Culture instability of auxotrophic amino acid producers

Abstract

The long-term dynamic characteristics of the L-lysine producer Corynebcterium glutamicum in continuous culture were studied over a range of specific growth rates. The double-auxotroph parent strain was found to be susceptible to a back mutation, or reversion, which negated the regulatory bypass that allows this strain to accumulate L-lysine in culture but also gives rise to a L-threonine auxotrophic requirement. Consequently, the revertant cells no longer over-produced L-lysine, nor were they limited in their growth by the low levels of L-threonine in the medium. All continuous culture experiments were eventually taken over by these revertants. The instability of the culture was found to be primarily due to the growth rate differential between the two competing populations, the (productive) parent auxotrophs and the (nonproductive) revertants. A deterministic mathematical model of the culture dynamics, incoroporating two limiting-substrate balances, satisfactorily described the takeover profiles. A linear stability analysis of the model equations identified that although long-term culture demise is inevitable, the dimensionless ratio of the two limiting substrates controls the rate of takeover by nonproductive cells. The analysis further demonstrated the importance of proper medium design in delaying the onset of takeover in cultures of this double-auxotroph strain. The theoretical medium design criterion was then confirmed experimentally by the stabilization of a fed-batch culture against revertant takeover for an extended fermentation time.