In vivo assembly of DNA-fragments in Physcomitrella patens ... · In vivo assembly of DNA-fragments in Physcomitrella patens as a biotech tool Henrik Toft Simonsen Associate Professor,

Mosspiration Biotech IVSProduction of natural fine chemicals using green cells

In vivo assembly of DNA-fragments in Physcomitrellapatens as a biotech tool

Henrik Toft Simonsen

Associate Professor, Photosynthetic Cell Factories, DTU

[email protected]

CEO and Founder, Mosspiration Biotech

www.mosspirationbiotech.com

DTU Bioengineering, Technical University of Denmark

Mosspiration Biotech IVS

The traditional way

http://2013.igem.org/Team:TU-Munich/Project/Physcomitrella Metabolic engineering of the moss Physcomitrella patens to produce the sesquiterpenoids

patchoulol and α/β-santalene. X Zhan et al. Frontiers in plant science, 2014

Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella

patens. SS Bach et al. Plant Isoprenoids: Methods and Protocols, 2014

DTU Bioengineering, Technical University of Denmark

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Several genes – large constructs

Sclareol production in the moss Physcomitrella patens and observations on growth and terpenoid biosynthesis

XW Pan, L Han, YH Zhang, DF Chen, HT Simonsen - Plant Biotechnology Reports, 2015

pUNI33-TPSsa3-2A-TPS113211,194 bp

DTU Bioengineering, Technical University of Denmark

Mosspiration Biotech IVS

Daniel G. Gibson et al. PNAS 2008;105:20404-20409

In-vivo assembly in yeast

Construction of a synthetic M. genitalium genome in yeast.

DTU Bioengineering, Technical University of Denmark

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Daniel G. Gibson Nucl. Acids Res. 2009;nar.gkp687

Twenty base-pair overlaps are sufficient for oligonucleotide assembly

in yeast.

DTU Bioengineering, Technical University of Denmark

Mosspiration Biotech IVS

In vivo assembly of DNA fragments – Novel transformation technology for moss – based on HR

In vivo assembly of DNA fragments. King et al., Scientific Reports 2016

Moss Genome

Promoter 1 Gene 1

Promoter 2 Gene 2

Promoter 3 Gene 3

Promoter 4Selection

gene

Moss genome crossover sequence – 1000 bp

12-100 bp crossover overhang

Moss genome crossover sequence – 1000 bp

DTU Bioengineering, Technical University of Denmark

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In-vivo 3 piece assembly illustrated by knock-out of cps/ks (ent-kaurene synthase)

1: ent-kaur-15-ene2: ent-kaur-16-ene

In vivo assembly of DNA fragments. King et al., Scientific Reports 2016

DTU Bioengineering, Technical University of Denmark

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Number of P. patens lines generated and comparison of transformation efficiency.

Resistant

lines

Fluorescent

lines

GC-MS

positive lines

Genotype-

positive lines(a)

Efficacy

[%](b)

linearized pBK3 20 14 6 2 10.0

In vivo ass. pBK3

12 bp overlap25 21 11 4 16.0

In vivo ass. pBK3

20 bp overlap18 14 6 4 22.2

In vivo ass. pBK3

50 bp overlap27 19 9 4 14.8

In vivo ass. pBK3

100 bp overlap19 12 8 4 21.1

PpENA1-Venus 32 32 c 6 18.8

AaADS 10 9 7 3 30.0

(a) Plant lines, in which the native locus replacement and proper assembly were confirmed.

(b) Defined as genotype-positive/resistant lines.

(c) No product was expected for the PpENA-Venus transformants.

DTU Bioengineering, Technical University of Denmark

Mosspiration Biotech IVS

In vivo assembly of DNA fragments A Novel transformation technology for moss

In vivo assembly of DNA fragments. King et al., Scientific Reports 2016

Moss Genome

Promoter 1 Gene 1

Promoter 2 Gene 2

Promoter 3 Gene 3

Promoter 4Selection

gene

Moss genome crossover sequence – 1000 bp

20-30 bp overhang

Moss genome crossover sequence – 1000 bp

DTU Bioengineering, Technical University of Denmark

Mosspiration Biotech IVS

Multiple in vivo assembly of DNA fragments for several Knock-outs in one go

Moss Genome

Promoter 1 Selection 1

Knock-out target 15’

KO target 13’

Promoter 2 Selection 2

Knock-out target 25’

KO target 23’

DTU Bioengineering, Technical University of Denmark

Mosspiration Biotech IVS

Thanks to …

@ Mosspiration BiotechHansol Bae CSO

@ Technical University of Denmark (DTU)Anantha Peramuna (PhD Student)Saira Ikram (PhD Student) - ArteMossKaren Martinez (PhD Student)Carmen Lopez (PhD Student)Freya Minnark-Oddsdottir (MSc student)Erling K. Rasmussen (BSc student)

@ University of CopenhagenSøren Brøgger Christensen (Prof.)Nina Rønsted (Prof.)

@ University of Calgary @ Michigan State UniversityDae-Kyun Ro Björn Hamberger

@ Firmenich @ Taxa BiotechnologiesVP Dong-Fang Chen Antony EvansMichel Schalk

@ University of Birmingham@ University of Freiburg Juliet CoatesRalf Reski Eleanor Vesty

Former members/students involved with mossChristina Lunde (NovoZymes A/S)Corinna Weitzel (Boehringer Ingelheim)Damian P. Drew (MD student, Adelaide)Brian C. KingTrine B. Andersen (looking for a postdoc)Søren Spanner Bach (NovoZymes A/S)Josephine Pinaeu (MSc student, Nancy)Bjørn Dueholm (PhD stud. UniSA, Aust.)Desire Fernandez (MSc Student)iGEM team UCPH 2015

Financial support: FTP x2, Carlsbergfondet, Fonden af 1870, Kong Christian D. X’s Fond, EU-Marie Curie, Det Strategiske Forskningsråd, Kræftens bekæmpelse, Malaysia Research Council.