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

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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.