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Systems metabolic engineering for hydroxytyrosol production in Escherichia coli.

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Τίτλος: Systems metabolic engineering for hydroxytyrosol production in Escherichia coli.
Συγγραφείς: Jiang W; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China., Chen Z; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China., Fan W; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China., Li C; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China., Li L; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China., Yu Z; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China., Li X; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China., Xu Q; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.
Πηγή: Applied and environmental microbiology [Appl Environ Microbiol] 2026 May 20; Vol. 92 (5), pp. e0245525. Date of Electronic Publication: 2026 Apr 17.
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 7605801 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1098-5336 (Electronic) Linking ISSN: 00992240 NLM ISO Abbreviation: Appl Environ Microbiol Subsets: MEDLINE
Imprint Name(s): Original Publication: Washington, American Society for Microbiology.
Ιατρικοί όροι (MeSH): Phenylethyl Alcohol*/analogs & derivatives , Phenylethyl Alcohol*/metabolism , Metabolic Engineering*/methods , Escherichia coli*/metabolism , Escherichia coli*/genetics, Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/enzymology ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Alcohol Dehydrogenase/genetics ; Alcohol Dehydrogenase/metabolism ; Carboxy-Lyases/genetics ; Carboxy-Lyases/metabolism ; Mixed Function Oxygenases/genetics ; Mixed Function Oxygenases/metabolism ; Fermentation
Περίληψη: Hydroxytyrosol (HT), the primary functional component of olive oil, presents significant challenges for microbial biosynthesis due to its inherent chemical properties and fermentation requirements. In this study, we describe the modular engineering of HT production in Escherichia coli (E. coli). We initially employed a highly efficient 4-hydroxyphenylacetic acid 3-monooxygenase (encoded by the HpaBC gene) from E. coli, phenylpyruvate decarboxylase (encoded by the ARO10 gene), and alcohol dehydrogenase (encoded by the ADH6 gene) from Saccharomyces cerevisiae (S. cerevisiae) to establish the de novo biosynthetic pathway for HT in E. coli. To enhance carbon flux toward HT production, we attenuated the competing metabolic pathway while reinforcing the synthetic route and optimized the gene copy numbers of key enzymes involved. Further strain engineering involved strengthening the membrane-bound pyridine nucleotide transhydrogenase (encoded by the pntAB gene) and incorporating a heterologous riboflavin biosynthesis pathway to improve the supply of the cofactors NADPH and FADH₂, culminating in the generation of the high-producing strain HT32-3. Then, a two-stage potential of hydrogen (pH) and two-stage dissolved oxygen (DO) control strategy was implemented to mitigate oxidative degradation of HT. In addition, a fed-batch supplementation process was developed to address the dependency of the key enzyme ARO10 on the cofactor vitamin B₁ (VB₁). These combined strategies enabled the achievement of a final HT titer of 9.22 g/L in a 5 L bioreactor. The present study provides a practical strategy for the modification of HT strains and a fermentation strategy, which lays the foundation for the development of engineered strains of high-value derivatives of tyrosine.IMPORTANCEIn this study, a strain with high hydroxytyrosol (HT) production capacity was constructed by means of metabolic engineering modification. A two-stage pH and two-stage DO fermentation strategy was developed based on its physicochemical properties. Combined with the VB1 replenishment strategy, the fermentation process of this strain was optimized, and the retention of HT was significantly improved, laying the foundation for the large-scale production of HT. This study explored the metabolic synthesis pathway and efficient fermentation strategy of HT, providing an innovative fermentation strategy and a practical strategy for the fermentation and production of HT and its related products.
Competing Interests: The authors declare no conflict of interest.
Contributed Indexing: Keywords: Escherichia coli; hydroxytyrosol; metabolic engineering
Substance Nomenclature: ML9LGA7468 (Phenylethyl Alcohol)
10597-60-1 (3,4-dihydroxyphenylethanol)
0 (Escherichia coli Proteins)
EC 4.1.1.43 (phenylpyruvate decarboxylase)
EC 1.1.1.1 (Alcohol Dehydrogenase)
EC 1.14.14.9 (4-hydroxyphenylacetate 3-monooxygenase)
EC 4.1.1.- (Carboxy-Lyases)
EC 1.- (Mixed Function Oxygenases)
Entry Date(s): Date Created: 20260417 Date Completed: 20260715 Latest Revision: 20260715
Update Code: 20260715
PubMed Central ID: PMC13188884
DOI: 10.1128/aem.02455-25
PMID: 41995320
Βάση Δεδομένων: MEDLINE
FullText Text:
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Header DbId: cmedm
DbLabel: MEDLINE
An: 41995320
AccessLevel: 3
PubType: Academic Journal
PubTypeId: academicJournal
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Items – Name: Title
  Label: Title
  Group: Ti
  Data: Systems metabolic engineering for hydroxytyrosol production in Escherichia coli.
– Name: Author
  Label: Authors
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  Data: <searchLink fieldCode="AU" term="%22Jiang+W%22">Jiang W</searchLink>; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.<br /><searchLink fieldCode="AU" term="%22Chen+Z%22">Chen Z</searchLink>; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.<br /><searchLink fieldCode="AU" term="%22Fan+W%22">Fan W</searchLink>; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.<br /><searchLink fieldCode="AU" term="%22Li+C%22">Li C</searchLink>; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.<br /><searchLink fieldCode="AU" term="%22Li+L%22">Li L</searchLink>; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.<br /><searchLink fieldCode="AU" term="%22Yu+Z%22">Yu Z</searchLink>; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.<br /><searchLink fieldCode="AU" term="%22Li+X%22">Li X</searchLink>; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.<br /><searchLink fieldCode="AU" term="%22Xu+Q%22">Xu Q</searchLink>; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, People's Republic of China.; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, People's Republic of China.
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  Data: <searchLink fieldCode="JN" term="%227605801%22">Applied and environmental microbiology</searchLink> [Appl Environ Microbiol] 2026 May 20; Vol. 92 (5), pp. e0245525. <i>Date of Electronic Publication: </i>2026 Apr 17.
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  Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22American+Society+for+Microbiology%22">American Society for Microbiology </searchLink><i>Country of Publication: </i>United States <i>NLM ID: </i>7605801 <i>Publication Model: </i>Print-Electronic <i>Cited Medium: </i>Internet <i>ISSN: </i>1098-5336 (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%2200992240%22">00992240 </searchLink><i>NLM ISO Abbreviation: </i>Appl Environ Microbiol <i>Subsets: </i>MEDLINE
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  Data: <i>Original Publication</i>: Washington, American Society for Microbiology.
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  Data: <searchLink fieldCode="MM" term="%22Phenylethyl+Alcohol%22">Phenylethyl Alcohol*</searchLink>/<searchLink fieldCode="MM" term="%22Phenylethyl+Alcohol+analogs+%26+derivatives%22">analogs & derivatives</searchLink> <br /><searchLink fieldCode="MM" term="%22Phenylethyl+Alcohol%22">Phenylethyl Alcohol*</searchLink>/<searchLink fieldCode="MM" term="%22Phenylethyl+Alcohol+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Metabolic+Engineering%22">Metabolic Engineering*</searchLink>/<searchLink fieldCode="MM" term="%22Metabolic+Engineering+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Escherichia+coli%22">Escherichia coli*</searchLink>/<searchLink fieldCode="MM" term="%22Escherichia+coli+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Escherichia+coli%22">Escherichia coli*</searchLink>/<searchLink fieldCode="MM" term="%22Escherichia+coli+genetics%22">genetics</searchLink><br /><searchLink fieldCode="MH" term="%22Saccharomyces+cerevisiae%22">Saccharomyces cerevisiae</searchLink>/<searchLink fieldCode="MH" term="%22Saccharomyces+cerevisiae+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Saccharomyces+cerevisiae%22">Saccharomyces cerevisiae</searchLink>/<searchLink fieldCode="MH" term="%22Saccharomyces+cerevisiae+enzymology%22">enzymology</searchLink> ; <searchLink fieldCode="MH" term="%22Escherichia+coli+Proteins%22">Escherichia coli Proteins</searchLink>/<searchLink fieldCode="MH" term="%22Escherichia+coli+Proteins+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Escherichia+coli+Proteins%22">Escherichia coli Proteins</searchLink>/<searchLink fieldCode="MH" term="%22Escherichia+coli+Proteins+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Alcohol+Dehydrogenase%22">Alcohol Dehydrogenase</searchLink>/<searchLink fieldCode="MH" term="%22Alcohol+Dehydrogenase+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Alcohol+Dehydrogenase%22">Alcohol Dehydrogenase</searchLink>/<searchLink fieldCode="MH" term="%22Alcohol+Dehydrogenase+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Carboxy-Lyases%22">Carboxy-Lyases</searchLink>/<searchLink fieldCode="MH" term="%22Carboxy-Lyases+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Carboxy-Lyases%22">Carboxy-Lyases</searchLink>/<searchLink fieldCode="MH" term="%22Carboxy-Lyases+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Mixed+Function+Oxygenases%22">Mixed Function Oxygenases</searchLink>/<searchLink fieldCode="MH" term="%22Mixed+Function+Oxygenases+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Mixed+Function+Oxygenases%22">Mixed Function Oxygenases</searchLink>/<searchLink fieldCode="MH" term="%22Mixed+Function+Oxygenases+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Fermentation%22">Fermentation</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: Hydroxytyrosol (HT), the primary functional component of olive oil, presents significant challenges for microbial biosynthesis due to its inherent chemical properties and fermentation requirements. In this study, we describe the modular engineering of HT production in Escherichia coli (E. coli). We initially employed a highly efficient 4-hydroxyphenylacetic acid 3-monooxygenase (encoded by the HpaBC gene) from E. coli, phenylpyruvate decarboxylase (encoded by the ARO10 gene), and alcohol dehydrogenase (encoded by the ADH6 gene) from Saccharomyces cerevisiae (S. cerevisiae) to establish the de novo biosynthetic pathway for HT in E. coli. To enhance carbon flux toward HT production, we attenuated the competing metabolic pathway while reinforcing the synthetic route and optimized the gene copy numbers of key enzymes involved. Further strain engineering involved strengthening the membrane-bound pyridine nucleotide transhydrogenase (encoded by the pntAB gene) and incorporating a heterologous riboflavin biosynthesis pathway to improve the supply of the cofactors NADPH and FADH₂, culminating in the generation of the high-producing strain HT32-3. Then, a two-stage potential of hydrogen (pH) and two-stage dissolved oxygen (DO) control strategy was implemented to mitigate oxidative degradation of HT. In addition, a fed-batch supplementation process was developed to address the dependency of the key enzyme ARO10 on the cofactor vitamin B₁ (VB₁). These combined strategies enabled the achievement of a final HT titer of 9.22 g/L in a 5 L bioreactor. The present study provides a practical strategy for the modification of HT strains and a fermentation strategy, which lays the foundation for the development of engineered strains of high-value derivatives of tyrosine.IMPORTANCEIn this study, a strain with high hydroxytyrosol (HT) production capacity was constructed by means of metabolic engineering modification. A two-stage pH and two-stage DO fermentation strategy was developed based on its physicochemical properties. Combined with the VB<subscript>1</subscript> replenishment strategy, the fermentation process of this strain was optimized, and the retention of HT was significantly improved, laying the foundation for the large-scale production of HT. This study explored the metabolic synthesis pathway and efficient fermentation strategy of HT, providing an innovative fermentation strategy and a practical strategy for the fermentation and production of HT and its related products.
– Name: Abstract
  Label: Competing Interests
  Group: Ab
  Data: The authors declare no conflict of interest.
– Name: SubjectMinor
  Label: Contributed Indexing
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  Data: <i>Keywords: </i>Escherichia coli; hydroxytyrosol; metabolic engineering
– Name: NumberCAS
  Label: Substance Nomenclature
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  Data: ML9LGA7468 (Phenylethyl Alcohol)<br />10597-60-1 (3,4-dihydroxyphenylethanol)<br />0 (Escherichia coli Proteins)<br />EC 4.1.1.43 (phenylpyruvate decarboxylase)<br />EC 1.1.1.1 (Alcohol Dehydrogenase)<br />EC 1.14.14.9 (4-hydroxyphenylacetate 3-monooxygenase)<br />EC 4.1.1.- (Carboxy-Lyases)<br />EC 1.- (Mixed Function Oxygenases)
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  Data: <i>Date Created: </i>20260417 <i>Date Completed: </i>20260715 <i>Latest Revision: </i>20260715
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  Data: 10.1128/aem.02455-25
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  Data: 41995320
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        Text: English
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        StartPage: e0245525
    Subjects:
      – SubjectFull: Saccharomyces cerevisiae genetics
        Type: general
      – SubjectFull: Saccharomyces cerevisiae enzymology
        Type: general
      – SubjectFull: Escherichia coli Proteins genetics
        Type: general
      – SubjectFull: Escherichia coli Proteins metabolism
        Type: general
      – SubjectFull: Alcohol Dehydrogenase genetics
        Type: general
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      – SubjectFull: Carboxy-Lyases genetics
        Type: general
      – SubjectFull: Carboxy-Lyases metabolism
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      – SubjectFull: Mixed Function Oxygenases genetics
        Type: general
      – SubjectFull: Mixed Function Oxygenases metabolism
        Type: general
      – SubjectFull: Fermentation
        Type: general
      – SubjectFull: Phenylethyl Alcohol analogs & derivatives
        Type: general
      – SubjectFull: Phenylethyl Alcohol metabolism
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      – TitleFull: Systems metabolic engineering for hydroxytyrosol production in Escherichia coli.
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