Akasaka Naoki, Ishii Yuri, Hidese Ryota, Sakoda Hisao, Fujiwara Shinsuke
Institute of Applied Microbiology, Marukan Vinegar Co. Ltd., 5-6 Koyo-cho West, Higashinada-ku, Kobe, Hyogo 658-0033, Japan.
Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
J Biosci Bioeng. 2014 Dec;118(6):607-15. doi: 10.1016/j.jbiosc.2014.05.024. Epub 2014 Jun 27.
Vinegar with increased amounts of branched-chain amino acids (BCAAs; valine, leucine and isoleucine) is favorable for human health as BCAAs decrease diet-induced obesity and hyperglycemia. To construct Gluconacetobacter europaeus which produces BCAAs, leucine responsive regulator (GeLrp) is focused and two Gelrp mutants were constructed. Wild-type KGMA0119 didn't produce significant amount of valine (0.13 mM) and leucine (0 mM) and strain KGMA7110 which lacks complete Gelrp accumulated valine (0.48 mM) and leucine (0.11 mM) but showed impaired growth, and it was fully restored in the presence of essential amino acids. Strain KGMA7203 was then constructed with a nonsense mutation at codon Trp132 in the Gelrp, which leads a specific deletion at an estimated ligand-sensing region in the C-terminal domain. KGMA7203 produced greater quantities of valine (0.80 mM) and leucine (0.26 mM) and showed the same growth characteristics as KGMA0119. mRNA levels of BCAAs biosynthesis genes (ilvI and ilvC) and probable BCAAs efflux pump (leuE) were determined by quantitative reverse-transcription PCR. Expression rates of ilvI and ilvC in the two Gelrp disruptants were greater than those in KGMA0119. leuE was highly expressed in KGMA7110 only, suggesting that the accumulation in KGMA7110 culture was caused by increased expression of the biosynthesis genes and abnormal enhanced export of amino acids resulting in impaired cell growth. In contrast, KGMA7203 would achieve the high level production through enhanced expression of the biosynthesis genes without enhancing that for the efflux pump. KGMA7203 was considered advantageous for production of vinegar with higher amounts of valine and leucine.
富含支链氨基酸（BCAAs；缬氨酸、亮氨酸和异亮氨酸）的醋对人体健康有益，因为支链氨基酸可减少饮食引起的肥胖和高血糖。为构建能产生支链氨基酸的欧洲葡糖醋杆菌，对亮氨酸响应调节因子（GeLrp）进行了研究，并构建了两个GeLrp突变体。野生型KGMA0119不产生大量的缬氨酸（0.13 mM）和亮氨酸（0 mM），而缺乏完整GeLrp的菌株KGMA7110积累了缬氨酸（0.48 mM）和亮氨酸（0.11 mM），但生长受到损害，在必需氨基酸存在的情况下其生长完全恢复。然后构建了菌株KGMA7203，其GeLrp中第132位密码子Trp发生无义突变，导致C端结构域中估计的配体感应区域出现特定缺失。KGMA7203产生了更多的缬氨酸（0.80 mM）和亮氨酸（0.26 mM），并表现出与KGMA0119相同的生长特性。通过定量逆转录PCR测定支链氨基酸生物合成基因（ilvI和ilvC）以及可能的支链氨基酸外排泵（leuE）的mRNA水平。两个GeLrp破坏株中ilvI和ilvC的表达率高于KGMA0119。leuE仅在KGMA7110中高表达，这表明KGMA7110培养物中的积累是由于生物合成基因表达增加和氨基酸异常增强的输出导致细胞生长受损。相比之下，KGMA7203将通过增强生物合成基因的表达而不增强外排泵的表达来实现高水平生产。KGMA7203被认为有利于生产含更高含量缬氨酸和亮氨酸的醋。
