Van Arsdell Scott W, Perkins John B, Yocum R Rogers, Luan Linda, Howitt C Linda, Chatterjee Nilu Prasad, Pero Janice G
OmniGene Bioproducts, Inc., 763D Concord Ave., Cambridge, Massachusetts 02138, USA.
Biotechnol Bioeng. 2005 Jul 5;91(1):75-83. doi: 10.1002/bit.20488.
In biotin biosynthesis, DAPA aminotransferase encoded by the bioA gene catalyzes the formation of the intermediate 7,8-diaminopelargonic acid (DAPA) from 7-keto-8-aminopelargonic acid (KAPA). DAPA aminotransferases from Escherichia coli, Serratia marcescens, and Bacillus sphaericus use S-adenosylmethionine (SAM) as the amino donor. Our observation that SAM is not an amino donor for B. subtilis DAPA aminotransferase led to a search for an alternative amino donor for this enzyme. Testing of 26 possible amino acids in a cell-free extract assay revealed that only l-lysine was able to dramatically stimulate the in vitro conversion of KAPA to DAPA by the B. subtilis DAPA aminotransferase. The K(m) for lysine and KAPA was estimated to be between 2 and 25 mM, which is significantly higher than the K(m) of purified E. coli BioA for SAM (0.15 mM). This higher requirement for lysine resulted in accumulation of KAPA during fermentation of B. subtilis biotin producing strains. However, this pathway bottleneck could be relieved by either addition of exogenous lysine to the medium or by introduction of lysine deregulated mutations into the production strains.
在生物素生物合成过程中，由bioA基因编码的DAPA转氨酶催化从7-酮-8-氨基壬酸（KAPA）形成中间体7,8-二氨基壬酸（DAPA）。来自大肠杆菌、粘质沙雷氏菌和球形芽孢杆菌的DAPA转氨酶使用S-腺苷甲硫氨酸（SAM）作为氨基供体。我们观察到SAM不是枯草芽孢杆菌DAPA转氨酶的氨基供体，这促使我们寻找该酶的替代氨基供体。在无细胞提取物测定中对26种可能的氨基酸进行测试后发现，只有L-赖氨酸能够显著刺激枯草芽孢杆菌DAPA转氨酶在体外将KAPA转化为DAPA。赖氨酸和KAPA的米氏常数（K(m)）估计在2至25 mM之间，这明显高于纯化的大肠杆菌BioA对SAM的K(m)（0.15 mM）。对赖氨酸的这种更高需求导致在枯草芽孢杆菌生物素生产菌株发酵过程中KAPA的积累。然而，通过向培养基中添加外源赖氨酸或通过在生产菌株中引入赖氨酸去调控突变，可以缓解这一途径瓶颈。
