Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel d-Mandelate Dehydrogenase with High Catalytic Activity

Abstract

d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD-dependent d-mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg, which was close to four times the activity of the probe. Meanwhile, the k/ K value of LhDMDH was 28.80 S·mM, which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA.

A genome-wide approach for identification and characterisation of metabolite-inducible systems

“…Furthermore, here, we report inducible systems (OapR/P H16_RS01330 and PhgR/P H16_RS05530 ) responding to βalanine and phenylglyoxylate. Both compounds play an important role as building blocks in chemical synthesis or food biotechnology 31,50 . Among the characterised inducible systems, different types of TR were identified, including LysR, AsnC, MocR, IclR and MarR.…”

One-Pot Synthesis of Phenylglyoxylic Acid from Racemic Mandelic Acids via Cascade Biocatalysis

“…coli BL21/pET28a- PpMR 10 , and E. coli BL21/pET28a- LbDMDH 10 preserved by our Lab were cultured in LB medium E.…”

“…On the basis of the results of genome mining, many paired primers shown in Supporting Information (SI), Table S1 were designed and synthesized, which were used to amplify the gene fragments of potential novel mandelate racemases. The cultivation, collection, and genomic DNA extraction of original strains were performed as reported before . Nextly, the gene fragments of potential mandelate racemase were respectively amplified based on these genomic DNA.…”

“…Phenylglyoxylic acid (PGA) are key building blocks in the chemical synthesis process and widely used to synthesize pharmaceutical intermediates or food additives. − Compared with chemical preparation generally involve toxic cyanide and complex processes, , microbial or enzymatic biosynthesis of PGA provides an eco-friendly approach to industrial-scale production. ,, It is reported that many microorganisms can selectively transform mandelic acid to PGA, including Gibberella fujikuroi, Alcaligenes bronchisepticus, Pseudomonas polycolor, and Pseudomonas putida. , d -Mandelate dehydrogenase (DMDH) plays a key role in the process of converting d -mandelic acid to PGA, and it is a member of ketopantoate reductase family. ,, Heterologous expression of DMDH genes from many microorganisms in Escherichia coli was capable to oxidize mandelic acid into PGA. , In our previous study, the PGA yield was about 40% from 50 mM racemic mandelic acid catalyzed by coculturing two engineered E. coli strains . Meanwhile, we have verified that the NAD + regeneration capacity could be enhanced by utilizing the pyruvic acid based on coculturing two engineered E.…”

“…P11444) is well-characterized and widely applied mandelate racemase, which k cat values toward d -mandelic acid and l -mandelic acid were 822 and 756 s –1 , , and many cascade reactions had been designed and applied using the Pp MR as the core catalyst. ,, However, the industrial application of Pp MR was impeded due to its poor soluble expression level. It had been reported that genome mining could be an economical strategy in discovering novel enzyme with higher expression level, ,− therefore it should also be used to discover novel mandelate racemase with higher expression level. In addition, the mature DNA synthesis and sequencing techniques have enabled the overproduction of a desired enzyme in microorganisms.…”