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.
Adol condensation mediated by L-threonine aldolase (LTA) is a promising route to synthesize β-hydroxy-α-amino acids, such as the anti-Parkinson's disease drug L- threo -3,4-dihydroxyphenylserine (L- threo -DOPS; droxidopa). However, the biocatalytic approach generally suffered from poor stereoselectivity at the C β chiral center. Herein, several LTAs from diverse microorganisms were examined for the stereoselective synthesis of L- threo -DOPS, and an LTA from Firmicutes bacterium ( Fb LTA) was identified with relatively high stereoselectivity. A semi-rational design was then employed to further enhance the stereoselectivity, resulting in the identification of beneficial mutations and the generation of beneficial mutants with markedly elevated stereoselectivity (up to 80 % de) over the wild-type (23 % de). Molecular dynamics (MD) simulation revealed that the significant improvement of stereoselectivity could be attributed to the structural reconstitution of substrate tunnels. Finally, the best mutant was applied to a scaled-up reaction, affording 1.3 g/L yield of L- threo -DOPS with 80 % de. This study provides new insights into the stereoselectivity control of LTAs and potential engineered LTAs for the manufacture of L- threo -DOPS.
l -syn-3-[4-(methylsulfonyl)] phenylserine is a key industrial intermediate for the synthesis of florfenicol, thiamphenicol , and other products. l -Threonine aldolase can synthesize l -syn-3-[4-(methylsulfonyl)] phenylserine from glycine and 4-methylsulphonyl benzaldehyde directly. However, the selectivity of free l -threonine aldolase was difficult to meet the requirement of industrialization. Here, we selected the suitable site to insert His tag as immobilized site to improve the selectivity of l -threonine aldolase based on the molecular dynamics simulation and prior protein engineering. The phenanthroline was coupled to the surface of magnetic nanoparticles coated with silicon and thus enabled the selective and oriented immobilization of His-tagged l -threonine aldolase. Due to magnetic nanoparticles coated with silicon as supports, it was easy to separate the residual reactant and impurities from the immobilized enzyme by magnetic attraction. Moreover, the immobilization was combined with the purification of free enzymes through directional immobilization, which avoided heterozyme linking supports and improved the utilization efficiency of supports. Our study demonstrates an effective approach: how to analyze and select enzyme-targeted ligation sites in conjunction with previous modifications of engineered bacteria, and the immobilized enzyme obtained also provided a good candidate for the industrial production of l -syn-3-[4-(methylsulfonyl)] phenylserine.
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