Optimization of a reduced enzymatic reaction cascade for the production of L-alanine

Tobias J Gmelch,Josef M Sperl,Volker Sieber

doi:10.1038/s41598-019-48151-y

Abstract

Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.

Highlights

Sustainability is a major focus of modern day research, fighting a world governed by the demand on oil
D-glucose is oxidized to D-gluconate under formation of NADH
D-gluconate undergoes dehydration forming 2-keto 3-deoxy D-gluconate (KDG) followed by a retro aldol reaction resulting in one molecule pyruvate and one molecule D-glyceraldehyde

Summary

Introduction

Sustainability is a major focus of modern day research, fighting a world governed by the demand on oil. In the last decades two inherently different process types evolved where either metabolically engineered whole cell approaches[7] are applied or purified enzymes from various organisms are combined to in vitro enzyme reaction cascades. Isolated biocatalysts are combined with the necessary cofactors in a controlled environment to produce valuable products[3,4,10] With this approach the advantages of well-established in vitro biocatalysis are combined with the power of multi-step in vivo pathways to yield sophisticated biomanufacturing platforms[14]. To identify the appropriate enzyme ratio and thereby avoid unnecessary expenses, one approach is a thorough kinetic analysis of the required biocatalysts including inhibition constants and pH effects as demonstrated by Beer et al.[19] To this knowledge based strategy, Liu www.nature.com/scientificreports/. For the final optimization of the enzyme ratio we combined the two approaches mentioned above and first determined the kinetic constants and titrated the biocatalysts based on their maximum activity to achieve an overall increased efficiency

Objectives

Methods

Results