Promoting FADH₂ Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli

Abstract

Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae, the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase (hpaB) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli

“…We integrated these two enzymes into the motA site of the genome for tandem expression using the P J23119 inducible promoter and two ribosome binding sites (RBS) (Figure A). The use of an IPTG-inducible promoter was to avoid imposing excessive metabolic burden on bacterial growth in the early stage, affecting growth or causing death . By adding sodium formate as a substrate to the culture medium, the exogenous NAD­(P)H regeneration system was successfully introduced.…”

“…For strains requiring IPTG induction, when the optical density at 600 nm (OD 600 ) reached 1.0, IPTG was added to a final concentration of 0.1 mM and the temperature was lowered to 30 °C. Lowering temperature helped in proper protein folding and reduced stress on cells expressing foreign proteins …”

Multienzymatic Cascade for Synthesis of Hydroxytyrosol via Two-Stage Biocatalysis

“…Since the high price of HT limits its use in the market, new approaches to access this important compound are constantly developed. − The present study was for the first time to produce high-value-added 5 from cheaper and more accessible materials 1 and l -threonine, making our approach more competitive than those that started with l -DOPA or l -tyrosine. Compared with metabolic engineering options to access 5 , our route required less catalytic processing (5 h), leading to a significantly greater space–time yield (0.88 g/L/h vs 0.26 g/L/h or 0.28 g/L/h). , Furthermore, our biocatalytic method, which employed resting cells as whole-cell catalysts in a buffered system, made it easier to purify the final product. These outcomes demonstrated the straightforward and ecofriendly use of this basic one-pot two-stage cascade biocatalysis for the efficient synthesis of 5 from low-cost material 1 in high yields.…”

“…Although l -tyrosine is cheaper and easier to obtain, the low activity and excessive oxidation of HpaBC render this route inappropriate for industrious purpose . Besides, microbial fermentation is also a promising way to produce HT from inexpensive materials. , However, the growth stage of the bacterial strain is not suitable for synthesis of HT because of its extensive antimicrobial activity. , Producing high-value-added HT from low-cost substrates is undoubtedly more competitive. Recently, a one-pot cascade biocatalytic approach for converting lignin-derived catechol and phenol into HT was reported, offering new and viable routes for the sustainable and environmentally friendly production of HT from low-cost sources .…”

Directed Evolution of 4-Hydroxyphenylpyruvate Biosensors Based on a Dual Selection System

“…They are expected to reach 130 million by 2030 [1]. Additionally, compounds such as tyrosol [2], salidroside [3] and salvianic acid A have gained prominence in the fields of medicine and food health. Phenols are typically and chemically extracted from plants for high product yields.…”