Constructing Genetically Engineered Escherichia coli for De Novo Production of L-Threo-3-Hydroxyaspartic Acid.

L-threo-3-hydroxyaspartic acid (L-THA) is a non-proteinogenic amino acid that has garnered significant attention due to its diverse biological activities. However, the synthesis of L-THA through enzymatic and whole-cell catalysis requires the expensive substrate L-aspartic acid or L-asparagine, and co-substrate α-ketoglutarate, which limits their large-scale application. Here, this is the first report of engineering E. coli as a cell factory for de novo production of L-THA from glucose by fermentation. Firstly, the asnO gene encoding asparagine hydroxylases from Streptomyces coelicolor was heterologously expressed in E. coli to yield the L-THA producing strain. The formation and configuration of L-THA were characterized by LC-MS and HPLC after FDAA derivatization. Secondly, the pathway genes aspC and asnB, which encode aspartate aminotransferase and asparagine synthase, respectively, were overexpressed to enhance L-THA titer from 49.9 to 90.84 mg/L. Thirdly, the efforts were made to improve the key precursor L-aspartic acid pool by overexpressing the aspartase encoding gene aspA and knocking out aspartate kinase (AK) III encoding gene lysC. The best strain CC03 was obtained and L-THA titer reached 278.3 mg/L in a shake flask, representing an approximately 5.6-fold increase compared to the original strain. Ultimately, 2.87 g/L L-THA was obtained after 32 h fed-batch fermentation. This research underscores the potential use of E. coli fermentation as a feasible platform for de novo biosynthesis of L-THA from glucose, which is amenable to industrial application.