Karl Friehs

Strategies for improving plasmid stability in genetically modified bacteria in bioreactors

Exploitation of recombinant organisms for the large-scale, commercial production of foreign proteins is often hampered by the problem of plasmid instability. A wide range of strategies have been reported for improving the stability of recombinant organisms. A combination of manipulating both the genetic design of recombinants and the conditions of culturing the organisms may be used to achieve stable host-vector associations during culture of recombinant organisms in bioreactors.

The green fluorescent protein is a versatile reporter for bioprocess monitoring.

The green fluorescent protein (GFP) of Aequorea victoria has become a convenient and versatile tool as a reporter protein in molecular cell biology and developmental biology. Here, it is shown that GFP may advantageously be used as a reporter system for bioprocess monitoring as well. Examples are given for monitoring fermentation as well as downstream processes for protein recovery. Thus, separation processes based on the application of affinity-fusion tags may be optimized in terms of the operational conditions by using GFP as a model target protein owing to facile screening by simple visual inspection. This item is discussed together with the presentation of a novel fusion tag with strong affinity for metal-chelate ligands: hisactophilin, a histidine-rich protein of Dictyostelium discoideum. This tag is of particular interest for affinity separation processes requiring multiple sites of interaction like aqueous and reverse micellar two-phase extraction as well as precipitation.

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.

Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors

Abstract Cultivation conditions for the extracellular production of a hybrid β-glucanase from Bacillus were established by using Escherichiacoli JM109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid β-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of β-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of β-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of β-glucanase [E. coli JM109(pLF3)] was tested on the fermenter scale.

Rapid determination of plasmid copy number

Plasmid copy number, the number of expression vectors per host cell, is a key variable in recombinant microbial cultivation. Therefore, it would be very helpful, if the plasmid copy number could be determined during the operating process period. A rapid quantification of this important process variable would even open the possibility of its use in process control. However current assays like gel electrophoresis, CsCl-gradient centrifugation, HPLC and other methods are time consuming and difficult to quantify. Indirect methods, like the correlation of copy number with, e.g. the activity of an enzyme, coded on the plasmid, are prone to errors due to the production kinetics, turnover rate and protein denaturation. Here, a method is presented, which enables the plasmid copy number to be determined in less than 30 min. This novel procedure based on plasmid isolation by means of a commercial DNA-isolation kit and quantification by capillary electrophoresis, should allow the copy number to be used in process control.

The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase

Abstract Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein. The kil gene was fused with the stationary-phase promoter of the fic gene of E. coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed. Using the gene for β-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase. Quasi-lysis and lethality were not observed. The primary effect of the induction of the kil gene was the overproduction of β-glucanase. The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil– control. The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective. This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene.

Improvement of downstream processing of recombinant proteins by means of genetic engineering methods

The rapid advancement of genetic engineering has allowed to produce an impressive number of proteins on a scale which would not have been achieved by classical biotechnology. At the beginning of this development research was focussed on elucidating the mechanisms of protein overexpression. The appearance of inclusion bodies may illustrate the success. In the meantime, genetic engineering is not only expected to achieve overexpression, but to improve the whole process of protein production. For downstream processing of recombinant proteins, the synthesis of fusion proteins is of primary importance. Fusion with certain proteins or peptides may protect the target protein from proteolytic degradation and may alter its solubility. Intracellular proteins may be translocated by means of fusions with signal peptides. Affinity tags as fusion complements may render protein separation and purification highly selective. These methods as well as similar ones for improving the downstream processing of proteins will be discussed on the basis of recent literature.

In situ dark field microscopy for on-line monitoring of yeast cultures

A new-type in situ probe has been developed to acquire dark field images of yeast in bioreactors. It has been derived from an in situ bright field microscope that is able to measure cell density in bioreactors during fermentation processes. The illumination part of the probe has been replaced with a dark field device, in which an aspheric condenser is used, so that high contrast dark field images can be obtained. The technique of second imaging is implemented to improve the sharpness of the images by means of a relay lens. This new in situ probe is expected to enable the evaluation of the cell viability without staining owing to modern image processing.

Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins

Aiming to facilitate the accessibility of recombinant proteins produced with Escherichia coli, extracellular expression may be achieved by means of bacteriocin release protein (BRP) coexpression. Different types of BRPs were tested in order to optimize protein secretion into the culture medium. Those included the well-studied BRPs of the Colicin E1 and Cloacin DF13 bacteriocins and variants thereof. BRP expression was stringently controlled by means of the arabinose inducible P(BAD) promoter, which accounts for a broad-range adjustment of expression strength. Using appropriate arabinose concentrations, a concentration range was determined, that allowed efficient secretion of the model proteins alkaline phosphatase and beta-lactamase, with 90-95% of the proteins released into the culture medium. Kinetic investigations into BRP expression and protein secretion revealed a rapid increase of extracellular protein concentration within 5-10 min past induction. Alternatively to fine-tuned BRP expression during cultivation, protein production and secretion could be decoupled by establishment of appropriate induction strategies and up to 90% of alkaline phosphatase was released into the culture medium within 3h after reaching maximum biomasss concentrations. Both, fine-tuned and growth decoupled BRP expression accounted for extracellular alkaline phosphatase concentrations of roughly 500 mg l(-1) of culture broth and selectivities of 50mg of this enzyme per gram of cell dry mass, respectively.

Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization

BackgroundThe classic AOX1 replacement approach is still one of the most often used techniques for expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Although this approach is largely successful, it frequently delivers clones with unpredicted production characteristics and a work-intense screening process is required to find the strain with desired productivity.ResultsIn this project 845 P. pastoris clones, transformed with a GFP expression cassette, were analyzed for their methanol-utilization (Mut)-phenotypes, GFP gene expression levels and gene copy numbers. Several groups of strains with irregular features were identified. Such features include GFP expression that is markedly higher or lower than expected based on gene copy number as well as strains that grew under selective conditions but where the GFP gene cassette and its expression could not be detected. From these classes of strains 31 characteristic clones were selected and their genomes sequenced. By correlating the assembled genome data with the experimental phenotypes novel insights were obtained. These comprise a clear connection between productivity and cassette-to-cassette orientation in the genome, the occurrence of false-positive clones due to a secondary recombination event, and lower total productivity due to the presence of untransformed cells within the isolates were discovered. To cope with some of these problems, the original vector was optimized by replacing the AOX1 terminator, preventing the occurrence of false-positive clones due to the secondary recombination event.ConclusionsStandard methods for transformation of P. pastoris led to a multitude of unintended and sometimes detrimental integration events, lowering total productivity. By documenting the connections between productivity and integration event we obtained a deeper understanding of the genetics of mutation in P. pastoris. These findings and the derived improved mutagenesis and transformation procedures and tools will help other scientists working on recombinant protein production in P. pastoris and similar non-conventional yeasts.