Influence of strain, feed profile and cultivation technique on the lipid structure and membrane function in Escherichia coli

Sammanfattning: The phospholipid and fatty acids content of the Escherichiacoli membrane were investigated during changes of feed rate,cultivation technique and host strains.During continuous cultivation it was shown that the leakageof periplasmic protein to the medium had an optimum at 0.3 h-1,resulting in transport of 20% of the total recombinant product.This corresponded to the accumulation of high amounts ofphosphatidylglycerol and unsaturated fatty acids, which arethus believed to be responsible for the increased fluidity. Theamount of cyclic fatty acids and cardiolipin were enriched atlower growth rates and marked the transition to conditionsresembling energy and/or carbon starvation.During fed-batch cultivation this pattern was visible butnot as pronounced. The fed-batch cultivation was characterisedby an overall and high level of cardiolipin and a lower degreeof membrane component changes. It was concluded that thedynamic character of this technique did not allow the steadystate membrane performance of continuous cultivation.A relaxed/stringent strain pair was cultivated in fed-batchcultivation and evaluated with respect to different membranestructure and function. The relaxed strain was characterised byheavy foaming during constant feed due to comparatively highercell lysis.During the shift to very low feed rate, the relaxed strainwas able to regulate both phospholipid and fatty acids leadingto a higher flux to saturated fatty acids and neutralphospholipids. The relaxed strain was also characterised by theinability to accumulate cardiolipin at low growth rate. Thedata suggest a more complicated mechanism responsible for theshift in membrane component accumulation at reduced substratefeed rates. This is not based directly on ppGpp, this compoundcould never be detected in these experiments, but is connectedto this formation since the relAﺷ strains have a verydifferent performance characteristics. The differences in lipidaccumulation was obvious for e.g. the performance duringsonication where the opposite behaviour was observed atdifferent feed rate. While the stringent strain showed thecharacteristic and rigid behaviour at low growth rate therelaxed strain was very sensible to sonication and was alsomore subjected to cell lysis.An evaluation of a shift in the model periplasmic protein,from b-lactamase to maltose binding protein, using a weakpromoter from the maltose operon, was investigated. The systemwas designed to operate with both cytoplasmic and periplasmictargeting of the protein. The data showed that the productionwith the periplasmic system, at different growth rates, ishigher at higher growth rate. This agrees with the data for theproduction with b-lactamase where the enrichment of unsaturatedfatty acids and phosphatidylglycerol increases by growth ratefrom 0.3h-1 and above and would thus lead to an increase influidity and transport.Key words:Escherichia coli, Phospholipid and fatty acidregulation, fed-batch, continuous cultivation, leakage, proteintransport.