The stressostat : A novel approach in adaptive laboratory evolution to improve end-product resistance
2023
Hartono, Sylviani | Meijerink, Marlisa F.A. | Abee, Tjakko | Smid, Eddy J. | van Mastrigt, Oscar
End-product inhibition in pH-controlled batch cultures, is the major limiting factor for bacterial biomass formation in the starter culture industry as well as in many other biotechnological processes. Adaptive laboratory evolution (ALE) has emerged over the past decades as a powerful tool for phenotype optimization, but none of the existing ALE methods could select for improved end-product resistance. Therefore, we developed the stressostat (STress Resistance Evolution in Substrate Surplus) as a novel continuous ALE method. Stressostat cultivation applies end-product concentrations as constant evolutionary pressure on microorganisms in the presence of substrate surplus. In this study, we improved the lactate resistance of Lactococcus lactis FM03P in 35 days of stressostat cultivations. The lactate concentrations increased over time from 530 to 675 mM, indicating the successful selection for variants with improved lactate resistance. Thirty-four variants were isolated and grouped into four clusters based on their growth rates at high lactate concentrations. In the high-throughput screening without pH control, most isolated variants could grow at high lactate concentrations (870–928 mM), while the wild type was completely inhibited. The variants grew slower than wild type at low lactate media indicating possible evolutionary trade-off. However, in pH-controlled batch cultivations, most variants produced more biomass than the wild type. In conclusion, stressostat cultivation is a valuable method to obtain L. lactis variants with improved end-product resistance and further characterization is needed to elucidate underlying resistance mechanisms and potential industrial applications.
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