Screening of novel cold-adapted microorganisms and exploitation of their useful gene resources

1.从阿拉斯加土壤和瑞士山地土壤中分离出27株在4℃条件下生长良好的菌株。它们被分为芽孢杆菌、假单胞菌和红球菌。菌株Ak26、SK2r6和SJ2r1被分类为假单胞菌，在培养基中产生单一的主要分泌蛋白，可以作为宿主在低温下胞外生产异源蛋白。假单胞菌ak26分泌的蛋白与荧光假单胞菌的溶血素协同调节蛋白（Hcp）同源。假单胞菌sp.SK2r6和假单胞菌sp.SJ2r1分泌的蛋白是荧光假单胞菌蛋白酶（PrtA）的同源物。这些PrtA同源物被认为是由I型分泌机制分泌的。我们构建了一个由冷适应细菌不动杆菌（Acinetobacter sp.strain no.）组成的宿主-载体系统。6，以及一系列在这种细菌和大肠杆菌中复制的穿梭载体。我们获得了在no.不动杆菌中起作用的启动子（P1和P3）。6 .产生异源蛋白。从南极海水中分离的嗜冷菌希瓦氏菌（Shewanella sp.Ac10）的耐热性α-淀粉酶被不动杆菌sp.菌株no.过量生产。6 .在低温（7℃）控制下的P3促进剂。该体系在37℃下产生的α-淀粉酶比大肠杆菌产生的α-淀粉酶多10倍左右。不动杆菌（Acinetobacter sp.）分泌α-淀粉酶。6占细胞外蛋白总量的35%。因此，本研究构建的表达系统有望用于大量生产耐热性异源蛋白。

1.We isolated 27 bacterial strains that grew well at 4℃ from Alaskan soil and Swiss mountain soil. They were classified into Bacillus, Pseudomonas, and Rhodococcus. The strains, Ak26, SK2r6, and SJ2r1, which were classified into Pseudomonas, produced a single major secreted protein in the culture medium and may be used as a host for extracellular production of heterologous proteins at low temperatures. The protein secreted by Pseudomonas sp.Ak26 was a homolog of hemolysin-coregulated protein(Hcp) from Pseudomonas fluorescens. The proteins secreted by Pseudomonas sp.SK2r6 and Pseudomonas sp.SJ2r1 were homologs of a protease (PrtA) from Pseudomonas fluorescens. These PrtA homologs were supposed to be secreted by the type I secretion machinery.2. We constructed a host-vector system comprised of a cold-adapted bacterium, Acinetobacter sp.strain no.6, and a series of shuttle vectors that replicate both in this bacterium and Escherichia coli. We obtained promoters (P1 and P3) that function in Acinetobacter sp.strain no.6 for the production of heterologous proteins. Thermolabile α-amylase of Shewanella sp.Ac10, a psychrotroph isolated from Antarctic seawater, was successfully overproduced by Acinetobacter sp.strain no.6 at low temperature (7℃) under the control of the P3 promoter. The amount of the α-amylase produced by this system was about ten times greater than that produced by E.coli at 37℃. The α-amylase secreted by Acinetobacter sp.stain no.6 amounted to 35% of the total extracellular proteins. Thus the expression system constructed in this study is expected to be useful for the production of thermolabile heterologous proteins in large quantities.