Biocatalytic reduction of carboxylic acids : production and application of carboxylate reductases in synthetic enzyme cascades
Weber, Douglas; Rother, Dörte (Thesis advisor); Lauterbach, Lars (Thesis advisor); Jupke, Andreas (Thesis advisor)
Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2022
The tetrahydroisoquinoline (THIQ) moiety is a "privileged scaffold" and it is found in numerous bioactive natural products. THIQ-containing compounds display various bioactivities and are therefore used for pharmaceutical applications. Chemical syntheses of these compounds are possible but they do not give high stereoselectivities. Besides, they often depend on the use of toxic or environmentally harmful chemicals. Thus, novel synthetic approaches towards THIQs are of significant interest. In vitro and in vivo biocatalysis provide viable methods of producing complex THIQs in high stereoselectivities and under mild conditions. For instance, a three-step (chemo)enzymatic cascade to access a 1,3,4-trisubstituted THIQ has been already established. In this approach, the commercially available 3-hydroxybenzaldehyde (3-OH-BA) serves as starting material. Although this aldehyde is quite cheap, it is still mainly obtained from petroleum resources. Thinking in a sustainable and economically competitive bioeconomy, alternative approaches employing renewable materials are well envisioned. Therefore, in this thesis, a four-step enzymatic cascade towards the same trisubstituted THIQ starting with 3-hydroxybenzoic acid (which can ultimately be obtained by microbial cell factories from renewable resources such as glycose and/or xylose) is proposed. The novelty of this cascade is the reduction of 3-hydroxybenzoic acid (3-OH-BZ) into 3-OH-BA by a carboxylate reductase (CAR). In this step, the implementation of an efficient cofactor regeneration system of both, ATP and NAD(P)H, is crucial. For this, an in vitro cofactor regeneration approach using only purified enzymes and a whole-cell approach were set-up and compared. Both approaches showed to be promising and high conversions of the acid into 3-OH-BA were achieved (>80%), but the whole-cell system showed superior performance because (i) it allowed the combination of the first and the second steps of the cascade in an one-pot system with excellent product yields (>99%, ee ≥95%) into the intermediate 3-hydroxyphenylacetylcarbinol (3-OH-PAC) and (ii) enhanced the substrate tolerance by the cells when compared to the system employing only purified enzymes. The third and fourth steps of the cascade were performed in a sequential mode due to cross-reactivities and formation of several by-products. (1R,2S)-metaraminol could be formed with good conversions (>90%, ic ≥95%) applying either purified or whole-cell amine transaminase from Bacillus megaterium (BmTA) or Chromobacterium violaceum (Cv2025). The cyclization step was performed using the norcoclaurine synthase variant from Thalictrum flavum (ΔTfNCS-A79I), leading to the formation of the 1,3,4-trisubstituted THIQ product with high product yields (>90%, ic >90%). In the end, the overall product yield of both in vitro and in vivo cascades were 72 and 87%, respectively, with similar ee and ic values. In conclusion, we were able to demonstrate the applicability of enzymatic cascades for the formation of a complex chiral compound bearing three chiral centers from low-cost renewable starting materials and furthermore show that some steps are transferable to other aromatic starting materials.
- DOI: 10.18154/RWTH-2022-10375
- RWTH PUBLICATIONS: RWTH-2022-10375