One-Pot Biosynthesis of l-Aspartate from Maleic Anhydride via a Thermostable Dual-Enzyme System under High Temperature

Abstract

l-Aspartate is an important chemical in the food and pharmaceutical industries. Herein, a dual-enzyme system was constructed to synthesize l-aspartate from maleic anhydride at 50 °C, which can reduce the byproduct production. Maleate transformed from maleic anhydride in the solution was converted into l-aspartate via fumarate catalyzed by maleate isomerase (MaiA) and thermostable aspartase (AspB), respectively. Because MaiA is a rate-limiting enzyme, enzyme activities of various MaiAs were compared, and the efficient and thermostable maleate isomerase AaMaiA from Alicyclobacillus acidoterrestris was chosen. The K cat/K m value of AaMaiA was 264.4 mM–1 min–1. AaMaiA and AspB were coexpressed in E. coli to produce l-aspartate. To improve the l-aspartate production rate, the ribosome binding site (RBS) sequence located upstream of AaMaiA was optimized and the Tat signal peptide was fused with AaMaiA. The conversion rate was 96% within 60 min, and the intermediate was not detected, the possible reason of which is that high temperature inhibits the activity of bacterial endogenous enzymes, but functional enzymes remain active. Cells from fermentation produced 243.6 g/L (1.83 M) of l-aspartate with a 2 M substrate. Our study revealed an effective method to produce l-aspartate without using gene knockout and provided a strategy for l-aspartate production in the industrial field.

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

“…21 This might be attributed to the high expression level and catalytic efficiency of the recombinant enzymes, which efficiently convert fumaric acid into l -aspartic acid before it enters other metabolic pathways, as observed in a previous report. 9 The results clearly demonstrated that the Q96E/A100M strain showed a significant improvement compared to the original strain, and this finding emphasizes the superior catalytic ability of the Q96E/A100M variant.…”

“…6b). In the study by Liu et al , 9 a recombinant strain (OD 600 = 20.0) was used to convert 2.0 mol L −1 maleic anhydride into l -aspartic acid within 6 h, with a conversion rate of 91.5% and a productivity of 2.0 g L −1 h −1 OD −1 . Additionally, the productivity of the original system was 7.2 g L −1 h −1 OD −1 (Fig.…”

“…Considering the solubility and cost advantage of maleic anhydride, a dual-enzyme catalytic method for the synthesis of L-aspartic acid from maleic anhydride has been developed. 9 This process achieved a high conversion rate of 91.5% at 50 °C and yielded a substantial amount of 244 g L −1 of L-aspartic acid. These developments in biocatalysis have shown promising results in enhancing the synthesis of L-aspartic acid, providing improved efficiency and more cost-effective production methods.…”

“…6b), representing a significant improvement over the state-of-the-art green processes. 9,21 The conversion of maleic acid to L-aspartic acid occurs within the cell, and they might be moved in and out cells through some transporters. Compared to the enzyme system, transporters are not the limiting factor in the current process (Fig.…”