An improved high cell density cultivation-iHCDC-strategy for leucine auxotrophic Escherichia coli K12 ER2507.

Fed-batch processes are commonly used tools in high cell density cultivation of Escherichia coli. However, the setup of a fed-batch process can be challenging, especially when working with genetically modified strains. This work deals with the design of a specific strategy for the leucine auxotrophic E. coli strain K12 ER2507 that is of interest for arabinose-inducible gene expression systems due to its ara-14 mutation. To set up this process the optimum yield coefficient of biomass from leucine was determined by use of a design of experiments tool. Unfortunately, the yield coefficient is negatively influenced by both leucine and glucose concentrations. Furthermore, an inhibitory effect of leucine reduced the specific growth rate even at low concentration. Under consideration of these problems, an improved high cell density cultivation (iHCDC) for E. coli K12 ER2507 was established. The start medium was set up without any C-source to omit a batch phase with high glucose concentration and substrate feeding was initiated directly upon inoculation. Due to the poor solubility of leucine in feed medium, the amino acid was dissolved in the base. With the improved high cell density cultivation process a final cell density of more than 90 g/L was obtained reproducibly.

Novel antimicrobial peptide of human epididymal duct origin.

HE2, a gene expressed specifically in human epididymis, gives rise to multiple mRNAs that encode a group of small cationic secretory peptides. Localization of HE2 within the defensin gene cluster and prediction that beta-defensin-like modules exist suggest that these peptides have antimicrobial activity and represent components of the innate epithelial defense system of the epididymal duct. Reverse transcription-polymerase chain reaction analysis confirmed the occurrence of eight human HE2-derived transcripts, including minor mRNA variants, that had previously been shown only in animal species. Employing isoform-specific antibodies against the predicted HE2 products, multiple 4- to 8-kDa peptides were detected in human epididymal epithelium, epididymal fluid, and ejaculate. N-terminal microsequencing has suggested a proteolytic processing of these peptides by a furin-like proprotein convertase, which cleaves a propiece from the longer precursor peptides. HE2alpha and HE2beta1, representing major peptide isoforms in the human epididymis, were recombinantly expressed, and their susceptibility to furin cleavage was demonstrated in vitro and in vivo. Processed recombinant peptides and chemosynthetic fragments were included in antimicrobial tests. In addition to the beta-defensin-like HE2beta1 with its expected antibacterial function, HE2alpha C-terminal fragments showed antibacterial activity against Escherichia coli, although it showed no significant similarity to beta-defensins nor to any other known protein family.