Cascade Synthesis of l-Homoserine Catalyzed by Lyophilized Whole Cells Containing Transaminase and Aldolase Activities: The Mathematical Modeling Approach

Abstract

Aldolase and transaminase coexpressed in Escherichia coli cells and lyophilized (i.e., lyophilized whole-cell biocatalyst (LWCB)) were used as biocatalysts for the one-pot cascade synthesis of L-homoserine with substrate cycling. The kinetic analysis of enzymes within lyophilized cells was performed to evaluate the behavior of the system. The best result among the performed fed-batch reactor experiments achieved was 640.8 mM (76.3 g L −1 ) of L-homoserine with a volume productivity of 2.6 g L −1 h −1 . This is comparable with the results of the same cascade synthesis using cell-free extracts (CFEs) and significantly better than the reports in the literature applying fermentation technology. The approach applied here can serve as guidance for the design of microbial cells with an optimal ratio of expressed enzymes that act as biocatalysts in the cascade, resulting in lower biocatalyst cost, no need for the addition of expensive coenzymes, and enhanced enzyme stability as compared with cell-free extracts.

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

“…Pyruvate aldolase reversibly mediates the C–C bond formation between pyruvate and aldehydes to produce 4-hydroxy-2-ketoacids that are less exploited in organic synthesis. − Pyruvate aldolase is a potential biocatalyst for condensing formaldehyde and pyruvate to produce 2-keto-4-hydroxybutyrate, a valuable building block for the synthesis of l -homoserine, 3-hydroxypropionic acid, and 1,3-propanediol, which are important starting materials in the manufacture of biocompatible plastic and polytrimethylene terephthalate. ,− In this regard, the high production of 2-keto-4-hydroxybutyrate from cheap materials is of great interest. Recently, 2-keto-4-hydroxybutyrate as an intermediate in metabolic pathways has been produced by several enzymes, such as 2-keto-3-deoxy- l -rhamnonate aldolase from Escherichia coli ( Ec YfaU), 2-keto-4-hydroxybutyrate aldolase from E.…”

Development of an aminotransferase‐driven biocatalytic cascade for deracemization of d,l‐phosphinothricin

“…However, the aminotransferases‐mediated amino transfer reactions between α‐amino acids and α‐keto acids were usually suffered unfavorable thermodynamic equilibrium because of the low equilibrium constant ( K eq = 1) (France et al, 2016; Zhu & Hua, 2009). Therefore, shifting reaction equilibrium to product was one of the most critical issues for the efficient transaminase reaction (Kelefiotis‐Stratidakis et al, 2019; Satyawali et al, 2019), and employing multienzyme cascade to circumvent this issue was highly required (Česnik Katulić et al, 2021; Simon et al, 2014).…”

Co‐Immobilization of a Multi‐Enzyme Cascade: (S)‐Selective Amine Transaminases, l‐Amino Acid Oxidase and Catalase

“…Multi‐enzyme cascades allow, e. g., more complex synthesis routes, the use of low concentrations of co‐substrates [11] and cofactors [12] enabled by their regeneration, the modification of reaction equilibria, the removal of co‐products, [13,14] or the further reaction of (unstable) intermediates, avoiding inhibition by an accumulation of products as well as additional isolation, protection, and deprotection steps [2] . As such, amine transaminases (ATAs) have often been combined with other enzymes, such as with an aldolase for simultaneous co‐product removal and substrate recycling using the co‐product, [15] or with a pyruvate decarboxylase for co‐product removal [14,16] …”

“…To date, many studies have addressed the immobilization of catalases [37,38,39] and d ‐AAOs, [40,41,42] and also the co‐immobilization of both, [43,44] whereas immobilization of l ‐AAOs [18,45] was performed in fewer studies mainly attributed to the obstacles in producing stable l ‐AAOs in soluble form [21,46] . In addition, although transaminases were immobilized in several studies, their co‐immobilization with other enzymes was reported only rarely, [15,47] and a combination with a l ‐AAO and a catalase was not demonstrated at all.…”