Abstract
The production of branched-chain amino acids (BCAAs) is still challenging, therefore we rationally engineered Corynebacterium glutamicum FA-1 to increase the l-leucine production by optimizing the aminotransferases. Based on this, we investigated the effects of the native aminotransferases, i.e., branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) and aspartate aminotransferase (AspB; encoded by aspB) on l-leucine production in C. glutamicum. The strain FA-1△ilvE still exhibited significant growth without leucine addition, while FA-1△ilvE△aspB couldn’t, which indicated that AspB also contributes to L-leucine synthesis in vivo and the yield of leucine reached 20.81 ± 0.02 g/L. It is the first time that AspB has been characterized for l-leucine synthesis activity. Subsequently, the aromatic aminotransferase TyrB and the putative aspartate aminotransferases, the aspC, yhdR, ywfG gene products, were cloned, expressed and characterized for leucine synthesis activity in FA-1△ilvE△aspB. Only TyrB was able to synthesize l-leucine and the l-leucine production was 18.55 ± 0.42 g/L. The two putative branched-chain aminotransferase genes, ybgE and CaIlvE, were also cloned and expressed. Both genes products function efficiently in BCAAs biosynthesis. This is the first report of a rational modification of aminotransferase activity that improves the l-leucine production through optimizing the aminotransferases.
Highlights
Branched-chain amino acids (BCAAs), i.e., L-leucine, L-valine and L-isoleucine, are three of eight essential amino acids [1] that cannot be synthesized in animals [2]
The synthesis of BCAAs comprises four reactions catalyzed by acetohydroxy acid synthase (AHAS; encoded by ilvBN), acetohydroxy acid isomeroreductase (AHAIR; encoded by ilvC), dihydroxyacid dehydratase
FA-1△ilvE still had weak activities for the formation of L-leucine, we studied the effects of many native aminotransferase genes from C. glutamicum FA-1 on BCAAs
Summary
Branched-chain amino acids (BCAAs), i.e., L-leucine, L-valine and L-isoleucine, are three of eight essential amino acids [1] that cannot be synthesized in animals [2]. Unlike the biosynthetic pathways of other amino acids, the biosynthetic pathway of BCAAs includes branched and parallel reactions catalyzed by identical enzymes (Figure 1) [5]. L-Isoleucine and L-valine are synthesized from 2-ketobutyrate and pyruvate respectively, while L-leucine is branched from the 2-ketoisovalaerate in the valine pathway [6]. The synthesis of BCAAs comprises four reactions catalyzed by acetohydroxy acid synthase (AHAS; encoded by ilvBN), acetohydroxy acid isomeroreductase (AHAIR; encoded by ilvC), dihydroxyacid dehydratase (DHAD; encoded by Molecules 2018, 23, 2102; doi:10.3390/molecules23092102 www.mdpi.com/journal/molecules ilvD) and branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) [7,8].
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