Increased Phenylalanine Production by Growing and Nongrowing Escherichia coli Strain CWML2 Christian Weikert, Uwe Sauer, and James E. Bailey* Institute of Biotechnology, ETH Zu ¨ rich, CH-8093 Zu ¨ rich, Switzerland Chemostat selection at low dilution rate in glycerol-limited minimal medium was previously employed to isolate the mutant Escherichia coli strain CWML2 which exhibits shorter lag phases, decreased acetate production, and higher specific growth rates and biomass yields in batch culture (Weikert, C.; Sauer, U.; Bailey, J. E. Microbiology 1997, 143, 1567-1574). In this study, CWML2 was analyzed for its biochemical production capabilities in batch culture and under nongrowing conditions. Both CWML2 and MG1655 were transformed with plasmid pSY130-14, which encodes feedback resistant mutants of the enzymes chorismate mutase P-prephenate synthase and 3-hydroxy-D-arabinoheptulosonate-7-phosphate dehydratase, to enable phenyla- lanine production. In batch culture, transformed CWML2 produced twice as much phenylalanine as did MG1655:pSY130-14. In contrast to the reference strain, substantial growth-independent production of phenylalanine was calculated for CWML2:pSY130-14 by using Luedeking-Piret kinetic analysis. Over a period of 30 h, nongrowing cells of CWML2:pSY130-14 exhibited a 2.5-fold higher specific pheny- lalanine production rate. The apparent capability of E. coli CWML2 to partly uncouple metabolic activity from growth suggests a potentially general advantage of this class of modified hosts for production of biochemicals. Introduction In nature, microorganisms are usually faced with severe and persistent nutrient shortage interrupted by short periods of abundant nutrient supply (Gottschal, 1992). As an adaptation to these conditions, most microorganisms have developed strategies for survival in a metabolically inert state (Chesbro et al., 1990; Jenkins et al., 1988; Matin, 1992). Upon encountering substrate availability, they resume growth at the maxi- mum possible rates, often accompained by extensive overflow metabolism and reduced bioenergetic efficiency (Russel and Cook, 1995). For many biotechnological applications, however, desirable process organisms should exhibit high metabolic activity under slow-growing or nongrowing conditions and yet maintain tight control of overflow metabolism. Therefore, control of substrate uptake and catabolism in the absence of growth is a key factor for improving the productivity and operational life span of microbial biocatalysts (Matin, 1992). Several strategies for improving productivity by reduc- ing biomass formation have been elucidated, including the utilization of slow-growing, immobilized (Bailey et al., 1987; Karkare et al., 1986), or stationary-phase cells (Tunner et al., 1992). Upon entry into stationary phase the cells undergo physiological changes that confer resistance against adverse physiological conditions (Ma- tin, 1991). Unfortunately these changes are associated with a reduction in their specific biocatalytic activity, as was shown in slow-growing chemostat culture (Flickinger and Rouse, 1993). Complete separation of biomass and product formation, however, would dramatically improve the commercial viability of many microbial processes, if suitable organisms could be identified or genetically engineered (Matin, 1991). Recently, we have been able to isolate Escherichia coli strains that exhibited generally improved physiological properties (Weikert et al., 1997). These strains were isolates from the dominant population of a glycerol- limited, long-term chemostat after 219 generations that was operated at an extremly low dilution rate of 0.05 h -1 . Proteome analysis using two-dimensional gel electro- phoresis revealed a very high degree of similarity be- tween the parental MG1655 strain and the chemostat isolates, thus proving the close relation of the isolated strains to their progenitor. One of the selected strains, E. coli CWML2, showed higher specific growth rates and increased biomass yields on glycerol, glucose, and the gluconeogenic substrate acetate (Weikert et al., 1997). This phenotypic property was combined with shorter lag phases and increased resistance to various stresses. Furthermore, this strain was shown to produce signifi- cantly higher concentrations of a heterologous secreted protein than the progenitor after transformation of both with the same expression vector (Weikert et al., 1998). In this paper, we examine the extent to which the improved physiological properties of E. coli strain CW- ML2 may be exploited for production of primary meta- bolites. The progenitor, wild-type E. coli MG1655, was used as the reference organism. As an example of a biotechnologically relevant metabolite, phenylalanine production was studied, both in batch culture and with nongrowing cells of each strain. For this purpose, cells were transformed with a plasmid encoding the feedback- resistant enzymes chorismate mutase, prephenate syn- * Corresponding author. Address: Institute of Biotechnology, ETH Ho ¨nggerberg, CH-8093 Zu ¨ rich, Switzerland. Telephone: +41/ 1/633 3170. Fax: +41/1/633 1051. 420 Biotechnol. Prog. 1998, 14, 420-424 S8756-7938(98)00030-7 CCC: $15.00 © 1998 American Chemical Society and American Institute of Chemical Engineers Published on Web 05/06/1998