Comparative Evaluation of the Asymmetric Synthesis of (S)-Norlaudanosoline in a Two-Step Biocatalytic Reaction with Whole Escherichia coli Cells in Batch and Continuous Flow Catalysis

Abstract

Opioids are important analgesics, and their pharmaceutical application is increasing worldwide. Many opioids are based on benzylisoquinoline alkaloids (BIA) and are still industrially produced from Papaver somniferum (opium poppy). (S)-norlaudanosoline ((S)-NLS) is a complex BIA and an advanced intermediate for diverse pharmaceuticals. The efficient synthesis of this scaffold could pave the way for a plant-independent synthesis platform. Although a promising biocatalytic route to (S)-NLS using norcoclaurine synthase (NCS) and ω-transaminase (TAm) has already been explored, the cost-effectiveness of this process still needs much improvement. Therefore, we investigated whether the synthesis could also be performed using whole cells to avoid the use of (partially) purified enzymes. With an optimized mixing ratio of TAm- and NCS-containing cells in batch biotransformations, 50 mM substrate was converted within 3 h with more than 90% yield and a high enantiomeric excess of the product (95%). To further increase the space–time yield, the cells were immobilized to enable their retainment in fixed-bed reactors. A comparison of glass beads, Diaion HP-2MG and alginate revealed that the addition of Diaion during bacterial growth led to the most active immobilisates. To facilitate sustained production of (S)-NLS, a fixed-bed setup was constructed based on lithographically printed columns from biocompatible PRO-BLK 10 plastic. The continuous production at two scales (5 mL and 50 mL columns) revealed insufficient system stability originating from biocatalyst leaching and inactivation. Thus, while the use of whole cells in batch biotransformations represents an immediate process improvement, the transfer to flow catalysis needs further optimization.