Synthetic phenylpropanoid pathway for the in vivo production of coniferyl alcohol in Escherichia coli

• Synthetischer Phenylpropanoidweg zur in vivo-Produktion von Coniferylalkohol in Escherichia coli

Kohl, Anna Christiane; Commandeur, Ulrich Heinrich (Thesis advisor); Blank, Lars M. (Thesis advisor)

Aachen : RWTH Aachen University (2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2023

Abstract

Lignans constitute a valuable substance group exhibiting many health-promoting effects on the human body as potential pharmaceuticals and nutraceuticals. One of the most popular lignans is (−) podophyllotoxin, which is applied in its semi-synthetic derivatives for the treatment of various types of cancer. The industrial demand for alternative production routes remains high, as the main extraction method still depends on endangered natural plant resources. Thus, the controlled microbial fermentation could provide the important lignans in sufficient and scalable amounts in the future. As one of the preconditions, the precursor for lignan synthesis coniferyl alcohol needs to be produced in industrial bulk. For a foremost cost-efficient and economically sustainable green synthesis of coniferyl alcohol, the fermentation from inexpensive resources such as glucose carried out in well-examined production strains like Escherichia coli (E. coli) can be advantageous also with respect to a possible combinable synthesis of lignans. The objective of this dissertation was to establish the microbial synthesis of coniferyl alcohol in E. coli from L-tyrosine or L-phenylalanine according to the plant phenylpropanoid pathway. In the context of metabolic engineering, different enzyme candidates for crucial central reaction steps concerning deamination, C3- and C4-hydroxylation and methylation were first evaluated. By division of the phenylpropanoid pathway into upstream and downstream pathway, the best enzyme combinations were then investigated for both modules. Furthermore, the complete and partly plasmid-based expression of the phenylpropanoid pathway was realized in E. coli BL21(DE3) or its genome-integrated derivatives containing variable gene copies and promoters. At last, two different cell assays were applied and compared.This thesis describes the first successful application of the TALs from Flavobacterium johnsoniae (FjTAL) and Saccharothrix espanaensis (Sesam8), the cytochrome P450 enzyme from Rhodopseudomonas palustris (CYP199A2) as C3H and the two methyltransferases from Zea mays for the production of coniferyl alcohol from L-tyrosine in E. coli. Noteworthily, caffeic acid was obtained from L-phenylalanine by using the bifunctionality of the CYP199A2 F185L mutant as C3- and C4-hydroxylase (C3H/C4H) and the TAL from Rhodotorula glutinis (RgTAL) for the first time. In the end, the highest amount of coniferyl alcohol (~ 850 µM) was achieved in growing E. coli cells of a genome-integrated strain. This coniferyl alcohol titer, produced under non-optimized conditions and in a prototype strain, is comparable to a value in one literature report, in which an L-tyrosine overproduction strain and adjusted cultivation conditions were used. Thus, the results from this dissertation lay a substantial foundation for the further development of a microbial coniferyl alcohol production platform.

Institutions

• Department of Biology [160000]
• Chair of Molecular Biotechnology [162910]