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

hets@dtu.dk

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

Mosspiration Biotech IVS

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

Mosspiration Biotech IVS

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

Mosspiration Biotech IVS

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

Mosspiration Biotech IVS

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.