title: Nonhierarchical Flux Regulation Exposes the Fitness Burden Associated with Lactate Production in Synechocystis sp. PCC6803.
abstract: Cyanobacteria are mostly engineered to be sustainable cell-factories by genetic manipulations alone. Here, by modulating the concentration of allosteric effectors, we focus on increasing product formation without further burdening the cells with increased expression of enzymes. Resorting to a novel 96-well microplate cultivation system for cyanobacteria, and using lactate-producing strains of Synechocystis PCC6803 expressing different l-lactate dehydrogenases (LDH), we titrated the effect of 2,5-anhydro-mannitol supplementation. The latter acts in cells as a nonmetabolizable analogue of fructose 1,6-bisphosphate, a known allosteric regulator of one of the tested LDHs. In this strain (SAA023), we achieved over 2-fold increase of lactate productivity. Furthermore, we observed that as carbon is increasingly deviated during growth toward product formation, there is an increased fixation rate in the population of spontaneous mutants harboring an impaired production pathway. This is a challenge in the development of green cell factories, which may be countered by the incorporation in biotechnological processes of strategies such as the one pioneered here.
authors.affiliation: Molecular Microbial Physiology Group, Faculty of Science, Swammerdam Institute for Life Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Wei
authors.initials: W
authors.lastname: Du
authors.affiliation: Molecular Microbial Physiology Group, Faculty of Science, Swammerdam Institute for Life Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: S Andreas
authors.initials: SA
authors.lastname: Angermayr
authors.affiliation: Molecular Microbial Physiology Group, Faculty of Science, Swammerdam Institute for Life Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Joeri A
authors.initials: JA
authors.lastname: Jongbloets
authors.affiliation: Systems Bioinformatics, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam , De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Douwe
authors.initials: D
authors.lastname: Molenaar
authors.affiliation: Systems Bioinformatics, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam , De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Herwig
authors.initials: H
authors.lastname: Bachmann
authors.affiliation: Molecular Microbial Physiology Group, Faculty of Science, Swammerdam Institute for Life Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: Hellingwerf
authors.affiliation: Molecular Microbial Physiology Group, Faculty of Science, Swammerdam Institute for Life Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Filipe
authors.identifier: 0000-0002-4268-8080
authors.initials: F
authors.lastname: Branco Dos Santos
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics*; D005635:Fructosediphosphates / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism*; D008353:Mannitol / Q000031:analogs & derivatives / Q000378:metabolism; D046939:Synechocystis / Q000378:metabolism*
chemical_list: D005635:Fructosediphosphates; D019344:Lactic Acid; D008353:Mannitol; C031765:2,5-anhydromannitol; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate
keywords: 96-well cultivation system; allosteric regulation; cyanobacteria; genetic instability; lactic acid