During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD ؉ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD ؉ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD ؉ ratio.The industrial importance of lactic acid bacteria is based on their ability to rapidly ferment sugars into lactic acid. For example, metabolism in the homolactic acid bacteria (the model organism is Lactococcus lactis) leads to Ͼ90% conversion of sugars to lactic acid. However, under certain conditions, this homolactic behavior is lost and increased amounts of other metabolites, such as formate or CO 2 , acetate, and ethanol, are produced in what is generally called mixed-acid fermentation. This behavior was first observed in glucose-limited chemostat cultures (22). Homolactic behavior was seen only during rapid growth in which significant amounts of glucose remained in the medium; mixed-acid fermentation was observed at lower rates of growth and true carbon-limited chemostat steady states. Such a mixed metabolism may also occur under carbon-excess conditions with certain sugars. Galactose metabolism of L. lactis results in a fermentation profile in which significant amounts of acetate and ethanol are produced (23), though lactic acid remains the major product (60% of the galactose consumed). A less pronounced shift toward mixed-acid metabolism is also observed during growth on maltose (11, 18). Although details of the biochemical pathways involved remain obscure, the use of pentose sugars involves significant acetate synthesis (9). Under conditions of carbon excess, sugar metabolism in L. lactis o...

Growth of Corynebacterium glutamicum on fructose was significantly less than that obtained on glucose, despite similar rates of substrate uptake. This was in part due to the production of overflow metabolites (dihydroxyacetone and lactate) but also to the increased production of CO 2 during growth on fructose. These differences in carbon-metabolite accumulation are indicative of a different pattern of carbon-flux distribution through the central metabolic pathways. Growth on glucose has been previously shown to involve a high flux (Ͼ 50% of total glucose consumption) via the pentose pathway to generate anabolic reducing equivalents. NMR analysis of carbon-isotope distribution patterns of the glutamate pool after growth on 1-13 C-or 6- 13C-enriched fructose indicates that the contribution of the pentose pathway is significantly diminished during exponential growth on fructose with glycolysis being the predominant pathway (80 % of total fructose consumption). The increased flux through glycolysis during growth on fructose is associated with an increased NADH/NAD ϩ ratio susceptible to inhibit both glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenase, and provoking the overflow of metabolites derived from the substrates of these two enzymes. The biomass yield observed experimentally is higher than can be estimated from the apparent quantity of NADPH associated with the pentose pathway and the flux through isocitrate dehydrogenase, suggesting an additional reaction yielding NADPH. This may involve a modified tricarboxylic acid cycle involving malic enzyme, expressed to significantly higher levels during growth on fructose than on glucose, and a pyruvate carboxylating anaplerotic enzyme.Keywords : Corynebacterium glutamicum; fructose metabolism; NMR analysis; NADH/NAD ϩ ratio; overflow metabolism.For several decades, Corynebacterium glutamicum and re-as to more efficiently supply the specific biosynthetic pathways with the necessary carbon precursors and coenzymes. lated species have been exploited industrially for the production of various amino acids. Improvements of the fermentation strateAlthough many enzymes of intermediary metabolism have been isolated from Brevibacterium flavum and characterised pregies employed and of the bacterial strains by genetic engineering techniques have been achieved, leading to progressively increas-dominantly by the team of Shiio [5Ϫ7], global carbon-flux models based on regulatory or energetic principles have only reing rates of production and/or yields [1]. These strategies have often been based upon overcoming the natural feedback regula-ceived attention recently. Over the last five years, various groups have examined how glucose is catabolised by the central pathtion mechanisms specific to each biosynthetic pathway [2Ϫ4] and have enabled a detailed understanding of these biochemical ways using NMR [8Ϫ10], enzymatic [11] and mathematical modelling [12,13] approaches. A general consensus opinion has sequences to be established. It is now apparent that further impr...

Apocarotenoids, such as α-, β-ionone, and retinol, have high commercial values in the food and cosmetic industries. The demand for natural ingredients has been increasing dramatically in recent years. However, attempts to overproduce β-ionone in microorganisms have been limited by the complexity of the biosynthetic pathway. Here, an Escherichia coli-based modular system was developed to produce various apocarotenoids. Incorporation of enzyme engineering approaches (N-terminal truncation and protein fusion) into modular metabolic engineering strategy significantly improved α-ionone production from 0.5 mg/L to 30 mg/L in flasks, producing 480 mg/L of α-ionone in fed-batch fermentation. By modifying apocarotenoid genetic module, this platform strain was successfully re-engineered to produce 32 mg/L and 500 mg/L of β-ionone in flask and bioreactor, respectively (>80-fold higher than previously reported).Similarly, 33 mg/L of retinoids was produced in flask by reconstructing apocarotenoid module, demonstrating the versatility of the "plug-n-play" modular system. Collectively, this study highlights the importance of the strategy of simultaneous modular pathway optimization and enzyme engineering to overproduce valuable chemicals in microbes. K E Y W O R D Sapocarotenoids, carotenoids, ionone, modular metabolic engineering, protein engineering, retinol

The metabolic characteristics of Lactococcus lactis IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.Lactococcus lactis is generally recognized as the model organism for the study of lactic acid bacteria, and the complete genome sequence of the IL1403 strain (3, 4) will undoubtedly consolidate this position. When growing on rapidly metabolized sugars, this species shows homolactic metabolism in which more than 90% of metabolized sugar is converted to lactic acid. However, under certain conditions, this homolactic metabolism is replaced by a shift in pyruvate metabolism towards alternative fermentation pathways. Under anaerobic conditions, this shift involves an increased flux through the pyruvate-formate lyase reaction and leads to mixed acid fermentation and the accumulation of formate, acetate, and ethanol. These products accumulate in only low quantities during homolactic metabolism but have been shown to account for the majority of carbon flux under circumstances in which the rate of glycolysis of sugars is significantly diminished.This shift in pyruvate metabolism has been correlated with the NADH/NAD ratio (11). When flux through glycolysis is high, the high NADH/NAD ratio favors lactate dehydrogenase activity and provokes the inhibition of glyceraldehyde-3P dehydrogenase activity upstream of pyruvate. Under such conditions, metabolite pools upstream of glyceraldehyde-3P dehydrogenase (notably triose-Ps) increase within the cell, due to the controlling influence of this enzyme on glycolytic flux (10). Triose-Ps have a negative allosteric effect on pyruvate-formate lyase activity, thereby greatly diminishing the in vivo activity of the enzyme. Sugars metabolized at diminished rates lead to the relaxation of this coordinated control and facilitate the shift towards the more energetically fav...