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Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. East China University of Science and Technology, China.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2015 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 34, E4689-E4696 p.Article in journal, Letter (Other academic) Published
Abstract [en]

There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used highthroughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed wholegenome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

Place, publisher, year, edition, pages
NATL ACAD SCIENCES , 2015. Vol. 112, no 34, E4689-E4696 p.
Keyword [en]
protein secretion;yeast cell factories, droplet microfluidics, random mutagenesis, systems biology
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-159295DOI: 10.1073/pnas.1506460112ISI: 000360005600010PubMedID: 26261321ScopusID: 2-s2.0-84940521020OAI: diva2:784171

QC 20160429

Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2016-06-22Bibliographically approved
In thesis
1. Droplet microfluidics for directed evolution of biocatalysts
Open this publication in new window or tab >>Droplet microfluidics for directed evolution of biocatalysts
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biocatalysts, biologically derived catalysts, are of great importance for a wide range of industrial applications. They are used in the production of for example foods, pharmaceuticals and biofuels. Improving biocatalysts commonly relies on directed evolution, i.e. mutagenesis to form diverse variants followed by functional screening in an iterative fashion.

Droplet microfluidics is an emerging technology that can be applied for high throughput screening. A key feature of droplet microfluidics is the ability to encapsulate discrete objects, such as single cells, in picoliter-sized droplets at rates of over 1000 cells per second. Each droplet serves as a reaction vessel, analogous to a microwell, where a single clone can be screened.

In this thesis, droplet microfluidics is employed for directed evolution of biocatalysts. In paper I, a multiplexed droplet microfluidic method for characterization of enzyme variants is presented and validated by measuring the kinetics of β-galactosidase inhibited by IPTG. In paper II-III, a method for directed evolution of cells with improved production of industrially important enzymes is presented. Two rounds of directed evolution yielded improved strains. The strains had up to 6 times increased enzyme expression levels and whole-genome sequencing revealed 300 mutations, many of which mapped to the protein secretory pathway. In Paper IV, a method for directed evolution of enzyme variants under conditions lethal to host cells is developed. The method is used to screen for α-amylase variants with improved activity or stability at pH4. In Paper V, a method to screen cyanobacteria cell factories is developed and we show that the method can enrich for a strain with high production of L-lactate. In Paper VI, the metabolism of yeast cells encapsulated in microfluidic droplets is studied and found to depend on the choice of emulsion incubation device.

Taken together, droplet microfluidics is a promising technology for directed evolution of biocatalysts with the potential to vastly increase throughput and cut costs. The technology could pave the way for process customized biocatalysts and help replace polluting processes with sustainable green chemistry.

Abstract [sv]

Biokatalysatorer, dvs. katalysatorer av biologiskt ursprung, är viktiga inom en mängd olika industrier. För att förbättra biokatalysatorer används vanligtvis riktad evolution. En biokatalysator muteras för att skapa diversifierade varianter, vilka sedan kan screenas på funktion för att välja ut förbättrade varianter i ett iterativt arbetsflöde.

Droppmikrofluidik är en ny teknik som kan användas för höghastighetsscreening. Tekniken innebär att objekt såsom enskilda celler kan inkapslas i droppar med pikoliter volymer i hastigheter över 1000 celler per sekund. Varje droppe fungerar som ett reaktionskärl, analogt till en mikrobrunn, där en enskild klon kan screenas.

I denna avhandling redovisas studier om användning av droppmikrofluidik för riktad evolution av biokatalysatorer. I Paper I presenteras en multiplex droppmikrofluidisk metod för att karakterisera enzymvarianter. Metoden valideras genom att mäta kinetiken av β-galactosidase under inhibition av IPTG.  Paper II-III behandlar en metod för riktad evolution av celler med förbättrad produktion av industriellt viktiga enzymer. Två rundor av riktad evolution utfördes och förbättrade stammar kunde isoleras. Stammarna hade upp till 6 gånger högre enzymuttryck och hel genomsekvensering avslöjade fler än 300 mutationer. Många av dessa mutationer var i gener involverade i cellens maskineri för proteinsekretion. I Paper IV presenteras en metod för riktad evolution av enzymvarianter i miljöer som är dödliga för värdceller. Metoden användes för att screena efter förbättrade α-amylase varianter vid pH4. Paper V redovisar en metod för att screena cyanobakterier efter produktion av kemikalier. Vi visar att metoden kan användas för att anrika en stam som producerar mycket L-lactat. I Paper VI studeras metabolismen hos jästceller som är inkapslade i mikrofluidiska droppar. Cell metabolismen påverkades kraftigt av vilket inkubationskärl som användes för förvaring av emulsionen.

Sammanfattningsvis är droppmikrofluidik en lovande teknologi för riktad evolution av biokatalysatorer i och med att den ger avsevärt förbättrad screeninghastighet och lägre kostnader. Teknologin skulle kunna underlätta utvecklingen av ändamålsspecifika biokatalysatorer som potentiellt kan ersätta miljöförstörande processer med hållbar grön kemi.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. 78 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:3
Droplet microfluidics, Enzymes, directed evolution, high throughput screening
National Category
Biocatalysis and Enzyme Technology
Research subject
urn:nbn:se:kth:diva-159286 (URN)978-91-7595-428-8 (ISBN)
Public defence
2015-02-20, Gard Aulan, Roslagstullsbacken, AlbaNova, KTH, Stockholm, 10:00 (English)

QC 20150129

Available from: 2015-01-29 Created: 2015-01-28 Last updated: 2015-01-29Bibliographically approved

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