Isolation of a gramicidin S hyperproducing strain of Bacillus brevis by use of a flourescence activated cell sorting system

Azuma, Tomoki; Harrison, Gail I.; Demain, Arnold L.

doi:10.1007/bf00174463

Cited by 32 publications

(18 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The degree of cellular heterogeneity observed in Figure 3 is an appropriate target for fluorescence-activated cell sorting (FACS), which can be used to selectively recover cells with the highest paclitaxel content for further analysis or the initiation of higher accumulating cell lines (10,13,16). However, the ultimate goal of sorting paclitaxel-accumulating subpopulations should be not only to increase production but also to reduce production variability.…”

Section: Resultsmentioning

confidence: 99%

“…Cell sorting can isolate a desired cell population at high purity (>95-99%) for the enhancement of cell culture properties, such as increased paclitaxel accumulation. This approach has been used to isolate subpopulations for strain improvement in bacterial cultures (13,16). Flow cytometric sorting has also been successfully applied to increase anthocyanin production in Aralia cordata (10) and berberine accumulation in Coptis japonica suspension cell cultures (8).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Flow Cytometric Identification of Paclitaxel-Accumulating Subpopulations

Naill

Roberts

2008

Biotechnol Progress

View full text Add to dashboard Cite

An immunofluorescence procedure was developed for paclitaxel quantification at the single cell level via flow cytometry in Taxus cuspidata suspension cultures. Intracellular staining was validated via fluorescence microscopy. Paclitaxel content of isolated cells and protoplasts was compared to total paclitaxel levels measured via HPLC. Paclitaxel accumulation was significantly increased by elicitation with methyl jasmonate (100 microM) on day 7 post-transfer as compared to unelicited cultures. Maximum accumulation was observed by day 12 post-transfer in both total paclitaxel (approximately 0.25 mg/L) and the percentage of paclitaxel-accumulating cells (approximately 95%). A similar trend was observed with isolated protoplasts, although protoplasts accumulated only ca. 40-75% of the paclitaxel present in single cells. In unelicited cell cultures, a small subpopulation (ca. 3-5%) of single cells was shown to accumulate paclitaxel. Although nearly all cells were observed to accumulate paclitaxel in methyl jasmonate-elicited cell cultures, a high degree of cell-to-cell variation was observed in paclitaxel content. The identified subpopulations represent targets for cell sorting, which may be applied to develop higher-accumulating cell lines. The quantification of single cell paclitaxel content is useful for characterizing production variability in cell cultures and can be utilized to develop rational strategies to increase paclitaxel production.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Flow Cytometric Identification of Paclitaxel-Accumulating Subpopulations

Naill

Roberts

2008

Biotechnol Progress

View full text Add to dashboard Cite

show abstract

“…Although we have deployed our biosensors for production optimization in 96-well plates, the greatest potential for biosensors is in pathway evaluation via fluorescence-activated cell sorting (FACS). FACS has been used for the optimization of biosynthetic pathways for more than two decades (31). Historically, the metabolic product must have possessed fluorescent properties or been amenable to fluorescent staining (31)(32)(33).…”

Section: Discussionmentioning

confidence: 99%

“…FACS has been used for the optimization of biosynthetic pathways for more than two decades (31). Historically, the metabolic product must have possessed fluorescent properties or been amenable to fluorescent staining (31)(32)(33). In contrast, genetically encoded biosensors have enabled FACS to be used for inconspicuous compounds.…”

Section: Discussionmentioning

confidence: 99%

Genetically encoded sensors enable real-time observation of metabolite production

Rogers

Church

2016

Proc. Natl. Acad. Sci. U.S.A.

164

114

View full text Add to dashboard Cite

Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules.biotechnology | directed evolution | biosensor | metabolic engineering | synthetic biology B iological production of valuable products such as pharmaceuticals or renewable chemicals holds the potential to transform the global economy. However, the rate at which bioengineers are able to engineer new living catalysts is hampered by an exceedingly slow design-build-test cycle. We describe a method to accelerate the design-build-test cycle for metabolic engineering by enabling the observation of product formation within microbes as it occurs.Biological production of a desired product is accomplished by guiding a low-cost starting material such as glucose through a series of intracellular enzymatic reactions, ultimately yielding a molecule of economic interest. The choice of culture conditions, the creation of enzyme variants, and the tuning of endogenous cellular metabolism create a vast universe of potential designs. Because of the complexity of biology, appropriate genetic designs and culture conditions are not known a priori. Even sophisticated modeling paradigms can result in very large design spaces (1, 2). Evaluating genetic designs or modulating process parameters to achieve a desired outcome is therefore a major bottleneck in the bioengineering design-build-test cycle.Current methods...

show abstract

“…Previously, targets for FACS screenings have included metabolites that are natural or synthetic photophores, such as carotenoids (Albrecht et al, 2000;An et al, 1991), the antibiotic gramicidin S (Azuma et al, 1992) or polyhydroxyalkanoates (Vidal-Mas et al, 2001). A common problem of FACS screenings is the high rate of false positives, in some cases beyond 99% (Sitnikov et al, 1995).…”

Section: Mutant Nomentioning

confidence: 99%