title: Enhanced production of branched-chain amino acids by Gluconacetobacter europaeus with a specific regional deletion in a leucine responsive regulator.
abstract: 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.
authors.affiliation: Institute of Applied Microbiology, Marukan Vinegar Co. Ltd., 5-6 Koyo-cho West, Higashinada-ku, Kobe, Hyogo 658-0033, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Akasaka
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Ishii
authors.affiliation: Research Center for Environmental Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, Sanda, Hyogo 669-1337, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Hidese
authors.affiliation: Institute of Applied Microbiology, Marukan Vinegar Co. Ltd., 5-6 Koyo-cho West, Higashinada-ku, Kobe, Hyogo 658-0033, Japan.
authors.forename: Hisao
authors.initials: H
authors.lastname: Sakoda
authors.affiliation: Research Center for Environmental Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, Sanda, Hyogo 669-1337, Japan; Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan. Electronic address: fujiwara-s@kwansei.ac.jp.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Fujiwara
mesh_terms: D000597:Amino Acids, Branched-Chain / Q000032:analysis / Q000096:biosynthesis*; D041821:Gluconacetobacter / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D007930:Leucine / Q000032:analysis / Q000378:metabolism*; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000378:metabolism
chemical_list: D000597:Amino Acids, Branched-Chain; D012329:RNA, Bacterial; D012333:RNA, Messenger; D007930:Leucine; D014633:Valine
keywords: Acetic acid bacteria; Acetohydroxyacid isomeroreductase; Acetohydroxyacid synthase; Branched-chain amino acids; Branched-chain amino acids efflux pump; Gluconacetobacter europaeus; Leucine responsive regulatory protein