Design and Assembly of a Biofactory for (2S)-Naringenin Production in Escherichia coli: Effects of Oxygen Transfer on Yield and Gene Expression

Daza, Laura E. Parra; Medina, Lina Suarez; Rangel, Albert Enrique Tafur; Fernández-Niño, Miguel; Mejía‐Manzano, Luis Alberto; González‐Valdez, José; Reyes, Luis H.; González-Barrios, Andrés Fernando

doi:10.3390/biom13030565

Cited by 3 publications

(1 citation statement)

References 40 publications

(55 reference statements)

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“…Naringenin chalcone, synthesized by CHS, is unstable but can be converted efficiently in vivo to stable (2S)-naringenin by chalcone isomerase (CHI). 6,23 Therefore, overexpressing CHI may increase the (2S)-naringenin titer by immediately converting naringenin chalcone before it can degrade. The TtgR-based biosensor was used to screen CHS mutants for increased catalytic activity because of the high specificity, S/N ratio, and broad detection range of the biosensor for (2S)-naringenin.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution

Tong,

Li,

Zhou

et al. 2024

ACS Synth. Biol.

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Chalcone synthase (CHS) catalyzes the rate-limiting step of (2S)-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2S)-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2S)-naringenin biosensor based on the TtgR operon was constructed in Escherichia coli and its detection range was expanded by promoter optimization to 0−300 mg/L, the widest range for (2S)-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, SjCHS1 S208N with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for de novo biosynthesis of (2S)-naringenin by SjCHS1 S208N was constructed in Saccharomyces cerevisiae, combined with CHS precursor pathway optimization, increasing the (2S)-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2S)-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution

Tong,

Li,

Zhou

et al. 2024

ACS Synth. Biol.

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Combinatorial optimization of pathway, process and media for the production of p-coumaric acid bySaccharomyces cerevisiae

Moreno-Paz,

van der Hoek,

Eliana

et al. 2023

Preprint

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Microbial cell factories are instrumental in transitioning towards a sustainable bio-based economy, offering alternatives to conventional chemical processes. However, fulfilling their potential requires simultaneous screening for optimal media composition, process and genetic factors, acknowledging the complex interplay between the organism’s genotype and its environment. This study employs statistical Design of Experiments (DoE) to systematically explore these relationships and optimize the production of p-coumaric acid (pCA) inSaccharomyces cerevisiae. Two rounds of fractional factorial designs were used to identify factors with a significant effect on pCA production, which resulted on a 168-fold improvement on pCA titer. Moreover, a significant interaction between the culture temperature and expression of ARO4 highlighted the importance of simultaneous process and strain optimization. The presented approach leverages the strengths of experimental design and statistical analysis and could be systematically applied during strain and bio-process design efforts to unlock the full potential of microbial cell factories.

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Saccharomyces cerevisiae biofactory to produce naringenin using a systems biology approach and a bicistronic vector expression strategy in flavonoid production

Mejía-Manzano,

Ortiz-Alcaráz,

Parra Daza

et al. 2024

Microbiol Spectr

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Naringenin is the central flavonoid in the biosynthesis of several bioactive compounds and presents a growing demand for its nutraceutical properties. Naringenin extraction from plants is non-viable due to low yields, and microbial platforms could represent a controlled and sustained alternative to produce it using several metabolic engineering tools. This study shows the naringenin production in Saccharomyces cerevisiae from glucose through a combined approach of systems biology, enzyme criteria selection, and a molecular engineering strategy. In silico prediction using a mixed integer linear programming (MILP) algorithm showed that the phenylpropanoid pathway was the shortest and most viable metabolic pathway. Two biscistronic constructs were generated using the PTV-1 2A peptide sequence, and a naringenin biofactory was assembled with the phenylalanine ammonia-lyase/tyrosine ammonia-lyase genes encoding phenylalanine/tyrosine ammonia-lyase ( Rhodobacter capsulatus ), 4-coumaroyl (4 Cl) encoding a p -coumaroyl-CoA ligase ( Solanum lycopersicum ), CHS encoding chalcone synthase ( Hypericum androsaemum ), and CHI encoding a chalcone isomerase ( Glycine max ). Naringenin productivity in batch fermentation was about 40.67 ± 3.47 µg/Lh with a 6.10 ± 0.52 mg/L titer (22.41 ± 1.91 µM) and a 3.26 ± 1.36 mg/g yield ( Y P / S ) with the detection of additional flavonoids. The obtained concentration is better than other related works in diverse engineered microorganisms. The results suggest a successful and optimizable alternative for the heterologous flavanone production in yeast combined with bicistronic expression mediated by a 2A peptide sequence for the first time. This strategy supports the production of extensive routes for other nutraceutical compounds. IMPORTANCE Flavonoids are a group of compounds generally produced by plants with proven biological activity, which have recently beeen recommended for the treatment and prevention of diseases and ailments with diverse causes. In this study, naringenin was produced in adequate amounts in yeast after in silico design. The four genes of the involved enzymes from several organisms (bacteria and plants) were multi-expressed in two vectors carrying each two genes linked by a short viral peptide sequence. The batch kinetic behavior of the product, substrate, and biomass was described at lab scale. The engineered strain might be used in a more affordable and viable bioprocess for industrial naringenin procurement.

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Design and Assembly of a Biofactory for (2S)-Naringenin Production in Escherichia coli: Effects of Oxygen Transfer on Yield and Gene Expression

Cited by 3 publications

References 40 publications

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution

Combinatorial optimization of pathway, process and media for the production of p-coumaric acid bySaccharomyces cerevisiae

Saccharomyces cerevisiae biofactory to produce naringenin using a systems biology approach and a bicistronic vector expression strategy in flavonoid production

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