Error-prone PCR of Vitreoscilla hemoglobin (VHb) to support the growth of microaerobic Escherichia coli

Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscill a has been previously used to improve recombinant cell growth and enhance p roduct formation under microaerobic conditions. It is very likely that the properties of VHb are not optimized for foreign hosts; therefore, we used e rror-prone PCR to generate a number of randomly mutated vhb genes to be exp ressed and studied in Escherichia coli. in addition, the mutated VHb protei ns also contained an extension of eight residues (MTMITPSF) at the amino te rminus. VHb mutants were screened for improved growth properties under micr oaerobic conditions and 15 clones expressing mutated hemoglobin protein wer e selected for further characterization and cultivated in a microaerobic bi oreactor to analyze the physiological effects of novel VHb proteins on cell growth. The expression of four VHb mutants, carried by pVM20, pVM50, pVM10 4, and pVM134, were able to enhance microaerobic growth of E. coli by appro ximately 22%, 155%, 50%, and 90%, respectively, with a concomitant decrease of acetate excretion into the culture medium. The vhb gene in pVM20 contai ns two mutations substituting residues Glu19(A17) and Glu137(H23) to Gly. p VM50 expresses a VHb protein carrying two mutations: His36(C1) to Arg36 and Gln66(E20) to Arg66. pVM104 and pVM134 express VHb proteins carrying the m utations Ala56(E10) to Gly and Ile24(B5) to Thr, respectively. Our experime nts also indicate that the positive effects elicited by mutant VHb-expressi on from pVM20 and pVM50 are linked to the peptide tail. Removal of the N-te rminal sequence reduced cell growth approximately 23% and 53%, respectively , relative to wild-type controls. These results clearly demonstrate that it is possible to obtain mutated VHb proteins with improved characteristics f or improving microaerobic growth of E. coli by using combined mutation tech niques, addition of a peptide tail, and random error-prone PCR. (C) 2000 Jo hn Wiley & Sons, Inc.