Novel hemoglobins to enhance microaerobic growth and substrate utilizationin Escherichia coli

Limited oxygen availability is a prevalent problem in microbial biotechnolo gy. Recombinant Escherichia coli expressing the hemoglobin from Vitreoscill a (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligene s eutrophus) (FHP) demonstrate significantly increased cell growth and prod uctivity under microaerobic conditions. We identify novel bacterial hemoglo bin-like proteins and examine if these novel bacterial hemoglobins can elic it positive effects similar to VHb and FHP and if these hemoglobins allevia te oxygen limitation under microaerobic conditions when expressed in E. col i. Several finished and unfinished bacterial genomes were screened using R. eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins. Novel hmp genes were identified in Pseudomonas aeruginosa, Salmon ella typhi, Klebsiella pneumoniae, Deinococcus radiodurans, and Campylobact er jejuni. Previously characterized hmp genes from E. coli and Bacillus sub tilis and the novel hmp genes from P. aeruginosa, S. typhi, C. jejuni, K pn eumoniae, and D. radiodurans were PCR amplified and introduced into a plasm id for expression in E. coli. Biochemically active hemoproteins were expres sed in all constructs, as judged by the ability to abduct carbon monoxide. Growth behavior and byproduct formation of E. coli K-12 MG1655 cells expres sing various hemoglobins were analyzed in microaerobic fed-batch cultivatio ns and compared to plasmid-bearing control and to E. coli cells expressing VHb. The clones expressing hemoglobins from E. coli, D. radiodurans, P. aer uginosa, and S. typhi reached approximately 10%, 27%, 23%, and 36% higher f inal optical density values, respectively, relative to the plasmid bearing E. coli control (A(600) 5.5). E. coli cells expressing hemoproteins from P. aeruginosa, S. typhi, and D. radiodurans grew to similar final cell densit ies as did the strain expressing VHb (A600 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E. coli strain. Addition ally, increased yield of biomass on glucose was measured for all recombinan t strains, and an approximately 2-fold yield enhancement was obtained with D. radiodurans hemoprotein-expressing E. coli relative to the E. coli contr ol carrying the parental plasmid without any hemoglobin gene.