One-Pot Biosynthesis of 2-Keto-4-hydroxybutyrate from Cheap C1 Compounds Using Rationally Designed Pyruvate Aldolase and Methanol Dehydrogenase

Abstract

One-carbon chemicals (C 1s) are potential building blocks as they are cheap, sustainable, and abiotic components. Methanol-derived formaldehyde can be another versatile building block for the production of 2-keto-4-hydroxyacid derivatives that can be used for amino acids, hydroxy carboxylic acids, and chiral aldehydes. To produce 2-keto-4-hydroxybutyrate from C 1s in an environment-friendly way, we characterized an aldolase from Pseudomonas aeruginosa PAO1 (PaADL), which showed much higher catalytic activity in condensing formaldehyde and pyruvate than the reported aldolases. By applying a structure-based rational approach, we found a variant (PaADLV121A/L241A) that exhibited better catalytic activities than the wild-type enzyme. Next, we constructed a one-pot cascade biocatalyst system by combining PaADL and a methanol dehydrogenase (MDH) and, for the first time, effectively produced 2-keto-4-hydroxybutyrate as the main product from pyruvate and methanol via an enzymatic reaction. This simple process applied here will help design a green process for the production of 2-keto-4-hydroxyacid derivatives.

Artificial Methylotrophic Cells via Bottom-Up Integration of a Methanol-Utilizing Pathway

“…Specifically, KHB aldolase has the potential to shift sugar-based metabolism toward industrially favorable biomanufacturing. This is due to the ability of KHB aldolase to catalyze the condensation of formaldehyde and pyruvate to form 2-keto-4-hydroxybutyrate, a vital building block for the synthesis of industrial intermediates such as l -homoserine and 1,3-propanediol . These intermediates can then be used to construct large carbon molecules of industrial value.…”

“…This is due to the ability of KHB aldolase to catalyze the condensation of formaldehyde and pyruvate to form 2-keto-4hydroxybutyrate, a vital building block for the synthesis of industrial intermediates such as L-homoserine and 1,3propanediol. 37 These intermediates can then be used to construct large carbon molecules of industrial value. In contrast, the sugar compounds ribulose-5-phosphate (Ru5P) and hexose-6-phosphate (hexose-6-p) involved in Hp metabolism serve as cellular metabolism precursors in the synthesis of biological molecules such as nucleotides and nucleic acids and have limited industrial relevance.…”

“…All samples for HPLC analysis were derivatized with Obenzylhydroxylamine hydrochloride, modified from the research as published. 37 Briefly, 50 μL of sample and 50 μL of 130 mM O-benzylhydroxylamine hydrochloride (dissolved in pyridine/methanol/water = 33:15:2) were mixed and incubated at 25 °C for 10 min. Methanol (1 mL) was added, and the solution was filtered (0.2 μm membrane filter) before HPLC analysis was conducted in duplicate injections.…”

Biotransformation of 2-keto-4-hydroxybutyrate via aldol condensation using an efficient and thermostable carboligase from Deinococcus radiodurans

“…The specific activity of the enzyme was defined as the produced amount of product per unit reaction time per enzyme amount. Formaldehyde, pyruvate, and 2-KHB were quantitatively determined by high performance liquid chromatography (HPLC) as described in our previous study (Jeong et al 2023 ).…”

“…Recently, we reported the use of pyruvate aldolase from Pseudomonas aeruginosa with maltose-binding protein (MBP-PaADL) for 2-KHB production from formaldehyde and pyruvate (Jeong et al 2023 ). In the previous study, MBP-PaADL produced the highest 2-KHB yield reported so far.…”