Engineering of Phosphoserine Aminotransferase Increases the Conversion of l‐Homoserine to 4‐Hydroxy‐2‐ketobutyrate in a Glycerol‐Independent Pathway of 1,3‐Propanediol Production from Glucose

Abstract

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4‐hydroxy‐2‐ketobutyrate, a key reaction in producing 1,3‐propanediol (1,3‐PDO) from glucose in a novel glycerol‐independent metabolic pathway. To this end, a computation‐based rational approach is used to change the substrate specificity of SerC from l‐phosphoserine to l‐homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerCR42W/R77W, is successfully improved by 4.2‐fold in comparison to the wild type when l‐homoserine is used as the substrate, while its activity toward the natural substrate l‐phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3‐PDO production, the “homoserine to 1,3‐PDO” pathway is constructed in E. coli by coexpression of SerCR42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L−1 1,3‐PDO in fed‐batch fermentation, which is 13‐fold higher than the wild‐type strain and represents an important step forward to realize the promise of the glycerol‐independent synthetic pathway for 1,3‐PDO production from glucose.

Recent advances in biological production of 1,3-propanediol: new routes and engineering strategies

“…73 Similarly, by overexpressing a mutated phosphoserine aminotransferase gene serC R42W/R77W and pdc, another E. coli strain was engineered to be able to accumulate 3.03 g L −1 1,3-PDO in fed-batch fermentation. 74 Further increasing the activities and specificities of the two key enzymes by protein engineering and the precursor and NADPH availability , lpdA E324G ; overexpression of gdp1, gpp2, glf, dhaB, gdrAB, dhaT…”

“…Moreover, most new producers with heterologous 1,3-PDO synthesis pathways still need to use expensive vitamin B 12 during fermentation. The establishment of non-natural pathways 74,76,78 circumvents the necessity of B 12 addition and also enables the conversion of different substrates into 1,3-PDO. For example, the introduction of 3-HP-based 1,3-PDO synthesis has enabled the conversion of glucose, glycerol, xylose, and acetate to 1,3-PDO by the same strain.…”

The Studies in Constructing Yeast Cell Factories for the Production of Fatty Acid Alkyl Esters

“…For example, the quantum mechanics-cluster approach is a popular technique for elucidating the enzymatic reaction mechanisms ( Himo, 2017 ); Caver Web is a commonly used tool for identification of access pathway and analysis of ligand transport ( Stourac et al, 2019 ); SoluProt ( Hon et al, 2021 ) and DeepSol ( Khurana et al, 2018 ) are widely used tools for predicting protein solubility. Virtual screening is an ideal tool to assess which proteins amongst a library of variants can better accommodate and catalyze a given substrate of interest ( Zhang et al, 2019 ).…”

Rational Proteomic Analysis of a New Domesticated Klebsiella pneumoniae x546 Producing 1,3-Propanediol

“…With the increase in the ThrB, the more threonine is produced. The K. pneumoniae can use threonine as a nitrogen source (Reitzer, 2005), and the cavity near ADP is very suitable for homoserine binding, so it can improve the catalytic activity by stabilizing the transition state (Fan et al, 2009;Zhang et al, 2019) and contribute to the 1,3-PDO production. The ligation of amino and carboxylate groups of small molecule metabolites is catalyzed by the ATP-grasp superfamily, which is widespread across primary metabolic processes (Zhang et al, 2008).…”