Lactococcus lactis as a Cell Factory for High-Level Diacetyl Production

Abstract

We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and ␣-acetolactate decarboxylase inactivation. Eighty percent of the carbon flux was found to be rerouted via ␣-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.Diacetyl has a strong, buttery flavor and is essential at low concentrations in many dairy products, such as butter, buttermilk, and fresh cheeses. It is also considered to be the most important off-flavor in the brewing process and in the wine industry.Diacetyl is a by-product of fermentation by many microorganisms. It is produced chemically by oxidative decarboxylation of the metabolic intermediate ␣-acetolactate (␣-AL). One molecule of ␣-AL is produced from two molecules of pyruvate by the condensing enzyme, ␣-AL synthase (ALS) (14) (Fig. 1). In dairy fermentation, ␣-AL is mainly produced by lactic acid bacteria as a result of the metabolism of citric acid, a minor component of milk (7). The ability to utilize citric acid is found only in some Leuconostoc species and specific variants of Lactococcus lactis, namely, the biovar diacetylactis. Due to the balancing of redox equivalents, sugars are converted via pyruvate to lactic acid, while the more oxidized substrate citric acid is converted into ␣-AL and subsequently into acetoin via ␣-AL decarboxylase (ALDB) (Fig. 1). Specific L. lactis strains isolated from dairy cultures that produce large amounts of ␣-AL from citric acid were shown to lack the ALDB enzyme (8). In dairy fermentation, these mutants are responsible for production of relatively high levels of diacetyl, the direct product of chemical decarboxylation of ␣-AL. New selection methods (4, 6) and deletion of the aldB gene by genetic engineering (15) have made these mutants more readily available.Based on the knowledge of the pathways involved in diacetyl production, several metabolic engineering strategies have been designed to improve diacetyl production by lactic acid bacteria. Since citric acid is only a minor component in milk, most efforts have been directed at converting lactose into diacetyl. Studies based on the overproduction of ALS (als [13] or ilvBN [2]), inactivation of lactate dehydrogenase (LDH) (3, 13), pyruvate formate-lyase (1), or ALDB (15), or a combination of these strategies (13, 15), have resulted in efficient conversion of lactose and glucose into acetoin, especially in the case of LDH inactivation (13).However, diacetyl production from all these engineered strains was low. Attempts to combine both LDH and ALDB inactivation in order to maximize the rerouting towards ␣-AL and diacetyl have so far been unsuccessful. Studies by Lopez de Felipe et al. (11) demonstrated that overproduction of the Streptococcus mutans NADH oxidase (NOX) in L. lactis resulted in a phenotype similar to that of the LDH-deficient strain described by Platteeuw et al. (12). In aerated cultures of L. lactis, more than 80% of the fermented su...

Natural sweetening of food products by engineering Lactococcus lactis for glucose production

“…Additionally, L. lactis is a suitable model organism for metabolic pathway engineering (Kleerebezem and Hugenholtz, 2003), since it has a relatively simple carbon metabolism and many molecular cloning tools are available (Kuipers et al, 1998;Leenhouts et al, 1996Leenhouts et al, , 1998. The metabolism of L. lactis has already been successfully engineered, e.g., for the production of the sweet amino acid L-alanine (Hols et al, 1999), the production of the buttery flavor diacetyl (Hugenholtz et al, 2000), the production of mannitol (Gaspar et al, 2004, Wisselink et al, 2005, and for the simultaneous overproduction of the vitamins folate and riboflavin (Sybesma et al, 2004). The aim of the present study was to disrupt glucose uptake and metabolism in L. lactis in such a way that, when growing on lactose it excretes glucose, which can be used as a natural sweetener in dairy products.…”

IS 981 -Mediated Adaptive Evolution Recovers Lactate Production by ldhB Transcription Activation in a Lactate Dehydrogenase-Deficient Strain of Lactococcus lactis

“…The las operon-encoded lactococcal Ldh protein converts pyruvate to lactate with high efficiency. Moreover, through the concomitant conversion of NADH to NAD ϩ , this reaction provides the electron sink required to maintain redox balance, which has been shown to be a critical determinant in the control of pyruvate flux in L. lactis (9,18,35,41).Construction of defined ldh disruption mutants of L. lactis has allowed redistribution of the lactococcal pyruvate pool toward products other than lactate (20,23,26,35,43,50). Under aerobic conditions, the ldh-deficient strains displayed an almost complete loss of lactate production and acetoin was found to be the main end product of fermentation, while the amounts of other metabolic end products like acetate, butanediol, ethanol, and formate appeared to depend on the fermentation conditions applied (23,43).…”

“…Construction of defined ldh disruption mutants of L. lactis has allowed redistribution of the lactococcal pyruvate pool toward products other than lactate (20,23,26,35,43,50). Under aerobic conditions, the ldh-deficient strains displayed an almost complete loss of lactate production and acetoin was found to be the main end product of fermentation, while the amounts of other metabolic end products like acetate, butanediol, ethanol, and formate appeared to depend on the fermentation conditions applied (23,43).…”

Genetically Engineered Lactobacilli for Technological and Functional Food Applications

“…Then, acetoin is formed by the activity of -acetolactate decarboxylase on -acetolactate and diacetyl results from a non-enzymatic oxidative decarboxilation of -acetolactate ( Figure 2). Most metabolic engineering approaches to produce diacetyl/acetoin by fermentation have been developed in the model LAB L. lactis, in which strains that divert an important part of pyruvate flux towards the production of -acetolactate have been constructed (Hugenholtz et al, 2000;Lopez de Felipe et al, 1998). ilvBN genes, encoding acetohydroxyacid synthase from L. lactis, have been expressed from the lactose operon in L. casei, an organism which shows marginal production of diacetyl/acetoin, resulting in increased diacetyl formation (Gosalbes et al, 2000).…”