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Elucidating Flux Regulation of the Fermentation Modes of Lactococcus Lactis a Mutlilevel Study

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Elucidating Flux Regulation of the Fermentation Modes of Lactococcus Lactis a Mutlilevel Study Downloaded from orbit.dtu.dk on: Oct 10, 2021 Elucidating Flux Regulation of the Fermentation Modes of Lactococcus lactis A Mutlilevel Study Chan, Siu Hung Joshua Link to article, DOI: 10.11581/DTU:00000016 Publication date: 2014 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Chan, S. H. J. (2014). Elucidating Flux Regulation of the Fermentation Modes of Lactococcus lactis: A Mutlilevel Study. Technical University of Denmark. https://doi.org/10.11581/DTU:00000016 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Elucidating Flux Regulation of the Fermentation Modes of Lactococcus lactis: A Mutlilevel Study PhD Thesis Siu Hung Joshua Chan November, 2014 Supervisors: Associate Professor Christian Solem Professor Peter Ruhdal Jensen Systems Biotechnology and Biorefining National Food Institute Technical University of Denmark Summary The long history of application to the dairy industry has established Lactococcus lactis (L. lactis), the lactic acid bacterium, as one of the most extensively characterized low GC organisms. The relatively simple metabolism of L. lactis has also made it an attractive target for metabolic engineering for the production of non-food related chemicals. Moreover, the status of being the first genetically modified organism to deliver immunoproteins alive to human has brought L. lactis considerable fame in biomedical research. Beside the exceptional industrial relevance of L. lactis, it is also an important subject for basic research in cellular metabolism because L. lactis exhibits an interesting metabolic shift. Under anaerobic conditions, on fast fermentable sugars, L. lactis produces lactate as the primary product, known as homolactic fermentation but on slowly fermentable sugars, significant amounts of formate, acetate and ethanol are formed, known as mixed-acid fermentation. This shift is termed the mixed- acid shift. This type of shift between a low-yield and a high-yield metabolism has drawn a lot of research focus and has similarly been observed in other bacteria, yeast and even tumor cells. Efforts have been put to find out the mechanism regulating the mixed-acid shift as well as to answer questions such as why L. lactis prefers such a switch. Until now, some pieces of evidence have been reported and several factors and models have been proposed as the keys to regulating the shift, including the expression level of certain genes in glycolysis and fermentation pathways, the levels of the cofactors NADH, NAD+, ATP and ADP, the balance between catabolism and anabolism, etc. In this project, we studied the mixed-acid fermentation of L. lactis by (i) examining the roles of the enzymes in the mixed-acid fermentation pathway under different growth conditions; (ii) testing the predicted effect of the cofactors NADH, NAD+ on the mixed-acid shift proposed in previous studies; (iii) looking into the connection between amino acid metabolism and the mixed-acid shift; and (iv) contrasting the difference regarding the mixed-acid shift between two widely studied laboratory strains of L. lactis, MG1363 that shifts significantly and IL1403 that does not shift. We have measured the promoter activities of several mixed-acid genes which suggested that the regulatory elements governing the transcriptional regulation of the mixed-acid genes in MG1363 and IL1403 were different. This led us to performing experimental control analysis of the role of pyruvate formate-lyase (PFL) in MG1363 and IL1403. The expression of PFL in MG1363 appeared to be optimized for growth rate when growing on maltose whereas overexpressing PFL in IL1403 was probably detrimental. ii The two homologous acetate kinases in MG1363 were also chacterized with respect to the transcription and enzyme activities. The isozymes were found to have complementary physiological roles that became important in acetate-producing or acetate-assimilating conditions respectively. The proposed roles of NADH and NAD+ on the mixed-acid shift were tested by perturbation via introducing activities of 2,3-butanediol dehydrogenase and supplying extracellular acetoin as an oxidizing agent. The additional NAD+- regenerating activities allowed a faster growth of MG1363 on maltose by shifting ethanol production into acetate production and also stimulated formate and acetate production in IL1403. Dependance of the mixed-acid fermentation of MG1363 on amino acid availability was observed and the impact of individual amino acids could differ significantly. Meanwhile, a computational method for combining metabolic flux analysis and elementary mode analysis was developed and applied to analyse a case of amino acid metabolism of L. lactis. iii Dansk resumé De mange års anvendelse af mælkesyrebakterien Lactococcus lactis (L. lactis) indenfor mejeriindustrien, har været medvirkende til at L. lactis er blevet en af de mest velkarakteriserede bakterier. Denne Gram positive bakterie, som har et lavt GC indhold, har en relativt simpel metabolisme og er let at modificere genetisk. Dette har gjort den til et attraktivt mål for ”metabolic engineering”, bl.a. med henblik på produktion af non-food relaterede kemikalier. Derudover har den status som den første genetisk modificerede organisme der i levende form er blevet anvendt til at levere immunproteiner til mennesker, og L. lactis er velkendt inden for biomedicinsk forskning. Ud over de vigtige roller som er blevet nævnt ovenfor, er L. lactis det også vigtig for grundforskning i cellulær metabolisme, bl.a. fordi L. lactis udviser et interessant metabolisk skift, som bevirker en ændring i produktdannelse. Let fermenterbare sukre giver anledning til hovedsagelig laktat (homolactic), medens sukre der omsættes langsommere giver anledning til andre organiske syrer og ethanol i tillæg til laktat (mixed-acid). Denne form for skift mellem en metabolisme med et lavt og et højt udbytte er der blevet forsket meget i, ikke kun i L. lactis men også i andre bakterier, gær og endda cancerceller. Indtil nu har mange beviser blevet rapporteret, og flere faktorer og modeller er blevet foreslået som nøglerne til regulering af skift, herunder udtrykket af visse gener i glycolyse og fermentering veje, niveauet for cofaktorer NADH, NAD+, ATP og ADP, balancen mellem katabolisme og anabolisme, etc. I dette projekt, vi studerede blandet syre fermentering af L. lactis ved (i) at undersøge de roller enzymer i blandet syre gæring vej under forskellige vækstbetingelser; (ii) testning af forudsagte virkning af cofaktorer NADH, NAD+ på blandede syre skift foreslået i tidligere undersøgelser; (iii) at se på sammenhængen mellem aminosyre metabolisme og det blandede syre skift; og (iv) kontrasterende forskellen med hensyn til blandet syre skift mellem to vidt studerede laboratoriestammer, L. lactis subsp. cremoris MG1363, der skifter betydeligt og L. lactis subsp. lactis IL1403, der ikke skifter. Vi har målt promotoraktiviteter i flere blandet syre gener, som antydede, at de regulatoriske elementen for transkriptionel regulering af det blandede syre gener i MG1363 og IL1403 var anderledes. Dette førte os til at udføre eksperimentel kontrol analyse af den rolle, pyruvat format lyase (PFL) i MG1363 og IL1403. Ekspressionen af PFL i MG1363 syntes at være optimeret til vækst, når de vokser på maltose henviser overekspression PFL i IL1403 var sandsynligvis skadelige. iv De to homologe acetat kinaser i MG1363 blev også chacterized med hensyn til transkription og enzymaktiviteter. De isozymer viste sig at have komplementære fysiologiske roller, som fik betydning i acetat-producerende eller acetat-assimilere betingelser hhv. De foreslåede roller af NADH og NAD+ på blandet syre skift blev testet ved perturbation via indføre aktiviteter af 2,3-butandiol dehydrogenase og leverer ekstracellulære acetoin som et oxidationsmiddel. De yderligere NAD+-regenererende aktiviteter have en hurtigere vækst i MG1363 på maltose ved at flytte ethanolproduktion i acetat produktion og også stimuleret formiat og acetat produktion i IL1403. Afhængighed af det blandede syre gæring af MG1363 på aminosyre tilgængelighed blev observeret og virkningen af individuelle aminosyrer kan afvige væsentligt. I mellemtiden var en beregningsmetode til at kombinere metabolisk flux analyse og elementær tilstand analyse udviklet og anvendt på metabolismen af L. lactis. v Acknowledgements It is impossible to complete my PhD study without the help of many people. First of all, I must express my sincerest gratitude to my supervisors, Christian Solem and Peter Ruhdal Jensen. Christian has taught me numerous principles, skills and knowledge in practical as well as theoretical molecular biology and cellular metabolism. From the very first day he has helped me, a person without any previous experience in biological experiments, to survive in the lab.
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