Metabolic engineering of Lactococcus lactis: the impact of genomics and metabolic modelling (original) (raw)
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Enzyme and Microbial Technology, 2000
Lactic acid bacteria display a relatively simple metabolism wherein the sugar is converted mainly to lactic acid. The extensive knowledge of metabolic pathways and the increasing information of the genes involved allows for the rerouting of natural metabolic pathways by genetic and physiological engineering. We discuss several examples of metabolic engineering of Lactococcus lactis for the production of important compounds, including diacetyl, alanine and exopolysaccharides.
Metabolic Engineering of Lactic Acid Bacteria (LAB)
Apple Academic Press eBooks, 2019
Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http ://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75 % of measured product formation rates.
Metabolic engineering as a tool for enhanced lactic acid production
Trends in biotechnology, 2014
Metabolic engineering is a powerful biotechnological tool that finds, among others, increased use in constructing microbial strains for higher lactic acid productivity, lower costs and reduced pollution. Engineering the metabolic pathways has concentrated on improving the lactic acid fermentation parameters, enhancing the acid tolerance of production organisms and their abilities to utilize a broad range of substrates, including fermentable biomass-derived sugars. Recent efforts have focused on metabolic engineering of lactic acid bacteria as they produce high yields and have a small genome size that facilitates their genetic manipulation. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating lactic acid producing organisms developed to address and overcome major challenges in the lactic acid production process.
METABOLIC ENGINEERING OF LACTIC ACID BACTERIA FOR THE PRODUCTION OF INDUSTR
Lactic acid bacteria are used worldwide in the industrial manufacture of fermented foods. Their most important application in this respect is in the dairy industry with an enormous variety of fermented dairy products, while next to that is the fermented meat and vegetable products industry. Besides food production, LAB are used in a variety of other industrial applications such as the production of lactic acid, high-value metabolites involved in flavor and texture development or health applications, probiotic products, and antimicrobial peptides. Characteristics such as a rather simple energy and carbon metabolism and a small genome size (~2-3 Mb), make LAB important candidates for metabolic engineering strategies. Such strategies have mainly focused on rerouting of pyruvate metabolism to produce important fermentation end-products e.g. sweeteners, flavors, aroma compounds [1,2], and on more complex biosynthetic pathways leading to the production of exopolysaccharides and vitamins , while attempts to manipulate the central carbon metabolism (CCM) are rather limited in number . Being one of the model organisms in microbial metabolism, Lactococcus lactis has been the main target of metabolic engineering among LAB. The knowledge of its complete genome sequence [6], the availability of numerous genetic tools for this microorganism , and its industrial relevance, facilitated its use in the development of efficient cell factories . The present work aims to give an overview of the recent advances in engineering the metabolism of LAB for the production of industrially important compounds.
Metabolic engineering of exopolysaccharide production in Lactococcus lactis
2002
Outline of this Thesis Chapter 1 Sugar Catabolism and its Impact on the Biosynthesis and Engineering of Exopolysaccharide Production in Lactic Acid Bacteria. 11 Chapter 2 Regulation of Exopolysaccharide Production in Lactococcus lactis subsp. cremoris by the Source of Sugar. 31 Chapter 3 Engineering of Carbon Distrubution between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis. 49 Chapter 4 Functional analysis of the Lactococcus lactis galU and galE Genes and their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis. 69 Chapter 5 Identification and Characterization of the Lactococcus lactis rfb Operon Required for dTDP-rhamnose Biosynthesis. 91 Chapter 6 Increased Exopolysaccharide Production in Lactococcus lactis by Increased Expression Levels of the NIZO B40 eps Gene Cluster.
Metabolic engineering of lactic acid bacteria for the production of nutraceuticals
Antonie van …, 2002
Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.
Molecular and metabolic adaptations of Lactococcus lactis at near-zero growth rates
Applied and environmental microbiology, 2015
This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in L. lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly...