Synthesis Of L-phosphinothricin Research Articles

Expression of L-phosphinothricin synthesis enzymes in Pichia pastoris for synthesis of L-phosphinothricin.

With the ban of highly toxic herbicides, such as paraquat and glyphosate, phosphinothricin (PPT) is becoming the most popular broad-spectrum and highly effective herbicide. The current PPT products in the market are usually a racemic mixture with two configurations, the D-type and L-type, of which only the L-PPT has the herbicidal activity. The racemic product is not atom economic, more toxic and may cause soil damage. Asymmetric synthesis of L-PPT has become a research focus in recent years, while biological synthesis methods are preferred for its character of environmental friendly and requiring less reaction steps when being compared to the chemical methods. We have developed a biological synthesis route to produce optically pure L-PPT from D,L-PPT in two steps using 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid as the intermediate. In this study, we expressed the glutamate dehydrogenase and glucose dehydrogenase using Pichia pastoris as the first time. After a series of optimization, the total L-PPT yield reached 84%. The developed synthesis system showed a high potential for future industrial application. Compare to the previous plasmid-carrying-E. coli expression system, the established method may avoid antibiotic usage and provided an alternative way for industrial synthesis of optically pure L-PPT.

Driving Transamination Irreversible by Decomposing Byproduct α-Ketoglutarate into Ethylene Using Ethylene-Forming Enzyme

The transformations of transaminases have been extensively studied as an approach to the production of chiral amino moieties. However, the low equilibrium conversion of the reaction is a critical disadvantage to transaminase application, and a strategy for shifting the reaction equilibrium is essential. Herein, we have developed a novel method to effectively prevent the reversibility of transamination by fully decomposing byproduct α-ketoglutarate into ethylene and carbon dioxide in situ using ethylene-forming enzyme (EFE). Two transaminases and one EFE were expressed in E. coli and purified to be used in the cascade reaction. After optimal reaction conditions were determined based on the enzymatic properties, a cascade reaction coupling transaminase with EFE was conducted and showed high efficiency in the synthesis of l-phosphinothricin. Finally, using this approach with only an equivalent amount of amino donor l-glutamate increased the conversions of various keto acids from 99%. This strategy shows great potential for transamination using glutamate as the amino donor.