An industrial clavulanic acid (CA) overproducer Streptomyces clavuligerus strain, namely DEPA, was engineered to further enhance its CA production. Single or multiple copies of ccaR, claR (pathway-specific activators), and cas2 (CA synthase) genes under the control of different promoters were introduced into this strain. CA titers of the resulting recombinants were analyzed by HPLC in a dynamic fashion and compared to the vector-only controls and a wild-type strain of S. clavuligerus while their growth was monitored throughout fermentation. The addition of an extra copy of ccaR, under control of its own promoter or constitutive ermE* promoter (P ermE* ), led to 7.6-and 2.3-fold increased volumetric levels of CA in respective recombinants, namely the AK9 and ID3 strains. Its highly stable multicopy expression by the glpF promoter (P glpF ) provided up to 25.9-fold enhanced volumetric CA titers in the respective recombinant, IDG3. claR expression controlled with its own promoter or ermE* and glpF-mediated amplification in an industrial strain brought about only about 1.2-fold increase in the volumetric CA titers. An extra copy of cas2 integration with P ermE* into the S. clavuligerus DEPA genome led to 3.8-fold higher volumetric CA production by GV61. Conclusively, multicopy expression of ccaR under P glpF can result in significantly improved industrial high-titer CA producers.
The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus.
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