presence of 1 mg/ml L-tyrosine as the substrate 63 mg/L L-DOPA has been obtained while with 1.2 mM CuSO 4 L-DOPA yield was 211 mg/L L-DOPA at the end of 24 hours. CuSO 4 , that has been added toactivate the tyrosinase enzyme inhibited cell growth when used at high concentrations.Ascorbic acid which has been added at a final concentration of 1 mg/ml to prevent theoxidation of the produced L-DOPA to dopachrome has inhibited the tyrosinase enzymesynthesized. When the non-phosphotransferase system carbon sources ribose, acetate orcitrate were used as the sole carbon source, there was no significant L-DOPA synthesis. Onthe other hand, the expression of tyrosinse from the pEKVx2 plasmid in the phage-free C.glutamicum MB001 (DE3), increased L-DOPA synthesis by 57%.
The increase in the number of elderly people suffering from the symptoms of Parkinson’sdisease is leading to an expansion in the market size of L-DOPA (3,4-dihydroxyphenyl-L-alanine) which is an amino acid used for the treatment of this disease. The need for betterqualityproductsthrougheconomicallyfeasibleprocessesmakesbiotechnologicalapproaches attractive. The current project uses metabolic engineering approaches tosynthesize L-DOPA in Corynebacterium glutamicum using the tyrosinase gene from the plantpathogen Ralstonia solanacearum.The gram-positive soil bacterium C. glutamicum is currently well known as an industrialworkhouse. Initially it was identified as a glutamate producer. Accelerated with the progressin metabolic engineering, which is based on the development and integration of geneticengineering tools, systems biology and omics-based global analysis techniques, it is nowpossible to expand its capabilities for the production of a huge number of bio-based valueadded chemicals and recombinant proteins. Based on the fact that L-DOPA is an amino acid,we have hypothesized that the metabolism of C. glutamicum can be expanded to produce L-DOPA.To this end, the codon optimized tyrosinase gene from R. solanacearum has been insertedwith the required ribosome binding site into the pEKEx2 plasmid, suitable for expression in C.glutamicum. The plasmid harboring the gene with the correct sequence has been named aspEKVx2. Then this plasmid has been transformed into C. glutamicum cells for L-DOPAsynthesis. In CGXII defined medium with 0.4 mM CuSO 4 in the presence of 1 mg/ml L-tyrosine as the substrate 63 mg/L L-DOPA has been obtained while with 1.2 mM CuSO 4 L-DOPA yield was 211 mg/L L-DOPA at the end of 24 hours. CuSO 4 , that has been added toactivate the tyrosinase enzyme inhibited cell growth when used at high concentrations.Ascorbic acid which has been added at a final concentration of 1 mg/ml to prevent theoxidation of the produced L-DOPA to dopachrome has inhibited the tyrosinase enzymesynthesized. When the non-phosphotransferase system carbon sources ribose, acetate orcitrate were used as the sole carbon source, there was no significant L-DOPA synthesis. Onthe other hand, the expression of tyrosinse from the pEKVx2 plasmid in the phage-free C.glutamicum MB001 (DE3), increased L-DOPA synthesis by 57%.
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Biyoteknoloji ve Uygulamalı Mikrobiyoloji, Hücre ve Doku Mühendisliği, Malzeme Bilimleri, Biyomalzemeler