Large scale microfluidic CRISPR screening for increased amylase secretion in yeast

Johansson, S. Andreas; Dulermo, Thierry; Jann, Cosimo; Smith, Justin D.; Pryszlak, Anna; Pignede, Georges; Schraivogel, Daniel; Colavizza, Didier; Desfougères, Thomas; Rave, Christophe; Farwick, Alexander; Merten, Christoph A.; Roy, Kevin R.; Wei, Wu; Steinmetz, Lars M.

doi:10.1039/d3lc00111c

Cited by 9 publications

(4 citation statements)

References 74 publications

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“…These enzymes and substrates have enabled high-throughput screens of signal peptides and genome engineering of strains to improve secretion efficiency, but are limited to optimising these subset of enzymes. 26, 29, 30 For the optimization of a more general POI, an invertase-based signal sequence trap method has been used to provide a survival based selection pressure for identifying optimal signal sequences within the genome. 9, 31 A potential limitation of this method however, is the requirement for a ∼60 kDa invertase C-terminal tag on a POI, which may limit secretion efficiency.…”

Section: Resultsmentioning

confidence: 99%

“…Overall, our system provides a rigorous way to analyse the effect of signal peptide sequence on protein secretion efficiency while varying promoter and terminator combinations in parallel, making it a useful tool in furthering our basic understanding of the synergistic effects of these different genetic elements upon this fundamental biological process. This system should also be applicable to high-throughput strain engineering, 30,51 and be useful in further parameterising genome-scale metabolic models of protein secretion. 52 Methods…”

Section: Discussionmentioning

confidence: 99%

“…While making significant progress on this problem, these methods still have drawbacks such as being substrate-dependent, labour intensive, or require large tags which may limit POI secretion efficiency. 4,8,9,26,[29][30][31] This highlights the need for a rapid and direct way to screen the secretion efficiency of any POI in a near native state, when assembled into a high-diversity SP and expression cassette library. Screening such diverse expression cassette libraries requires a high-throughput method to select the best cassette combinations for POI secretion.…”

Section: Introductionmentioning

confidence: 99%

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High-throughput optimisation of protein secretion in yeast via an engineered biosensor

Williams,

Luo,

Smith

et al. 2024

Preprint

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Secretion of high value proteins and enzymes is fundamental to the synthetic biology economy; allowing continuous fermentation during production and protein purification without cell lysis. Most eukaryotic protein secretion is encoded by an N-terminal signal peptide; however, the strong impact of signal peptide sequence variation on the secretion efficiency of a given protein is not well defined. Despite high natural signal peptide sequence diversity, most recombinant protein secretion systems employ only a few well characterised signal peptides. Additionally, the selection of promoters and terminators can significantly affect secretion efficiency, yet screening numerous genetic constructs for optimal sequences remains inefficient. Here, we have adapted a yeast G-protein coupled receptor biosensor, to measure the concentration of a peptide tag that is co-secreted with any protein of interest. Protein secretion efficiency can thus be quantified via the induction of a fluorescent reporter that is upregulated downstream of receptor activation. This enables high-throughput screening of over 6000 combinations of promoters, signal peptides and terminators, assembled using one-pot Combinatorial Golden Gate cloning. We demonstrate this biosensor can quickly identify best combinations for secretion and quantify secretion levels.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High-throughput optimisation of protein secretion in yeast via an engineered biosensor

Williams,

Luo,

Smith

et al. 2024

Preprint

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“…The enzyme's stability in a more extreme environment makes it suitable for bioremediation applications. In another example, Johansson et al , 77 have developed a workflow to boost recombinant protein production of the secreted α-amylase, starch degrading enzyme, used for bioethanol production. By combining droplet microfluidic screening with a large-scale CRISPR library to their workflow, they were able to control gene expression (increase or decrease expression) of 345 genes in S. cerevisiae , where the resulted shift in expression led to an increase in enzyme production.…”

Section: Applications Of Integrated Synthetic Biology and Microfluidi...mentioning

confidence: 99%

Integrating microfluidics and synthetic biology: advancements and diverse applications across organisms

Leal-Alves,

Deng,

Kermeci

et al. 2024

Lab Chip

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A Microfluidic Multiplex Sorter for Strain Development

Leal‐Alves,

Dumont,

Deng

et al. 2024

Adv Materials Technologies

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Selecting strains with superior traits from strain improvement strategies is challenging, as it involves navigating the fitness landscape by applying selective pressures that drive variants from peaks of improvement to valleys over time. In recent years, the screening and selection is conducted via droplet microfluidic methods due to its high throughput capabilities. However, the oft‐used binary strategy, targeting only the high levels of improved traits, may not reflect the overall enhancement. A multiplexed sorting method capable of applying an additional threshold to sort traits by phenotypic strength is reported. The novel approach uses a droplet‐digital microfluidic sorter to screen different volumes of droplets using the same device design and sorting parameters. This method is used to sort glucoamylase enzyme mutants with two levels of activity (medium and high) from libraries of diastatic yeast that have been mutated with non‐genetically modified techniques. Using the multiplex system, medium‐performing strains with enhanced (up to 60%) fermentation kinetics in synthetic beverage media, which would have been missed with a binary screening approach, are identified. The multiplex sorting strategy efficiently finds strains with superior fermentation traits in the fitness landscape without requiring extensive screening rounds and mutations.

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Large scale microfluidic CRISPR screening for increased amylase secretion in yeast

Abstract: Large scale perturbation of gene expression in yeast using CRISPR libraries, coupled with high-throughput screening using fluorescence-based sorting of microfluidic droplets, to identify genes important for increased α-amylase secretion.

Cited by 9 publications

References 74 publications

High-throughput optimisation of protein secretion in yeast via an engineered biosensor

High-throughput optimisation of protein secretion in yeast via an engineered biosensor

Integrating microfluidics and synthetic biology: advancements and diverse applications across organisms

A Microfluidic Multiplex Sorter for Strain Development

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