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Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli.

Kim SC, Min BE, Gyu Hwang H, Seo SW, Jung GY - Sci Rep (2015)

Bottom Line: To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5'-untranslated region (5'-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels.The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield.Thus, this work demonstrates that pathway optimization by 5'-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria.

View Article: PubMed Central - PubMed

Affiliation: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 790-784, Korea.

ABSTRACT
L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5'-untranslated region (5'-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5'-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria.

No MeSH data available.


Related in: MedlinePlus

Carbon flux redistribution at the PEP node by fine-tuning of ppsA expression.(a) Pathway optimization for fine-tuning the expression levels of the ppsA gene using UTR Designer. J23100 indicates a strong constitutive promoter from the Registry of Standard Biological Parts (BBa_J23100; http://partsregistry.org). (b) Comparisons of predicted expression levels from UTR Designer and specific enzyme activities of ppsA variants. (c) The specific L-tyrosine productivity of each ppsA variant after 24 h cultivation in M9 minimal medium. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.
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f2: Carbon flux redistribution at the PEP node by fine-tuning of ppsA expression.(a) Pathway optimization for fine-tuning the expression levels of the ppsA gene using UTR Designer. J23100 indicates a strong constitutive promoter from the Registry of Standard Biological Parts (BBa_J23100; http://partsregistry.org). (b) Comparisons of predicted expression levels from UTR Designer and specific enzyme activities of ppsA variants. (c) The specific L-tyrosine productivity of each ppsA variant after 24 h cultivation in M9 minimal medium. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.

Mentions: Although L-tyrosine productivity of E. coli was enhanced by amplification of the L-tyrosine biosynthetic pathway, we hypothesized that maximum L-tyrosine production would not be achieved by this method because of the metabolic imbalance between cell growth and L-tyrosine production around the PEP node. PEP is a starting material in the L-tyrosine pathway and is also used as precursor of pyruvate that enters into the TCA cycle (Fig. 2a). Accordingly, an excessive increase of flux toward the L-tyrosine pathway results in a decreased L-tyrosine productivity by cells due to growth inhibition caused by reduced flux to the TCA cycle. Optimization of flux distribution at the PEP node must therefore be optimized in order for maximum L-tyrosine productivity to be achieved.


Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli.

Kim SC, Min BE, Gyu Hwang H, Seo SW, Jung GY - Sci Rep (2015)

Carbon flux redistribution at the PEP node by fine-tuning of ppsA expression.(a) Pathway optimization for fine-tuning the expression levels of the ppsA gene using UTR Designer. J23100 indicates a strong constitutive promoter from the Registry of Standard Biological Parts (BBa_J23100; http://partsregistry.org). (b) Comparisons of predicted expression levels from UTR Designer and specific enzyme activities of ppsA variants. (c) The specific L-tyrosine productivity of each ppsA variant after 24 h cultivation in M9 minimal medium. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561953&req=5

f2: Carbon flux redistribution at the PEP node by fine-tuning of ppsA expression.(a) Pathway optimization for fine-tuning the expression levels of the ppsA gene using UTR Designer. J23100 indicates a strong constitutive promoter from the Registry of Standard Biological Parts (BBa_J23100; http://partsregistry.org). (b) Comparisons of predicted expression levels from UTR Designer and specific enzyme activities of ppsA variants. (c) The specific L-tyrosine productivity of each ppsA variant after 24 h cultivation in M9 minimal medium. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.
Mentions: Although L-tyrosine productivity of E. coli was enhanced by amplification of the L-tyrosine biosynthetic pathway, we hypothesized that maximum L-tyrosine production would not be achieved by this method because of the metabolic imbalance between cell growth and L-tyrosine production around the PEP node. PEP is a starting material in the L-tyrosine pathway and is also used as precursor of pyruvate that enters into the TCA cycle (Fig. 2a). Accordingly, an excessive increase of flux toward the L-tyrosine pathway results in a decreased L-tyrosine productivity by cells due to growth inhibition caused by reduced flux to the TCA cycle. Optimization of flux distribution at the PEP node must therefore be optimized in order for maximum L-tyrosine productivity to be achieved.

Bottom Line: To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5'-untranslated region (5'-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels.The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield.Thus, this work demonstrates that pathway optimization by 5'-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria.

View Article: PubMed Central - PubMed

Affiliation: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 790-784, Korea.

ABSTRACT
L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5'-untranslated region (5'-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5'-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria.

No MeSH data available.


Related in: MedlinePlus