Publication: Metabolic engineering of Corynebacterium glutamicum for L-DOPA production
Abstract
Mikrobiyal sistemler aracılığıyla aromatik amino asitlerden aromatik bileşiklerin üretimi ilgi çekici ve sürdürülebilir bir biyoteknolojik yaklaşımdır. Bu çalışmada, metabolizma mühendisliği stratejileri kullanılarak Corynebacterium glutamicum'da 3,4-dihidroksifenil-l-alanin (L-DOPA) üretimi amaçlanmıştır. L-DOPA, Parkinson hastalığının tedavisinde en sık kullanılan ilaçtır. Bu doğrultuda, Ralstonia solanacearum’dan alınan tirozinaz enzimi, C. glutamicum hücrelerinde heterolog olarak eksprese edilmiş ve fermentatif üretimde harici olarak eklenen L-tirozin ile birlikte 255 ± 20 mg/ L L-DOPA elde edilmiştir. Ayrıca, tam hücre biyotransformasyonu ile tirozinaz enzimini eksprese eden hücrelerde 6.6 mgDCW ile 400 ± 10 mg/ L L-DOPA üretimi sağlanmıştır. L-DOPA üretiminin sürdürülebilirliğini test etmek amacıyla fındık kabuğu hidrolizatı tek karbon kaynağı olarak ilk kez değerlendirilmiş ve 20 ± 1 mg/ L L-DOPA üretilmiştir. L-DOPA üretimi amacıyla yapılan ilk denemelerde, yabanıl tür hücrelerde harici L-tirozin kullanılmıştır. Harici L-tirozin kullanımının önüne geçmek amacıyla C. glutamicum rasyonel olarak tasarlanmıştır. L-tirozin aşırı üretimi şu şekilde sağlandı: (i) prefenat dehidrataz (pheA), antranilat sentaz (trpE) ve fenilalanin aminotransferaz (pat) genlerinde translasyonel başlangıç kodon değişimleri, (ii) shikimat dehidrojenaz ve 3-dehidrokinat dehidratazı kodlayan aroE ve qsuC genlerinin aşırı ekspresyonu, (iii) ptsG geninin silinmesi ve inositol permeaz (iolT2) ve glukokinazın (glcK) genlerinin aşırı ekspresyonu yoluyla glukoz alımını sağlayan PtsG sisteminin inaktivasyonu, (iv) C. glutamicum ARO02 suşunda L-tirozin sentezi için gereken öncü havuzunu artırmak için ksilozun kullanılması. Üretimi daha da arttırmak için, L-tirozin yolağındaki tanımlanamayan aminotransferaz tahmin edilmiş ve L-tirozin biyosentezindeki rolünden yararlanılmıştır. Ticari olarak temin edilebilen L-tirozini üreten C. glutamicum ATCC 21573 suşunun genomu dizilenmiş ve olası rasyonel tasarım modifikasyonları hakkında ipucu elde etmek için genom çapında analiz yapılmıştır. Son olarak, tasarlanan suşta en yüksek L-tirozin titresi 3.6 ± 0.10 g/ L olarak bulunmuştur. Bu suş, L-DOPA üretimi için kullanıldığında, 185 ± 9 mg/ L'lik bir titre elde edilmiştir.
Production of aromatic compounds derived from aromatic amino acids using microbial systems is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for 3,4-dihydroxyphenyl-l-alanine (L-DOPA) production is described. L-DOPA is the most commonly used drug for the treatment of Parkinson’s disease. In this work, Ralstonia solanacearum tyrosinase was heterologously expressed in C. glutamicum cells by fermentative production to get 255 ± 20 mg/ L of L-DOPA with externally added L-tyrosine. Tyrosinase expressing cells were also used for whole-cell biotransformation to get 400 ± 10 mg/ L of L-DOPA with 6.6 mgDCW. Furthermore, production with hazelnut husk hydrolysate as the sole carbon source was evaluated for sustainable L-DOPA production for the first time, and 20 ± 1 mg/ L of L-DOPA was produced. Initial L-DOPA production efforts used external L-tyrosine with wild-type cells. To circumvent external L-tyrosine addition, C. glutamicum was then rationally designed. L-tyrosine over-production was achieved by: (i) translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes, (ii) overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, (iii) inactivation of PtsG system for glucose uptake by deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK), and (iv) utilization of xylose to increase precursor pool for L-tyrosine synthesis in the chassis of C. glutamicum, ARO02. To further enhance production, the unidentified aminotransferase in L-tyrosine pathway was predicted and its role in L-tyrosine biosynthesis was exploited. The genome of the commercially available L-tyrosine producing strain C. glutamicum ATCC 21573 was sequenced and genome-wide analysis was performed to get clues on possible modifications for the rational design. Finally, the highest L-tyrosine titer was 3.6 ± 0.10 g/ L in the designed strain. When this strain was used for L-DOPA production, a titer of 185 ± 9 mg/ L was obtained.
Production of aromatic compounds derived from aromatic amino acids using microbial systems is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for 3,4-dihydroxyphenyl-l-alanine (L-DOPA) production is described. L-DOPA is the most commonly used drug for the treatment of Parkinson’s disease. In this work, Ralstonia solanacearum tyrosinase was heterologously expressed in C. glutamicum cells by fermentative production to get 255 ± 20 mg/ L of L-DOPA with externally added L-tyrosine. Tyrosinase expressing cells were also used for whole-cell biotransformation to get 400 ± 10 mg/ L of L-DOPA with 6.6 mgDCW. Furthermore, production with hazelnut husk hydrolysate as the sole carbon source was evaluated for sustainable L-DOPA production for the first time, and 20 ± 1 mg/ L of L-DOPA was produced. Initial L-DOPA production efforts used external L-tyrosine with wild-type cells. To circumvent external L-tyrosine addition, C. glutamicum was then rationally designed. L-tyrosine over-production was achieved by: (i) translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes, (ii) overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, (iii) inactivation of PtsG system for glucose uptake by deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK), and (iv) utilization of xylose to increase precursor pool for L-tyrosine synthesis in the chassis of C. glutamicum, ARO02. To further enhance production, the unidentified aminotransferase in L-tyrosine pathway was predicted and its role in L-tyrosine biosynthesis was exploited. The genome of the commercially available L-tyrosine producing strain C. glutamicum ATCC 21573 was sequenced and genome-wide analysis was performed to get clues on possible modifications for the rational design. Finally, the highest L-tyrosine titer was 3.6 ± 0.10 g/ L in the designed strain. When this strain was used for L-DOPA production, a titer of 185 ± 9 mg/ L was obtained.