Robust at-line quantification of poly(3-hydroxyalkanoate) biosynthesis by flow cytometry using a BODIPY 493/503-SYTO 62 double-staining

Karmann, Stephanie; Follonier, Stéphanie; Bassas-Galià, Mònica; Panke, Sven; Zinn, Manfred

doi:10.1016/j.mimet.2016.10.003

Cited by 23 publications

(37 citation statements)

References 34 publications

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“…[176] Using the flow cytometer, the fluorescence signals can be measured at the single-cell level. [179][180][181][182][183] This flow-based quantification enables monitoring of the population heterogeneity in cell size and PHA accumulation within a short time (10 min for about 10 6 cells) with high sensitivity. [183] Thus, FACS can be used to isolate the desired variants of poly(3HB) producing E. coli.…”

Section: Pha Synthase Engineeringmentioning

confidence: 99%

“…[179][180][181][182][183] This flow-based quantification enables monitoring of the population heterogeneity in cell size and PHA accumulation within a short time (10 min for about 10 6 cells) with high sensitivity. [183] Thus, FACS can be used to isolate the desired variants of poly(3HB) producing E. coli. [182] This type of screening is simple and powerful, but it only depends on the amount of PHA accumulation regardless of the monomer compositions.…”

Section: Pha Synthase Engineeringmentioning

confidence: 99%

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Microbial Polyhydroxyalkanoates and Nonnatural Polyesters

et al. 2020

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Microorganisms produce diverse polymers for various purposes such as storing genetic information, energy, and reducing power, and serving as structural materials and scaffolds. Among these polymers, polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized and accumulated intracellularly as a storage material of carbon, energy, and reducing power under unfavorable growth conditions in the presence of excess carbon source. PHAs have attracted considerable attention for their wide range of applications in industrial and medical fields. Since the first discovery of PHA accumulating bacteria about 100 years ago, remarkable advances have been made in the understanding of PHA biosynthesis and metabolic engineering of microorganisms toward developing efficient PHA producers. Recently, nonnatural polyesters have also been synthesized by metabolically engineered microorganisms, which opened a new avenue toward sustainable production of more diverse plastics. Herein, the current state of PHAs and nonnatural polyesters is reviewed, covering mechanisms of microbial polyester biosynthesis, metabolic pathways, and enzymes involved in biosynthesis of short‐chain‐length PHAs, medium‐chain‐length PHAs, and nonnatural polyesters, especially 2‐hydroxyacid‐containing polyesters, metabolic engineering strategies to produce novel polymers and enhance production capabilities and fermentation, and downstream processing strategies for cost‐effective production of these microbial polyesters. In addition, the applications of PHAs and prospects are discussed.

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Section: Pha Synthase Engineeringmentioning

confidence: 99%

Section: Pha Synthase Engineeringmentioning

confidence: 99%

Microbial Polyhydroxyalkanoates and Nonnatural Polyesters

et al. 2020

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“…The intracellular polymer content of a bacterial culture can be quantified with different techniques, such as gas chromatography (GC) [ 10 , 11 ], fluorescence microscopy [ 12 , 13 , 14 ], or flow cytometry (FCM) [ 15 , 16 ]. Acomprehensive review about qualitative and quantitative methods for PHA analytics has been published recently [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

The Bistable Behaviour of Pseudomonas putida KT2440 during PHA Depolymerization under Carbon Limitation

2017

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Poly(hydroxyalkanoates) (PHAs) are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor PHA accumulation, whereas carbon limitation triggers PHA depolymerization when all other essential nutrients are present in excess. We studied the population dynamics of Pseudomonas putida KT2440 at the single cell level during different physiological conditions, favoring first PHA polymerization during growth on octanoic acid, and then PHA depolymerization during carbon limitation. PHAs accumulate intracellularly in granules, and were proposed to separate preferentially together with nucleic acids, leading to two daughter cells containing approximately equal amounts of PHA. However, we could show that such P. putida KT2440 cells show bistable behavior when exposed to carbon limitation, and separate into two subpopulations: one with high and one with low PHA. This suggests an asymmetric PHA distribution during cell division under carbon limitation, which has a significant influence on our understanding of PHA mobilization.

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“…In this respect, a successful collaboration between HES-SO Va lais Wallis and HES-SO Fribourg [10] led to the development of a quick and specific analytical method to follow the production of His-tagged proteins. [22] The new method is based on the binding of a specific fluorescent probe to the recombinant proteins released after cell lysis, with a read-out based on fluorescence polarization. Proof of principle was obtained that this assay can be integrated in a lab-on-valve (LOV) setup with automated lysis and mixing, followed by detection in a microfluidic platform (Fig.…”

Section: Hes-so Valais-wallis -Institute Of Life Te Chnologiesmentioning

confidence: 99%

“…Recently, we described a robust method for the quantification of cellular PHA contents using flow cytometry: the cells are double-stained by BODIPY 493/503 and SYTO 62 to mark PHA and DNA, respectively. [22] The advantage of this method is that PHA in cells can be clearly distinguished from hydrophobic debris (e.g. lysed cells), and most importantly, the time for at-line PHA analysis was less than 10 minutes (Fig.…”

Section: Hes-so Valais-wallis -Institute Of Life Te Chnologiesmentioning

confidence: 99%