One-Pot Biosynthesis of l-Aspartate from Maleate via an Engineered Strain Containing a Dual-Enzyme System.

l-Aspartate has been widely used in medicine and the food and chemical industries. In this study, maleate isomerase (MaiA) and aspartase (AspA) were coupled and coexpressed in an engineered strain in which the byproduct metabolic pathway was inactivated. The engineered strain containing the dual-enzyme system (pMA) was employed to bioproduce l-aspartate from maleate with a conversion of 98%. We optimized the activity ratio of double enzymes through ribosome binding site (RBS) regulation and molecular modification of MaiA, resulting in an engineered strain: pMA-RBS4-G27A/G171A. The conversion of l-aspartate biotransformed from maleate using the pMA-RBS4-G27A/G171A strain was almost 100%. It required 40 min to complete the whole-cell catalysis, without the intermediate product and byproduct, compared to 120 min before optimization. The induction timing and the amount of inducer in a 5-liter fermentor were optimized for scale-up of the production of l-aspartate. The amount of produced l-aspartate using the cells obtained by fermentation reached 419.8 g/liter (3.15 M), and the conversion was 98.4%. Our study demonstrated an environmentally responsible and efficient method to bioproduce l-aspartate from maleate and provided an available pathway for the industrial production of l-aspartate. This work should greatly improve the economic benefits of l-aspartate, which can now be simply produced from maleate by the engineered strain constructed based on dual-enzyme coupling. l-Aspartate is currently produced from fumarate by biological methods, and fumarate is synthesized from maleate by chemical methods in industry. We established a biosynthesis method to produce l-aspartate from maleate that is environmentally responsible, convenient, and efficient. Compared to conventional l-aspartate production, no separation and purification of intermediate products is required, which could greatly improve production efficiency and reduce costs. As environmental issues are attracting increasing attention, conventional chemical methods gradually will be replaced by biological methods. Our results lay an important foundation for the industrialization of l-aspartate biosynthesis from maleate.