Publications

• Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases.

  Yu D., Xu F., Gage D., Zhan J.

  BbBSLS and BbBEAS were dissected and reconstituted in Saccharomyces cerevisiae. The intermodular linker is essential for the reconstitution of the separate modules. Module 1 can be swapped between BbBEAS and BbBSLS, while modules 2 and 3 control the product profiles. BbBSLS is a flexible enzyme th ... >> More

  BbBSLS and BbBEAS were dissected and reconstituted in Saccharomyces cerevisiae. The intermodular linker is essential for the reconstitution of the separate modules. Module 1 can be swapped between BbBEAS and BbBSLS, while modules 2 and 3 control the product profiles. BbBSLS is a flexible enzyme that also synthesizes beauvericins. << Less

  Chem. Commun. (Camb.) 49:6176-6178(2013) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae.

  Yu D., Xu F., Zi J., Wang S., Gage D., Zeng J., Zhan J.

  Two fungal cyclooligomer depsipeptide synthetases(CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, r ... >> More

  Two fungal cyclooligomer depsipeptide synthetases(CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, respectively. The titers of beauvericins (33.8 ± 1.4 mg/l) and bassianolide (21.7± 0.1 mg/l) in the engineered S. cerevisiae BJ5464-NpgA strains were comparable to those in the native producer B. bassiana. Feeding D-hydroxyisovaleric acid (D-Hiv) and the corresponding L-amino acid precursors improved the production of beauvericins and bassianolide. However, the high price of D-Hiv limits its application in large-scale production of these cyclooligomer depsipeptides. Alternatively, we engineered another enzyme, ketoisovalerate reductase (KIVR) from B. bassiana, into S. cerevisiae BJ5464-NpgA for enhanced in situ synthesis of this expensive substrate. Co-expression of BbBEAS and KIVR in the yeast led to significant improvement of the production of beauvericins.The total titer of beauvericin and its congeners (beauvericins A-C) was increased to 61.7 ± 3.0 mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC7159. Supplement of L-Val at 10 mM improved the supply of ketoisovalerate, the substrate of KIVR, which consequently further increased the total titer of beauvericins to 105.8 ± 2.1 mg/l. Using this yeast system,we functionally characterized an unknown CODS from Fusarium venenatum NRRL 26139 as a beauvericin synthetase, which was named as FvBEAS. Our work thus provides a useful approach for functional reconstitution and engineering of fungal CODSs for efficient production of this family of anticancer molecules. << Less

  Metab. Eng. 18:60-68(2013) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Harnessing fungal nonribosomal cyclodepsipeptide synthetases for mechanistic insights and tailored engineering.

  Steiniger C., Hoffmann S., Mainz A., Kaiser M., Voigt K., Meyer V., Suessmuth R.D.

  Nonribosomal peptide synthetases represent potential platforms for the design and engineering of structurally complex peptides. While previous focus has been centred mainly on bacterial systems, fungal synthetases assembling drugs like the antifungal echinocandins, the antibacterial cephalosporins ... >> More

  Nonribosomal peptide synthetases represent potential platforms for the design and engineering of structurally complex peptides. While previous focus has been centred mainly on bacterial systems, fungal synthetases assembling drugs like the antifungal echinocandins, the antibacterial cephalosporins or the anthelmintic cyclodepsipeptide (CDP) PF1022 await in-depth exploitation. As various mechanistic features of fungal CDP biosynthesis are only partly understood, effective engineering of NRPSs has been severely hampered. By combining protein truncation, <i>in trans</i> expression and combinatorial swapping, we assigned important functional segments of fungal CDP synthetases and assessed their <i>in vivo</i> biosynthetic capabilities. Hence, artificial assembly line components comprising of up to three different synthetases were generated. Using <i>Aspergillus niger</i> as a heterologous expression host, we obtained new-to-nature octa-enniatin (4 mg L^-1) and octa-beauvericin (10.8 mg L^-1), as well as high titers of the hybrid CDP hexa-bassianolide (1.3 g L^-1) with an engineered ring size. The hybrid compounds showed up to 12-fold enhanced antiparasitic activity against <i>Leishmania donovani</i> and <i>Trypanosoma cruzi</i> compared to the reference drugs miltefosine and benznidazole, respectively. Our findings thus contribute to a rational engineering of iterative nonribosomal assembly lines. << Less

  Chem. Sci. 8:7834-7843(2017) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase.

  Xu F., Butler R., May K., Rexhepaj M., Yu D., Zi J., Chen Y., Liang Y., Zeng J., Hevel J., Zhan J.

  <h4>Background</h4>Creating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the d ... >> More

  <h4>Background</h4>Creating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the domain functions stands as a major obstacle in the field. Of particular interest is understanding the basis for a class of methyltransferase domains (MT) that are found embedded within the adenylation domain (A) of fungal NRPS systems instead of in an end-to-end architecture.<h4>Results</h4>The MT domain from bassianolide synthetase (BSLS) was removed and the truncated enzyme BSLS-ΔMT was recombinantly expressed. The biosynthesis of bassianolide was abolished and <i>N</i>-desmethylbassianolide was produced in low yields. Co-expression of BSLS-ΔMT with standalone MT did not recover bassianolide biosynthesis. In order to address the functional implications of the protein insertion, we characterized the <i>N</i>-methyltransferase activity of the MT domain as both the isolated domain (MT_BSLS) and as part of the full NRPS megaenzyme. Surprisingly, the MT_BSLS construct demonstrated a relaxed substrate specificity and preferentially methylated an amino acid (L-Phe-SNAC) that is rarely incorporated into the final product. By testing the preference of a series of MT constructs (BSLS, MT_BSLS, cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MT_BSLS by crosslinking or embedding the MT within an A domain narrowed the substrate specificity of the methyltransferase toward L-Leu-SNAC, the preferred substrate for the BSLS megaenzyme.<h4>Conclusions</h4>The embedding of MT into the A2 domain of BSLS is not required for the product assembly, but is critical for the overall yields of the final products. The substrate specificity of MT is significantly affected by the protein context within which it is present. While A domains are known to be responsible for selecting and activating the biosynthetic precursors for NRPS systems, our results suggest that embedding the MT acts as a secondary gatekeeper for the assembly line. This work thus provides new insights into the embedded MT domain in NRPSs, which will facilitate further engineering of this type of biosynthetic machinery to create structural diversity in natural products. << Less

  J. Biol. Eng. 13:65-65(2019) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.