Maciej Wolak
Current Organizations / Institutes
The organizations/institutes from the scholar's latest article
Research Subjects
Maciej Wolak has a total of 6 co-authors. Here are the top co-authors by number of shared publications:
- Agata Matera
Department of Food Chemistry and Biocatalysis, Wroclaw University of Environmental and Life Sciences, 50-375 Wroclaw, Poland
1 shared publication - Ewa Huszcza
Department of Food Chemistry and Biocatalysis, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
1 shared publication - Jarosław Popłoński
Department of Food Chemistry and Biocatalysis, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
1 shared publication - Kinga Dulak
Department of Food Chemistry and Biocatalysis, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
1 shared publication - Kinga Hyla
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, 51-630 Wrocław, Poland
1 shared publication - Sandra Sordon
Department of Food Chemistry and Biocatalysis, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
1 shared publication
Latest Publications
- Open Access
P450 Fusion Protein Expressed in E. coli for Regioselective Hydroxylation of Flavonoids
Plant cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the regioselective hydroxylation of aromatic compounds. However, their expression in bacterial hosts is hampered by poor solubility, membrane anchoring and the requirement for redox partners. In this work, we report the design and characterization of modular expression systems that enable the functional production of SbCYP82D1.1 from Scutellaria baicalensis (SbF6H) in Escherichia coli. Both independent expression and synthetic fusion systems were evaluated by combining a CYP with a compatible reductase (ATR2_tr from Arabidopsis thaliana) to catalyze the conversion of chrysin into baicalein. A combinatorial library of N-terminal variants, host strains, media, and induction strategies was constructed and screened. Among the tested host, E. coli DH 10-beta provided the highest product titers, particularly when cultures were supplemented with 5-aminolevulinic acid. Truncation of the native transmembrane anchor significantly improved catalytic performance, whereas the addition of the heterologous MALLLAVF tag decreased activity. Fusion systems outperformed separate expression formats, showing approximately two-fold higher activity, with the flexible glycine–serine linker (L_GS) supporting the highest hydroxylation product formation. The corresponding fusion construct showed an apparent conversion of 0.1 mM chrysin to baicalein of up to 90% under the applied whole-cell reaction and analytical conditions, although this value should be interpreted with caution due to the concurrent instability of baicalein observed in all reactions and culture conditions. This result nevertheless indicates a marked improvement in whole-cell baicalein formation compared with previously reported bacterial systems. Together, these results demonstrate that rational N-terminal engineering combined with fusion protein design can enable efficient bacterial expression of plant CYPs, representing a promising step toward scalable production of hydroxylated flavonoids.
Open AccessP450 Fusion Protein Expressed in E. coli for Regioselective Hydroxylation of Flavonoids
Plant cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the regioselective hydroxylation of aromatic compounds. However, their expression in bacterial hosts is hampered by poor solubility, membrane anchoring and the requirement for redox partners. In this work, we report the design and characterization of modular expression systems that enable the functional production of SbCYP82D1.1 from Scutellaria baicalensis (SbF6H) in Escherichia coli. Both independent expression and synthetic fusion systems were evaluated by combining a CYP with a compatible reductase (ATR2_tr from Arabidopsis thaliana) to catalyze the conversion of chrysin into baicalein. A combinatorial library of N-terminal variants, host strains, media, and induction strategies was constructed and screened. Among the tested host, E. coli DH 10-beta provided the highest product titers, particularly when cultures were supplemented with 5-aminolevulinic acid. Truncation of the native transmembrane anchor significantly improved catalytic performance, whereas the addition of the heterologous MALLLAVF tag decreased activity. Fusion systems outperformed separate expression formats, showing approximately two-fold higher activity, with the flexible glycine–serine linker (L_GS) supporting the highest hydroxylation product formation. The corresponding fusion construct showed an apparent conversion of 0.1 mM chrysin to baicalein of up to 90% under the applied whole-cell reaction and analytical conditions, although this value should be interpreted with caution due to the concurrent instability of baicalein observed in all reactions and culture conditions. This result nevertheless indicates a marked improvement in whole-cell baicalein formation compared with previously reported bacterial systems. Together, these results demonstrate that rational N-terminal engineering combined with fusion protein design can enable efficient bacterial expression of plant CYPs, representing a promising step toward scalable production of hydroxylated flavonoids.