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Production of cinnamic and p-hydroxycinnamic acid from sugar mixtures with engineered Escherichia coli.

Vargas-Tah A, Martínez LM, Hernández-Chávez G, Rocha M, Martínez A, Bolívar F, Gosset G - Microb. Cell Fact. (2015)

Bottom Line: These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroGfbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer.In this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates.These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Morelos, 62210, México. avargas@ibt.unam.mx.

ABSTRACT

Background: The aromatic compounds cinnamic acid (CA) and p-hydroxycinnamic acid (pHCA) are used as flavoring agents as well as precursors of chemicals. These compounds are present in plants at low concentrations, therefore, complex purification processes are usually required to extract the product. An alternative production method for these aromatic acids is based on the use of microbial strains modified by metabolic engineering. These biotechnological processes are usually based on the use of simple sugars like glucose as a raw material. However, sustainable production processes should preferably be based on the use of waste material such as lignocellulosic hydrolysates.

Results: In this study, E. coli strains with active (W3110) and inactive phosphoenolpyruvate:sugar phosphotransferase system (PTS) (VH33) were engineered for CA and pHCA production by transforming them with plasmids expressing genes encoding phenylalanine/tyrosine ammonia lyase (PAL/TAL) enzymes from Rhodotorula glutinis or Arabidopsis thaliana as well as genes aroGfbr and tktA, encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and transketolase, respectively. The generated strains were evaluated in cultures with glucose, xylose or arabinose, as well as a simulated lignocellulosic hydrolysate containing a mixture of these three sugars plus acetate. Production of CA was detected in strains expressing PAL/TAL from A. thaliana, whereas both CA and pHCA accumulated in strains expressing the enzyme from R. glutinis. These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroGfbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer. To improve pHCA production, a mutant with inactive pheA gene was generated, causing an 8-fold increase in the yield of this aromatic acid from the sugars in a simulated hydrolysate.

Conclusions: In this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates. These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.

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Related in: MedlinePlus

Yields of CA and pHCA from carbon sources by strains grown in glucose, xylose, arabinose or simulated hydrolysate (SH).
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Fig3: Yields of CA and pHCA from carbon sources by strains grown in glucose, xylose, arabinose or simulated hydrolysate (SH).

Mentions: In cultures employing glucose as the carbon source, strains WPJRg and VPJRg produced both CA and pHCA, whereas strains WPJAt and VPJAt produced only CA (Figures 2 and 3). This is the result of the distinct affinities for substrates displayed by the two expressed PAL/TAL enzymes, as it will be discussed below. Under these growth conditions, a clear improvement in specific productivity and yield from glucose for CA and pHCA was observed when comparing strains with the PTS− glucose+ and PTS+ phenotypes. The positive effect of PTS inactivation results from avoiding phosphoenolpyruvate consumption during glucose import. In strain VH33, PTS activity is replaced by glucose transport dependent on the GalP glucose/galactose symporter and ATP-dependent phosphorylation by glucokinase. The improved performance on aromatics production when employing glucose as carbon source has been reported for other PTS− glucose+ strains and the particular level of improvement is dependent on specific product and culture conditions [13,26].Figure 2


Production of cinnamic and p-hydroxycinnamic acid from sugar mixtures with engineered Escherichia coli.

Vargas-Tah A, Martínez LM, Hernández-Chávez G, Rocha M, Martínez A, Bolívar F, Gosset G - Microb. Cell Fact. (2015)

Yields of CA and pHCA from carbon sources by strains grown in glucose, xylose, arabinose or simulated hydrolysate (SH).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4305220&req=5

Fig3: Yields of CA and pHCA from carbon sources by strains grown in glucose, xylose, arabinose or simulated hydrolysate (SH).
Mentions: In cultures employing glucose as the carbon source, strains WPJRg and VPJRg produced both CA and pHCA, whereas strains WPJAt and VPJAt produced only CA (Figures 2 and 3). This is the result of the distinct affinities for substrates displayed by the two expressed PAL/TAL enzymes, as it will be discussed below. Under these growth conditions, a clear improvement in specific productivity and yield from glucose for CA and pHCA was observed when comparing strains with the PTS− glucose+ and PTS+ phenotypes. The positive effect of PTS inactivation results from avoiding phosphoenolpyruvate consumption during glucose import. In strain VH33, PTS activity is replaced by glucose transport dependent on the GalP glucose/galactose symporter and ATP-dependent phosphorylation by glucokinase. The improved performance on aromatics production when employing glucose as carbon source has been reported for other PTS− glucose+ strains and the particular level of improvement is dependent on specific product and culture conditions [13,26].Figure 2

Bottom Line: These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroGfbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer.In this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates.These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Morelos, 62210, México. avargas@ibt.unam.mx.

ABSTRACT

Background: The aromatic compounds cinnamic acid (CA) and p-hydroxycinnamic acid (pHCA) are used as flavoring agents as well as precursors of chemicals. These compounds are present in plants at low concentrations, therefore, complex purification processes are usually required to extract the product. An alternative production method for these aromatic acids is based on the use of microbial strains modified by metabolic engineering. These biotechnological processes are usually based on the use of simple sugars like glucose as a raw material. However, sustainable production processes should preferably be based on the use of waste material such as lignocellulosic hydrolysates.

Results: In this study, E. coli strains with active (W3110) and inactive phosphoenolpyruvate:sugar phosphotransferase system (PTS) (VH33) were engineered for CA and pHCA production by transforming them with plasmids expressing genes encoding phenylalanine/tyrosine ammonia lyase (PAL/TAL) enzymes from Rhodotorula glutinis or Arabidopsis thaliana as well as genes aroGfbr and tktA, encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and transketolase, respectively. The generated strains were evaluated in cultures with glucose, xylose or arabinose, as well as a simulated lignocellulosic hydrolysate containing a mixture of these three sugars plus acetate. Production of CA was detected in strains expressing PAL/TAL from A. thaliana, whereas both CA and pHCA accumulated in strains expressing the enzyme from R. glutinis. These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroGfbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer. To improve pHCA production, a mutant with inactive pheA gene was generated, causing an 8-fold increase in the yield of this aromatic acid from the sugars in a simulated hydrolysate.

Conclusions: In this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates. These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.

Show MeSH
Related in: MedlinePlus