Cascade Synthesis of l-Homoserine Catalyzed by Lyophilized Whole Cells Containing Transaminase and Aldolase Activities: The Mathematical Modeling Approach

Morana Česnik Katulić,Martina Sudar,Zvjezdana Findrik Blažević,Đurdica Vasić-Rački,Simon J. Charnock,Pere Clapés,Yuyin Qi,Karel Hernández

doi:10.1021/acs.iecr.1c02343

Abstract

Aldolase and transaminase coexpressed in Escherichia coli cells and lyophilized (i.e., lyophilized whole-cell biocatalyst (LWCB)) were used as biocatalysts for the one-pot cascade synthesis of l-homoserine with substrate cycling. The kinetic analysis of enzymes within lyophilized cells was performed to evaluate the behavior of the system. The best result among the performed fed-batch reactor experiments achieved was 640.8 mM (76.3 g L–1) of l-homoserine with a volume productivity of 2.6 g L–1 h–1. This is comparable with the results of the same cascade synthesis using cell-free extracts (CFEs) and significantly better than the reports in the literature applying fermentation technology. The approach applied here can serve as guidance for the design of microbial cells with an optimal ratio of expressed enzymes that act as biocatalysts in the cascade, resulting in lower biocatalyst cost, no need for the addition of expensive coenzymes, and enhanced enzyme stability as compared with cell-free extracts.

Highlights

Biotechnological processes have the potential to produce specific products in high yields with low energy consumption and minimal waste generation.[1−3] It is often the case that biocatalytic processes explored in the laboratory show favorable opportunities from the standpoint of green chemistry and demonstrate limitations in terms of their economic potential.[4]
We aim to demonstrate the behavior of the same reaction system when both enzymes required for the catalysis are overexpressed in Escherichia coli cells, i.e., lyophilized whole-cell biocatalyst (LWCB)
The key difference between the cell-free extracts (CFEs) and LWCB is the presence of a cell wall and membrane that can act as natural support for enzymes

Summary

Introduction

Biotechnological processes have the potential to produce specific products in high yields with low energy consumption and minimal waste generation.[1−3] It is often the case that biocatalytic processes explored in the laboratory show favorable opportunities from the standpoint of green chemistry and demonstrate limitations in terms of their economic potential.[4]. Adequate tools must be used to speed up the development time and to decrease the resources needed for process optimization.[7,8] Many bright examples in the recent literature illustrate the application of mathematical modeling in the development of biocatalytic processes, from those simpler[9−12] to significantly more complex, such as the cascade reactions.[13−18] Advanced modeling techniques increase the rate of the development and industrialization of new biocatalytic processes They enable the formation of mathematical relationships between the system variables, allowing the prediction of the reaction scenarios without the need of conducting experiments in the reactor. For wider implementation of cascades in the industry, it is necessary to optimize them with respect to Received: June 17, 2021 Published: September 16, 2021

Objectives

Results

Conclusion