Gerd Miksch

Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors.

Abstract. The gene for phytase from Escherichia coli was sequenced and compared with the appA gene. It was found to be a mutant derivative of the appA gene. After fusion with a C-terminal His-tag, phytase was purified by affinity chromatography and the enzymatic properties were analyzed. To develop a system for overexpression and extracellular production of phytase in E. coli, factors affecting the expression and secretion such as promoter type, host strain and selection pressure were analyzed. Using a secretion system based on the controlled expression of the kil gene, the expression of phytase was improved and the enzyme was released into the culture medium at a high level. An effective fermentation strategy based on fed-batch operation was developed.

A 4.6 kb DNA region of Rhizobium meliloti involved in determining urease and hydrogenase activities carries the structural genes for ureas (ureaA, ureB, ureC) interrupted by other open reading frames

A 4.6 kb DNA region of the Rhizobium meliloti strain AK631 was found to contain seven open reading frames (ORFs), all oriented in the same direction. The putative gene products of four of these ORFs were highly homologous to UreA, UreB and UreC of Klebsiella aerogenes, Proteus mirabilis, Proteus vulgaris and Canavalia ensiformis. The overall organisation of the DNA region analysed was ORF1, ureA (ORF2), ORF3, ureB (ORF4), ORF5, ORF6 and ureC (ORF7), indicating that the organisation of the urease structural genes in R. meliloti differs from that of other urease genes so far characterized. ORF1 was incomplete; only the 3′ end of the coding region was present. The six complete ORFs coded for polypeptides of 11.1 (UreA), 8.9 (ORF3), 10.8 (UreB), 15.0 (ORF5), 13.8 (ORF6) and 60.7 kDa (UreC). No sequence homology to known polypeptides could be detected for the gene products of ORF1, ORF3, ORF5 and ORF6. Using a lacZ fusion and insertional mutagenesis it was shown that the seven ORFs identified were all located in the same transcription unit. For mutational analysis a resistance gene cassette was introduced into each of the complete ORFs resulting in apolar mutations. Mutations in ureA, ureB and ureC, but not in ORF3, ORF5 and ORF6, abolished urease activity in R. meliloti. The determination of hydrogen uptake in these R. meliloti mutants revealed that only ORF6 and ureB are necessary for hydrogen uptake.

A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli

To develop a rapid reporter system for the screening of stationary-phase promoters in Escherichia coli, the expression pattern of the green fluorescent protein (GFP) during bacterial cultivation was compared with that of the commonly used β-galactosidase. Using GFP with enhanced fluorescence, the expression pattern of both reporter systems GFP and β-galactosidase were similar and showed a typical induction of gene activity of the reporter genes, i.e. increase of expression at the transition from exponential to stationary phase. The expression was affected by the culture medium, i.e. in contrast to the complex medium (LB medium), the stationary-phase specific induction was only observed in synthetic medium (M9) when amino acids were added, whereas there was generally no induction in MOPS medium. To develop a rapid screening method on agar plates for stationary-phase promoters, a photographic approach was used, continued with computational image treatment. A screening method is presented which enables an on-line monitoring of gene activity.

High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium

Abstract The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome of Klebsiella planticola with high transposition frequency. A Bacillus hybrid β-glucanase located on an RSF1010-derived plasmid was mobilized from E.coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments it was shown that the production of β-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter used for the expression of the kil gene the secretion of β-glucanase into the medium started at the transition from the exponential to the stationary phase, as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins.

Secretion of Homologous and Heterologous Recombinant Proteins in Escherichia Coli and Other Gram-Negative Bacteria by Using a New Secretion System

Since Gram-negative bacteria are not able to transport proteins containing a leader sequence across the outer membrane, recombinant proteins cannot be secreted into the culture medium. On the other hand the extracellular production of overexpressed proteins is highly desirable, since downstream processing would be much easier. In recent years we developed an expression/secretion system for E. coli based on the membrane permeabilizing effect of the kil peptide of E. coli. Using this secretion system we demonstrate here the successful extracellular production of several important industrial enzymes. For some enzymes the results were confirmed bei experiments in bioreactors. Furthermore, a method was described to transfer the secretion system to other Gram-negative bacterial species. Using Klebsiella planticola and Acetobacter methanolicus it was shown that recombinant proteins can be produced extracellularly in an unexpectedly high level.