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Droplet microfluidics for directed evolution of biocatalysts
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:3
Keyword [en]
Droplet microfluidics, Enzymes, directed evolution, high throughput screening
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-159286ISBN: 978-91-7595-428-8 (print)OAI: oai:DiVA.org:kth-159286DiVA: diva2:784107
Public defence
2015-02-20, Gard Aulan, Roslagstullsbacken, AlbaNova, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20150129

Available from: 2015-01-29 Created: 2015-01-28 Last updated: 2015-01-29Bibliographically approved
List of papers
1. Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors
Open this publication in new window or tab >>Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors
2013 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 13, no 9, 1754-1761 p.Article in journal (Refereed) Published
Abstract [en]

Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine and industrial bioprocess technology. We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzyme substrate inhibitor systems in a single experiment utilizing an integrated droplet picoinjector for bioanalysis. This device enables the scanning of multiple fluorescently-barcoded inhibitor concentrations and substrate conditions in a single, highly time-resolved experiment yielding the Michaelis constant (K-m), the turnover number (k(cat)) and the enzyme inhibitor dissociation constants (k(i), k(i)'). Using this device we determine K-m and k(cat) for beta-galactosidase and the fluorogenic substrate Resorufin beta-D-galactopyranoside (RBG) to be 442 mu M and 1070 s(-1), respectively. Furthermore, we examine the inhibitory effects of isopropyl-beta-D-thiogalactopyranoside (IPTG) on beta-galactosidase. This system has a number of potential applications, for example it could be used to screen inhibitors to pharmaceutically relevant enzymes and to characterize engineered enzyme variants for biofuels production, in both cases acquiring detailed information about the enzyme catalysis and enzyme inhibitor interaction at high throughput and low cost.

Keyword
Beta-Galactosidase, Microchip, Systems, Assays, Chip, Flow
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-121627 (URN)10.1039/c3lc41398e (DOI)000316962700012 ()2-s2.0-84875790638 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130503

Available from: 2013-05-03 Created: 2013-05-03 Last updated: 2017-12-06Bibliographically approved
2. High-throughput screening for industrial enzyme production hosts by droplet microfluidics
Open this publication in new window or tab >>High-throughput screening for industrial enzyme production hosts by droplet microfluidics
Show others...
2014 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 4, 806-813 p.Article in journal (Refereed) Published
Abstract [en]

A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.

Keyword
Saccharomyces-Cerevisiae, Directed Evolution, Microdroplets, Selection, Systems, Assays
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-141316 (URN)10.1039/c3lc51202a (DOI)000330046100024 ()2-s2.0-84893020696 (Scopus ID)
Funder
Swedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140214

Available from: 2014-02-14 Created: 2014-02-13 Last updated: 2017-12-06Bibliographically approved
3. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
Open this publication in new window or tab >>Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
Show others...
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
Keyword
protein secretion;yeast cell factories, droplet microfluidics, random mutagenesis, systems biology
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-159295 (URN)10.1073/pnas.1506460112 (DOI)000360005600010 ()26261321 (PubMedID)2-s2.0-84940521020 (Scopus ID)
Note

QC 20160429

Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2017-12-05Bibliographically approved
4. High-­throughput screening for improved enzymes in environments lethal to host cells
Open this publication in new window or tab >>High-­throughput screening for improved enzymes in environments lethal to host cells
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-159306 (URN)
Note

QS 2015

Available from: 2015-01-29 Created: 2015-01-29 Last updated: 2015-01-29Bibliographically approved
5. Single-cell screening of secreted lactate production in cyanobacteria
Open this publication in new window or tab >>Single-cell screening of secreted lactate production in cyanobacteria
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-159307 (URN)
Note

QS 2015

Available from: 2015-01-29 Created: 2015-01-29 Last updated: 2015-01-29Bibliographically approved
6. Controlling cell metabolic state in droplet microfluidics
Open this publication in new window or tab >>Controlling cell metabolic state in droplet microfluidics
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-159308 (URN)
Note

QS 2015

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

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