Developing a green and efficient biosynthesis system for l-aspartic acid by addressing enzyme imbalance and catalysis-extraction circulation processes

Abstract

L-Aspartic acid is a naturally occurring compound widely used in various industries, such as pharmaceuticals, food, and cosmetics. In industry, L-aspartic acid is synthetized from fumaric acid using cells containing aspartase, and extracted by isoelectric point titration with sulfuric acid. A method for producing L-aspartic acid has been developed, which involved the coupling of maleate cis–trans isomerase (MaiA) and aspartase. However, two main challenges hinder the implementation of the “one-pot biosynthesis” method for L-aspartic acid production from maleic acid, poor stability of MaiA and enzyme imbalance (significant difference in the catalytic ability between MaiA and aspartase). To enhance the synthesis efficiency, we performed molecular modifications on the rate-limiting enzyme MaiA to enhance its catalytic ability, and optimize the expression elements to rectify the imbalance in enzyme utilization. As a result, a variant of MaiA with significantly improved thermostability and an expression system that exhibited superior catalytic performance were obtained. By employing whole-cell catalysis using 3.2 mol L⁻¹ maleic anhydride and Escherichia coli with an OD₆₀₀ of 3.0, an impressive conversion rate of 99.5% at 8 h was achieved, which represents a significant improvement over the original system (60.5% conversion rate at 12 h). Moreover, a catalysis-extraction circulation process was developed to solve the environmental pollution problem of sulfuric acid deposition in the preparation of L-aspartic acid. After the extraction process was optimized, the process of extraction of L-aspartic acid by using maleic anhydride was successfully implemented, with a yield of 90.1% and a product purity of 98%. This study represents an important step toward improving the biosynthesis of L-aspartic acid, reducing costs by more than 60% and reducing wastewater discharge and environmental pollution. The results of this study have the potential to benefit various industries that rely on L-aspartic acid as a key ingredient in their products.