TY - BOOK

T1 - Expression and Engineering of Biosynthetic Enzymes in Fungi

AU - Feng, Jin

N1 - Doctoral thesis

PY - 2021

Y1 - 2021

N2 - Fungal heterologous expression is a powerful tool for the production of natural products. In this presented work, we aim to further expand the application of fungal heterologous systems. In addition to iterative PKS, extensively studied in the past, fungal hosts are also expected to be capable of expressing modular PKS, which have never been expressed in fungi. Thus, DEBS1-TE, a minimal bimodular PKS for the triketide 1 biosynthesis, was taken as a representative model system to conduct expression in Aspergillus oryzae in this work. In the re-engineered DEBS1-TE biosynthesis in A. oryzae, the supply pathways of propionate-related precursors were established. DEBS1-TE was codon-optimized and activated to be active in A. oryzae. With this engineered A. oryzae, the heterologous expression of a DEBS1-TE was functionally achieved to produce 1 (0.6 mg/L) in a filamentous fungal host for the first time. Then a set of titer optimizations of 1 was attempted, including fermentation conditions, overexpression of biosynthetic genes, and blockage of degradation pathways. It led to the 12-fold increase of the overall titer. Using the new A. oryzae system, we attempted to explore the possibility of fusion expression of modular and iterative PKSs. Unfortunately, there was no new product detectable. Another aim of this work was to identify the biosynthetic pathway of brasilane-type sesquiterpenes brasilane A 2, D 3, and E 4 by A. oryzae heterologous expression. The bra biosynthetic pathway, including a terpene cyclase (BraA), an N-acetylglucosamine transferase (BraB) and a cytochrome P450 monooxygenase (BraC), were identified. With the biochemical characterization of enzymes in vitro, BraB displayed a broad scope of acceptor substrates in vitro.

AB - Fungal heterologous expression is a powerful tool for the production of natural products. In this presented work, we aim to further expand the application of fungal heterologous systems. In addition to iterative PKS, extensively studied in the past, fungal hosts are also expected to be capable of expressing modular PKS, which have never been expressed in fungi. Thus, DEBS1-TE, a minimal bimodular PKS for the triketide 1 biosynthesis, was taken as a representative model system to conduct expression in Aspergillus oryzae in this work. In the re-engineered DEBS1-TE biosynthesis in A. oryzae, the supply pathways of propionate-related precursors were established. DEBS1-TE was codon-optimized and activated to be active in A. oryzae. With this engineered A. oryzae, the heterologous expression of a DEBS1-TE was functionally achieved to produce 1 (0.6 mg/L) in a filamentous fungal host for the first time. Then a set of titer optimizations of 1 was attempted, including fermentation conditions, overexpression of biosynthetic genes, and blockage of degradation pathways. It led to the 12-fold increase of the overall titer. Using the new A. oryzae system, we attempted to explore the possibility of fusion expression of modular and iterative PKSs. Unfortunately, there was no new product detectable. Another aim of this work was to identify the biosynthetic pathway of brasilane-type sesquiterpenes brasilane A 2, D 3, and E 4 by A. oryzae heterologous expression. The bra biosynthetic pathway, including a terpene cyclase (BraA), an N-acetylglucosamine transferase (BraB) and a cytochrome P450 monooxygenase (BraC), were identified. With the biochemical characterization of enzymes in vitro, BraB displayed a broad scope of acceptor substrates in vitro.

U2 - 10.15488/11466

DO - 10.15488/11466

M3 - Doctoral thesis

CY - Hannover

ER -