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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

The L-tyrosine biosynthetic pathway engineering strategy.(a) Each gene was under the control of synthetic expression design that substitutes native promoter and 5′-UTR with synthetic constitutive promoter and designed 5′-UTR specific to target gene. (b) Dashed lines indicate feedback regulation, ‘X’ denotes deletion of the tyrR gene, and thick red arrows represent overexpression of genes in the L-tyrosine synthetic pathway. Abbreviations: Glc, glucose; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SHIK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; CHA, chorismate; PPA, prephenate; HPP, 3-hydroxyphenylpyruvate; L-tyr, L-tyrosine; KPP, keto-phenylpyruvate; L-Phe, L-phenylalanine. (c) Comparison of L-tyrosine productivity of wild-type (W3110) and rationally engineered (SCK1) E. coli strain. The specific productivity of L-tyrosine was increased to 0.0014 g/h/g DCW in the SCK1 strain while the wild type strain did not produce L-tyrosine. The y-axis represents specific productivity of L-tyrosine (g/h/g DCW) in each strain. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.
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f1: The L-tyrosine biosynthetic pathway engineering strategy.(a) Each gene was under the control of synthetic expression design that substitutes native promoter and 5′-UTR with synthetic constitutive promoter and designed 5′-UTR specific to target gene. (b) Dashed lines indicate feedback regulation, ‘X’ denotes deletion of the tyrR gene, and thick red arrows represent overexpression of genes in the L-tyrosine synthetic pathway. Abbreviations: Glc, glucose; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SHIK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; CHA, chorismate; PPA, prephenate; HPP, 3-hydroxyphenylpyruvate; L-tyr, L-tyrosine; KPP, keto-phenylpyruvate; L-Phe, L-phenylalanine. (c) Comparison of L-tyrosine productivity of wild-type (W3110) and rationally engineered (SCK1) E. coli strain. The specific productivity of L-tyrosine was increased to 0.0014 g/h/g DCW in the SCK1 strain while the wild type strain did not produce L-tyrosine. The y-axis represents specific productivity of L-tyrosine (g/h/g DCW) in each strain. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.

Mentions: To construct an L-tyrosine-overproducing strain (SCK1), a strong and constitutive promoter, BBA_J23100 from the Registry of Standard Biological Parts (http://partsregistry.org), was switched with each original promoter to increase the transcription levels of all genes in the L-tyrosine biosynthetic pathway. Additionally, a 5′-UTR sequence was designed by UTR Designer to achieve the maximum expression level of each gene in the tyrosine pathway (Fig. 1a). Based on the N-terminal coding sequence of each gene, UTR Designer generated different 5′-UTR sequences that can obtain maximum expression levels (Supplementary Table S1). Since the 5′-UTR sequences can be different depending on coding sequences that we input, we selected the sequence with the highest expression level for each gene and replaced it from the original UTR sequence of the gene on a chromosome using Red recombination. In case of aroB where we could not achieve high translation rate with native coding sequence, we further optimized the coding sequence with same codon preference to achieve high translation rate (Supplementary Table S1). Thus, each gene in the pathway was expressed as a monocistronic transcript under the control of the synthetic constitutive promoter and the 5′-UTR using inherent terminator (Fig. 1b). The tyrR gene encoding the TyrR protein was additionally knocked out to remove transcriptional regulation of the L-tyrosine pathway. Feedback-resistant variants of AroGfbr [D146N] and TyrAfbr [M53I; A354V] were then substituted for the wild-type enzymes to deregulate feedback inhibition by L-tyrosine8. In other studies, overexpression was performed using inducible promoter such as T7 promoter on a plasmid and only several target genes were engineered among all genes involved in tyrosine biosynthetic pathway8444546. However, in this study, each gene was under the control of constitutive strong promoters and synthetic 5′-UTRs on the chromosome to eliminate problems related with marker and plasmid origin incompatibilities as well as to eliminate the need for antibiotics and inducers15.


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)

The L-tyrosine biosynthetic pathway engineering strategy.(a) Each gene was under the control of synthetic expression design that substitutes native promoter and 5′-UTR with synthetic constitutive promoter and designed 5′-UTR specific to target gene. (b) Dashed lines indicate feedback regulation, ‘X’ denotes deletion of the tyrR gene, and thick red arrows represent overexpression of genes in the L-tyrosine synthetic pathway. Abbreviations: Glc, glucose; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SHIK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; CHA, chorismate; PPA, prephenate; HPP, 3-hydroxyphenylpyruvate; L-tyr, L-tyrosine; KPP, keto-phenylpyruvate; L-Phe, L-phenylalanine. (c) Comparison of L-tyrosine productivity of wild-type (W3110) and rationally engineered (SCK1) E. coli strain. The specific productivity of L-tyrosine was increased to 0.0014 g/h/g DCW in the SCK1 strain while the wild type strain did not produce L-tyrosine. The y-axis represents specific productivity of L-tyrosine (g/h/g DCW) in each strain. 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

f1: The L-tyrosine biosynthetic pathway engineering strategy.(a) Each gene was under the control of synthetic expression design that substitutes native promoter and 5′-UTR with synthetic constitutive promoter and designed 5′-UTR specific to target gene. (b) Dashed lines indicate feedback regulation, ‘X’ denotes deletion of the tyrR gene, and thick red arrows represent overexpression of genes in the L-tyrosine synthetic pathway. Abbreviations: Glc, glucose; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SHIK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; CHA, chorismate; PPA, prephenate; HPP, 3-hydroxyphenylpyruvate; L-tyr, L-tyrosine; KPP, keto-phenylpyruvate; L-Phe, L-phenylalanine. (c) Comparison of L-tyrosine productivity of wild-type (W3110) and rationally engineered (SCK1) E. coli strain. The specific productivity of L-tyrosine was increased to 0.0014 g/h/g DCW in the SCK1 strain while the wild type strain did not produce L-tyrosine. The y-axis represents specific productivity of L-tyrosine (g/h/g DCW) in each strain. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.
Mentions: To construct an L-tyrosine-overproducing strain (SCK1), a strong and constitutive promoter, BBA_J23100 from the Registry of Standard Biological Parts (http://partsregistry.org), was switched with each original promoter to increase the transcription levels of all genes in the L-tyrosine biosynthetic pathway. Additionally, a 5′-UTR sequence was designed by UTR Designer to achieve the maximum expression level of each gene in the tyrosine pathway (Fig. 1a). Based on the N-terminal coding sequence of each gene, UTR Designer generated different 5′-UTR sequences that can obtain maximum expression levels (Supplementary Table S1). Since the 5′-UTR sequences can be different depending on coding sequences that we input, we selected the sequence with the highest expression level for each gene and replaced it from the original UTR sequence of the gene on a chromosome using Red recombination. In case of aroB where we could not achieve high translation rate with native coding sequence, we further optimized the coding sequence with same codon preference to achieve high translation rate (Supplementary Table S1). Thus, each gene in the pathway was expressed as a monocistronic transcript under the control of the synthetic constitutive promoter and the 5′-UTR using inherent terminator (Fig. 1b). The tyrR gene encoding the TyrR protein was additionally knocked out to remove transcriptional regulation of the L-tyrosine pathway. Feedback-resistant variants of AroGfbr [D146N] and TyrAfbr [M53I; A354V] were then substituted for the wild-type enzymes to deregulate feedback inhibition by L-tyrosine8. In other studies, overexpression was performed using inducible promoter such as T7 promoter on a plasmid and only several target genes were engineered among all genes involved in tyrosine biosynthetic pathway8444546. However, in this study, each gene was under the control of constitutive strong promoters and synthetic 5′-UTRs on the chromosome to eliminate problems related with marker and plasmid origin incompatibilities as well as to eliminate the need for antibiotics and inducers15.

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