Optimization of Manganese Oxidation Engineering Bacteria BL21-pET-copA

Abstract Our group have identified wild highly efficient manganese-oxidizing strains and constructed the manganese oxidation engineering bacteria BL21-pET-copA. This research construct a strain of engineering bacteria ATCC-pET-copA&infA which doesn`t contain the essential genes infA via auxotrophic complementary technology, then five strains of engineered bacteria with domain deletions and three strains with multipoint mutations were constructed. The colony ratio of the bacterium ATCC-pET-copA&infA still reached approximately 80% after 40 generations of inheritance. The growth status of the engineering bacteria was good and not affected by antibiotics. After seven days of culture, the highest manganese removal rate of domain deletion bacteria BL21-pET-copA-△1&2 was 80.06% under Cu(II)-free conditions. In the presence of Cu(II), the manganese removal rate of domain-deleted engineered bacteria CopA-△1 was the highest, 91.65%. The multipoint mutant strain BL21-pET-copA442H − N&494H−N had the highest manganese oxidation rates of 93.25% with Cu(II) and 81.47% without Cu(II). The auxotrophic complementary technology, domain deletion and gene site-directed mutagenesis was used to optimize manganese-oxidizing bacteria, which improved the manganese disposal rate and stability of manganese-oxidizing bacteria and reflected the potential of engineering bacteria for practical applications.