Abstract
Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. Most if not all AFAAs are produced under aerobic conditions which is energy-intensive. To establish a cost-effective anaerobic process for production of AFAAs, it holds great promise to develop a new pathway enabling the conversion of oxoloacetate into aspartate through direct amination which is catalyzed by aspartate dehydrogenase (AspDH). Compared with the well studied aspartate aminotransferase and aspartate ammonia-lyase, only a few AspDHs are characterized till date, and failure to reproduce the high activity of AspDH from Rastonia eutropha documented in the literature encouraged us to screen and characterize novel AspDHs from different origins. Interestingly, the AspDHs from Klebsiella pneumoniae 34618 (KpnAspDH) and Delftia sp. Cs1–4 (DelAspDH) showed successful soluble expression. KpnAspDH and DelAspDH containing C-terminal hexa-histidine tags were purified and characterized for their catalytic properties. Notably, in addition to its high reductive amination activity, DelAspDH exhibited considerable stability as compared to the other source of AspDHs. This work thus provides novel enzyme resource for engineering strains capable of producing AFAAs under anaerobic conditions.
Highlights
Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications
The dominate pathway used in most industrial AFAA producing microbes (e.g. Corynebacterium glutamicum and Escherichia coli)[3,4,5,6] was oxaloacetate (OAA) amination catalyzed by aspartate aminotransferase (EC:2.6.1.1) through the transfer of amino group from glutamate
To establish an efficient OAA amination system for AFAA fermentation, two aspartate dehydrogenase (AspDH) from R. eutropha (ReuAspDH) and B. japonicum (BjaAspDH), with very high reported catalytic activities were first evaluated in our system
Summary
Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. In addition to its high reductive amination activity, DelAspDH exhibited considerable stability as compared to the other source of AspDHs. This work provides novel enzyme resource for engineering strains capable of producing AFAAs under anaerobic conditions. The dominate pathway used in most industrial AFAA producing microbes (e.g. Corynebacterium glutamicum and Escherichia coli)[3,4,5,6] was oxaloacetate (OAA) amination catalyzed by aspartate aminotransferase (EC:2.6.1.1) through the transfer of amino group from glutamate. OAA can be converted to aspartate through the reductive TCA cycle where it is first reduced to malate and fumarate before its conversion to aspartate by aspartate ammonia-lyase (EC:4.3.1.1) Another pathway, which is less studied, is the direct amination of OAA by aspartate dehydrogenase (AspDH, EC:1.4.1.21)[6]. OAA amination through aspartate aminotransferase may not be very efficient under anoxic conditions because a less active TCA cycle would not give forth to a sufficient glutamate/2-ketoglutarate pool[12]
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