TY - BOOK

T1 - Rational engineering of an iterative polyketide synthase

AU - Schmidt, Katharina

PY - 2024/3/19

Y1 - 2024/3/19

N2 - The focus of the presented work is the engineering of polyketide-derived natural products in different stages of their biosynthesis. Within this context, one project centres on the engineering of a polyketide synthase (PKS) to enable the production of unnatural products, while another project focusses on unravelling intricacies of biosynthetic pathways and the understanding of tailoring enzymes. The iterative PKS TenS is known to produce the pentaketide-tyrosine hybrid tenellin. Products of iterative PKS can differ in reduction level, methylation pattern and chain length. Thus, programming must take place. In TenS, the programming of the chain length is known to be determined by the KR domain. Structure prediction of the KR domain enabled the identification of a relevant substrate binding helix, which was the focus of in vivo swap experiments. Swap experiments were conducted with analogous substrate binding helices from related PKS, which produce similar hexa- and heptaketides. The hybrid PKS products were analysed and indicated an influence of the helix swap on the programming. Further, the simultaneous swap of only four rationally selected amino acids showed a small effect on the chain length, as well as mutation of single isolated amino acids during a following alanine scan of the helix. The fungus Hypoxylon lienhwacheense is a producer of the tropolone alkyl citrate conjugates lienhwalides A-C and cordyanhydride B, consisting of three alkyl citrate units linked linearly. Genome analysis revealed two distinct gene clusters proposed to be responsible for the biosynthesis of tropolones and alkyl citrates, respectively. Both clusters were analysed bioinformatically, followed by the elucidation of both pathways by a combination of heterologous expression in the host Aspergillus oryzae NSAR1 and protein isolation with following in vitro assays. For alkyl citrates, the biosynthesis was revealed to be analogous to other known fungal alkyl citrate biosynthetic pathways. Furthermore, the discovery of a related alkylcitrate of unknown origin hints on the biosynthetic pathway of cordyanhydride B. The formation of pentaketide tropolones was revealed to be catalysed by a set of four key enzymes. Lastly, the coupling of the two natural product classes was investigated, but remains mysterious and requires investigations in the future.

AB - The focus of the presented work is the engineering of polyketide-derived natural products in different stages of their biosynthesis. Within this context, one project centres on the engineering of a polyketide synthase (PKS) to enable the production of unnatural products, while another project focusses on unravelling intricacies of biosynthetic pathways and the understanding of tailoring enzymes. The iterative PKS TenS is known to produce the pentaketide-tyrosine hybrid tenellin. Products of iterative PKS can differ in reduction level, methylation pattern and chain length. Thus, programming must take place. In TenS, the programming of the chain length is known to be determined by the KR domain. Structure prediction of the KR domain enabled the identification of a relevant substrate binding helix, which was the focus of in vivo swap experiments. Swap experiments were conducted with analogous substrate binding helices from related PKS, which produce similar hexa- and heptaketides. The hybrid PKS products were analysed and indicated an influence of the helix swap on the programming. Further, the simultaneous swap of only four rationally selected amino acids showed a small effect on the chain length, as well as mutation of single isolated amino acids during a following alanine scan of the helix. The fungus Hypoxylon lienhwacheense is a producer of the tropolone alkyl citrate conjugates lienhwalides A-C and cordyanhydride B, consisting of three alkyl citrate units linked linearly. Genome analysis revealed two distinct gene clusters proposed to be responsible for the biosynthesis of tropolones and alkyl citrates, respectively. Both clusters were analysed bioinformatically, followed by the elucidation of both pathways by a combination of heterologous expression in the host Aspergillus oryzae NSAR1 and protein isolation with following in vitro assays. For alkyl citrates, the biosynthesis was revealed to be analogous to other known fungal alkyl citrate biosynthetic pathways. Furthermore, the discovery of a related alkylcitrate of unknown origin hints on the biosynthetic pathway of cordyanhydride B. The formation of pentaketide tropolones was revealed to be catalysed by a set of four key enzymes. Lastly, the coupling of the two natural product classes was investigated, but remains mysterious and requires investigations in the future.

U2 - 10.15488/16648

DO - 10.15488/16648

M3 - Doctoral thesis

CY - Hannover

ER -