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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

Fermentation profiles of E. coli strains cultivated in complex medium.Data for the (a) wild-type (W3110; open symbols) and (b) SCK5 (closed symbols) E. coli strains are shown. pH adjustments were made at 6-h intervals. The left y-offset and right y-axis represent glucose and L-tyrosine concentrations (g/L), respectively. The left y-axis represents OD600 and the x-axis represents culture time (h). Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures. Symbols: circle, OD600; triangle, glucose; rectangle, L-tyrosine.
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f3: Fermentation profiles of E. coli strains cultivated in complex medium.Data for the (a) wild-type (W3110; open symbols) and (b) SCK5 (closed symbols) E. coli strains are shown. pH adjustments were made at 6-h intervals. The left y-offset and right y-axis represent glucose and L-tyrosine concentrations (g/L), respectively. The left y-axis represents OD600 and the x-axis represents culture time (h). Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures. Symbols: circle, OD600; triangle, glucose; rectangle, L-tyrosine.

Mentions: Although metabolic flux was optimized by controlling the expression level of ppsA, maximal L-tyrosine production in E. coli also requires optimization of the culture conditions. Cell growth and carbon uptake are inhibited in media with a low pH (resulting from the accumulation of L-tyrosine). For this reasons, the high L-tyrosine-producing E. coli strain engineered in this study (SCK5) produced only up to 0.3265 g/L L-tyrosine in batch cultures grown in M9 minimal medium. With pH control, L-tyrosine production was increased to 0.5155 g/L (Supplementary Table S2). We further tested the SCK5 E. coli strain as well as the other engineered ppsA variants in complex medium, after which L-tyrosine production was measured. As in the case of M9 minimal medium, L-tyrosine productivity among the ppsA variants grown in complex medium was highest in the SCK5 strain (Supplementary Fig. S2) and the highest titer was 1 g/L. After culturing in complex medium with pH adjustment, the L-tyrosine titer and productivity increased to 3 g/L, 0.0354 g/h/g DCW and 0.102 g L-tyrosine/g glucose yield from the SCK5 strain (Fig. 3b) compared with negligible L-tyrosine production by the wild type W3110 E. coli strain (Fig. 3a). However, glucose uptake by the SCK5 strain was reduced after ~48 h in culture when L-tyrosine started to be precipitated. We are not sure why glucose uptake is inhibited after ~48 h. One possibility is that tyrosine precipitation may be accompanied by cell aggregation48 and reduce the performance of the batch culture. Collectively, our findings indicate that L-tyrosine was produced to capacity in the engineered SCK5 E. coli strain, and that this capacity is limited by low pH as a result of L-tyrosine accumulation.


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)

Fermentation profiles of E. coli strains cultivated in complex medium.Data for the (a) wild-type (W3110; open symbols) and (b) SCK5 (closed symbols) E. coli strains are shown. pH adjustments were made at 6-h intervals. The left y-offset and right y-axis represent glucose and L-tyrosine concentrations (g/L), respectively. The left y-axis represents OD600 and the x-axis represents culture time (h). Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures. Symbols: circle, OD600; triangle, glucose; rectangle, L-tyrosine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Fermentation profiles of E. coli strains cultivated in complex medium.Data for the (a) wild-type (W3110; open symbols) and (b) SCK5 (closed symbols) E. coli strains are shown. pH adjustments were made at 6-h intervals. The left y-offset and right y-axis represent glucose and L-tyrosine concentrations (g/L), respectively. The left y-axis represents OD600 and the x-axis represents culture time (h). Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures. Symbols: circle, OD600; triangle, glucose; rectangle, L-tyrosine.
Mentions: Although metabolic flux was optimized by controlling the expression level of ppsA, maximal L-tyrosine production in E. coli also requires optimization of the culture conditions. Cell growth and carbon uptake are inhibited in media with a low pH (resulting from the accumulation of L-tyrosine). For this reasons, the high L-tyrosine-producing E. coli strain engineered in this study (SCK5) produced only up to 0.3265 g/L L-tyrosine in batch cultures grown in M9 minimal medium. With pH control, L-tyrosine production was increased to 0.5155 g/L (Supplementary Table S2). We further tested the SCK5 E. coli strain as well as the other engineered ppsA variants in complex medium, after which L-tyrosine production was measured. As in the case of M9 minimal medium, L-tyrosine productivity among the ppsA variants grown in complex medium was highest in the SCK5 strain (Supplementary Fig. S2) and the highest titer was 1 g/L. After culturing in complex medium with pH adjustment, the L-tyrosine titer and productivity increased to 3 g/L, 0.0354 g/h/g DCW and 0.102 g L-tyrosine/g glucose yield from the SCK5 strain (Fig. 3b) compared with negligible L-tyrosine production by the wild type W3110 E. coli strain (Fig. 3a). However, glucose uptake by the SCK5 strain was reduced after ~48 h in culture when L-tyrosine started to be precipitated. We are not sure why glucose uptake is inhibited after ~48 h. One possibility is that tyrosine precipitation may be accompanied by cell aggregation48 and reduce the performance of the batch culture. Collectively, our findings indicate that L-tyrosine was produced to capacity in the engineered SCK5 E. coli strain, and that this capacity is limited by low pH as a result of L-tyrosine accumulation.

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