catching up with the times: development of advanced...

Dr. Kyle J. Lauersen

Bielefeld University

Department of Biology

Center for Biotechnology CeBiTec

Algae Biotechnology & Bioenergy Group

Prof. Dr. Olaf Kruse

Catching up with the times:

Development of advanced genetic tools permits sustainable production of novel high and medium value products from eukaryotic microalgae

1

2

Trace minerals CO2

(Organic carbon) H2O

H2

O2

Pigments

Oils (TAG/PUFA)

Starch

Inputs Outputs

Biomass Fermentation Feed Biogass

Recombinant products

3

http://www.keweenawalgae.mtu.edu Jason Oyadomari, reproduced with permission

Finlandia University, Hancock, MI Michigan Technological University, Houghton, MI

4

Chloroplast engineering: - Prokaryotic-like and low GC - Recombinant protein production titres up to 5% TSP - Products contained in chloroplast - reducing environment

Nuclear engineering: - High GC content - All eukaryotic PTM possible subcellular targeting and secretion

Chlamydomonas reinhardtii

5

Challenges in nuclear transgene expression

~50% colonies contain incomplete vector (breakage) Range of expression due to random insertion in genome High-throughput screening for expression strains necessary

Lauersen, K. J., Kruse, O., and J. H. Mussgnug. 2015. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology and Biotechnology. 99(8), 3491-3503 6

SpeI AgeI

NdeI

BglII ZraI EcoRV

EcoRI AvrII HindIII XhoI KpnI

MCS

StrepII-TAA

i Reporter Selection marker i 3’ 3’ R H R H

ParomomycinR

HindIII XhoI

HygromycinR 1011 bp

845 bp

NdeI BglII

ZraI EcoRV EcoRI

Clover i2

mRuby2 i2

mCerulean3 i2

mVenus i2

gLuc i2

1064 bp

1091 bp

1097 bp

1097 bp

890 bp

NdeI BglII

N-term

EcoRV EcoRI

Targeting peptides and fusion proteins

C-term

pOptimized vector concept

No secretion Secretion

7 Lauersen et al. (2013) Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii. Journal of Biotechnology 167(2), 101-110.

8

Parental

pOpt

CFP YFP GFP DsRed Filter set

Lauersen, K. J., Kruse, O., and J. H. Mussgnug. 2015. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology and Biotechnology. 99(8), 3491-3503

Agar plate

Confocal

9 Lauersen et al. 2015. Applied Microbiology and Biotechnology

Putting pOpt to work Two applications of high-throughput reporter screening

Lauersen, K. J., Kruse, O., and J. H. Mussgnug. 2015. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology and Biotechnology. 99(8), 3491-3503 11

12

PTS2 PTS1

Protein X

[(S/A/C)-(K/R/H)-(L/M)] [(R/K)-(L/V/I)-X5-(H/Q)-(L/A)]

PTS2 PTS1

Peroxisomal targeting is mediated by two different signal peptide types

or

Strategy allows systematic screening for targeting

or

Enzymes of glyoxylate cycle in C. reinhardtii targeted to peroxisomes Important for metabolic network modeling

Lauersen et al., 2016. Algal Research

amiRNA gLuc

Rapid screening of amiRNA constructs facilitated by secreted luciferase

Hu et al., 2014 Plant J.

Luciferase signal TAP

Iodine vapour (starch) TAP-N

Black colour = starch present Light colour = less or no starch

gLuc control

sta6

UVM4 parent

ami1

ami2

ami3

ami4

ami5

Going beyond reporters: 1. Sesquiterpene production from C. reinhardtii 2. Modifying FA chain length in vivo

Going beyond reporters: 1. Sesquiterpene production from C. reinhardtii 2. Modifying FA chain length in vivo

MEP pathway

Cytosol Chloroplast

G3P C3

+ Pyruvate C3

DXP C5

MEP

CDP-ME

CDP-MEP

ME-cPP

HMBPP

IPP C5

DMAPP C5

GGPP C20

Carotenoids Chlorophylls

C45 (PQ pool)

DXS

DXR

MCT

CMK

MDS

HDS

HDR

IDI

GGPPs

PSY

GGR

SPPs

CO2 Mito

Nucleus

FPPs

FPP C15

Sesquiterpene products

Transporter?

C15

C45

UQ

C30

sterols

ER

Green algae

Heterologous terpene synthase expression

21

i i i i i2

Pogostemon cablin (Patchouli)

Single enzymatic step converts FPP –> Patchoulol

Patchoulol synthase (PcPs) – YFP fusion

Noticeable odour of patchoulol from fluorescent mutant cultures

Sesquiterpene (C15) production from C. reinhardtii Example: Patchoulol First demonstrated in December 2014

Patchoulol synthase (PcPs)

Farnesyl pyrophosphate (FPP) Patchoulol

0

0.001

0.002

0.003

0.004

0.005

Flu

ore

sce

nce

/ c

ell

Colony number

Fluorescence screen colony selection

Cultivate and dilute

Fluorescence and cell density measurements

1 week after transformation

7500

27500

47500

Flu

ore

sce

nce

Cell density

M/Z 138.00, 222.00, Patchoulol molar mass: 222.36

Two phase cultivation with dodecane

Allows direct analysis by GC MS

20150201_Julian_Kyle_Pat_alpha-humule... 01/02/2016 7:37:56 PM

RT: 13.0 - 21.1

13 14 15 16 17 18 19 20 21

Time (min)

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Re

lative

Ab

un

da

nce

19.8

14.119.8

14.0

14.1

NL: 3.20E5

m/z= 137.50-138.50+146.50-147.50+221.50-222.50 MS 20150201_Julian_Kyle_Pat_alpha-humulene_03_PcPs_18-2

NL: 3.20E5

m/z= 137.50-138.50+146.50-147.50+221.50-222.50 MS 20150201_julian_kyle_pat_alpha-humulene_04_pcps_5-3

NL: 3.20E5

m/z= 137.50-138.50+146.50-147.50+221.50-222.50 MS 20150201_julian_kyle_pat_alpha-humulene_05_wt

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1.2E+06

1.4E+06

1.6E+06

1.8E+06

2.0E+06

0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045 0.005

Pe

ak a

rea/

DB

M

Expression (fluorescence / cell)

0

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i

ii

iii

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vi

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Pat

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/g

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i i+ii iii iv v vi

Re

lati

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%)

27

3XPcPs_YFP – largest ever heterologous protein in C. reinhardtii ~224 kDa

Going beyond reporters: 1. Sesquiterpene production from C. reinhardtii 2. Modifying FA chain length in vivo

PsaD tesA YFP

tesA – originally a secreted protein from E. coli Patrick Jones (ICL) – PhotoFuel Removing N-terminal target peptide resulted in modified fatty acid composition in E. coli (1995)

Mod. from: Choi et al. (2013)

YFP chloro overlay DIC

GSG

29

+N -N +N -N UVM4 tesA

0.0

0.2

0.4

0.6

0.8

1.0

0 1 2 3 4 5 6

CD

W (

g/L)

Cultivation time (d)

0

5

10

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20

25

30

35

0 1 2 3 4 5 6

Celldensity10^6/m

LCul va on me/d

UVM4+N UVM4-N

TesA+N TesA-N

UVM4preculture TesApreculture

30

0

5

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UVM4+N

TesA+N

UVM4-N

TesA-N

lipid

co

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of

bio

mas

s [%

]

lipid content of UVM4 and TesA under +N and -N

TL

NL

PL

31

Polar lipids +N -N

32

0.0

0.5

1.0

1.5

2.0

UVM4+ TesA+

mg

/ g

dry

bio

mas

s

C14:0

0.0

0.5

1.0

1.5

2.0

2.5

UVM4- TesA-

mg

/ g

dry

bio

mas

s

C12:0 C14:0

Neutral lipids (and FFA) under -N

UVM4 tesA

C12:0 0.06 mg/g 0.39 mg/g

C14:0 0.46 mg/g 2.03 mg/g

33

0.0

0.5

1.0

1.5

2.0

2.5

UVM4- TesA-

mg

/ g

dry

bio

mas

s C12:0 C14:0

High-throughput screening agar plate level – robust reporters Multiple transgene expression Rapid gLuc-ami-RNA Sesquiterpenoid produced from C. reinhardtii (~1 mg g-1 DBM) FA modification in C. reinhardtii C12:0 + C14:0 in polar and neutral lipids

35

36

http://www.keweenawalgae.mtu.edu Jason Oyadomari, reproduced with permission

Finlandia University, Hancock, MI Michigan Technological University, Houghton, MI

Dr. Kyle J. Lauersen Prof. Dr. Olaf Kruse

April 6, 2016 This project has received funding in part from the European Union’s

Horizon 2020 research and innovation programme under grant

agreement No 640720

EU-HORIZON 2020 Collaborative Research Project “PHOTOFUEL”

i i i i i2

Terpene synthase – Fluorescent protein fusion

General strategy

Metabolic engineering of C. reinhardtii for terpenoid production

Generate transformants

more

fluorescence =

more synthase =

more product

Screen for fluorescence

pOpt

Modified from Lauersen et al. 2015

i i i i i2

Patchoulol synthase (PcPs) – YFP fusion

i i i i i2 i i

YFP PcPs ispA

0

50000

100000

150000

200000

250000

300000

350000

400000

PcPs PcPs +CrFPPs

PcPs + ispA PcPs + CFP ispaPcPs

Pe

ak a

rea

M/Z

13

8, 2

22

Fusion with bacterial FPP synthase (ispA) did increase patchoulol yields

Paro

Paro

40

Adding a new layer of value to algal cultivation Product secretion into culture media

ATG

Carbonic anhydrase 1 secretion signal (C. reinhardtii) ‘cCA’

H R i 3’ gLuc LpIBP

TAA

Lauersen et al. (2013) Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii. Journal of Biotechnology 167(2), 101-110.

Gaussia princeps luciferase

42

Lolium perenne ice binding protein

HSP70A-RBCS2-i1 constitutive promoter

Lauersen et al. (2013) Ice recrystallization inhibition mediated by a nuclear-expressed and secreted recombinant ice binding protein in the microalga Chlamydomonas reinhardtii.

Applied Microbiology and Biotechnology 97(22), 9763-9772.

43

Activity in culture media

Bioluminescence signal

detectable directly in

media

Spatial separation of

biomass and

recombinant product

44

45

Control gLucLpIBP WT

Secreted ice binding protein in ice cream

-15 ˚C 49% sucrose

Andreas Leiter, Dr. Volker Gaukel

1 mm

46

47

48

A little history…

NIT1 nitrate reductase Mainly from 1989-1995 complements NO3 reductase auxotrophy

Random insertion mutagenesis

pUC /pBluescript…

Codon optimized GOI

High GC selection marker ß-tubulin

Selectable marker i coGOI

Genomic GC content of ~64%

Requirement for endogenous regulatory elements

Codon optimized synthetic genes required

Integration into genome via non-homologous end-joining

49

First steps towards algae-specific transgene expression – nuclear transformations

Paromomycin R

Hygromycin R

0.E+00

5.E+05

1.E+06

2.E+06

2.E+06

3.E+06

0

2

4

6

8

10

12

14

16

18

0 24 48 72 96 120 144 168 192 216 240 264

GC

MS

Peak

Are

a (m

/z 1

38

.00

, 22

2.0

)

Cel

l de

nsi

ty (

*10

^6/m

L)

Time (h)

Cell density Peak area M/Z 138, 222

11 day cultivation 100 µE – TAP (acetate only) Patchoulol, elution time 19.6 min, mass: 138.00, 222.00

Product accumulation in dodecane is stable and occurs until anabolic processes cease in the cell

gLuc CFP Paro

Leica-Binocular MZFLIII system

Lauersen, K. J., Kruse, O., and J. H. Mussgnug. 2015. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology and Biotechnology. 99(8), 3491-3503 52

Parental

Cytoplasmic

Secretion

CFP YFP GFP DsRed Filter set

mVenus

cCA

PsaD

53

Photosystem I reaction center subunit II N-terminal targeting peptide. 36 aa. Chloroplast stroma

C. reinhardtii carbonic anhydrase 1 secretion signal. 21 aa. ER-Golgi

Scale bars 5 µm

Parental strain exhibits no signal in fluorescent channels (other than chlorophyll)

Cytoplasmic

RBCS1

Simian Virus 40 nuclear localization signal (NLS). 7 aa. Nucleus

54

cDNA of C. reinhardtii Rubisco small subunit 1. 185 aa. Pyrenoid

Peroxisome targeting sequence type 1-like (PTS1-like) of C. reinhardtii malate synthase. 10 aa. Peroxisome?

PTS1-like

NLS

Scale bars 5 µm

AtpA

ATP synthase subunit alpha N-terminal targeting peptide. 45 aa. Mitochondria

mCerulean3

Paro

Hyg

PTS1-like

RBCS1

PsaD

+

+

+

Scale bars 5 µm 55

Fluorescent protein fusion allows reliable screening

More fluorescence / cell (expression) correlates to increased productivity in culture

How to improve yields? – Precursor enzyme co overexpression

– Gene loading – co transformation or fusions

i i i i i2

MEP pathway Cytosol Chloroplast

G3P C3

+ Pyruvate C3

DXP C5

MEP

CDP-ME

CDP-MEP

ME-cPP

HMBPP

IPP C5

DMAPP C5

GGPP C20

Carotenoids Chlorophylls

C45 (PQ pool)

DXS

DXR

MCT

CMK

MDS

HDS

HDR

IDI

GGPPs

PSY

GGR

SPPs

CO2 Mito

Nucleus

FPPs

FPP C15

Sesquiterpene products

Transporter?

C15

C45

UQ

C30

sterols

ER

Green algae

Heterologous terpene synthases

i i i i2

C. reinhardtii FPP synthase – CFP fusion (genomic-hybrid)

i i i i2 i i i i i i i i i

E. coli FPP synthase (ispA) – CFP fusion

Hyg

Hyg

i i2

CFP control

Hyg

+

i i i i i2

Patchoulol synthase (PcPs) – YFP fusion

Paro

59

i i i i2

C. reinhardtii FPP synthase – CFP fusion (genomic-hybrid)

i i i i2 i i i i i i i i i

E. coli FPP synthase (ispA) – CFP fusion

Hyg

Hyg

i i2

CFP control

Hyg

+

i i i i i2

Patchoulol synthase (PcPs) – YFP fusion

Paro

0

200

400

600

800

Patc

ho

ulo

l µ

g/g

DB

M

0

100

200

300

400

Rela

tive

pro

du

cti

vit

y

(%)

Two types of proteins regulate FA chain length

Acyltransferases – transfer acyl-ACP directly to G3P

Thioesterases – split an ester into acid and alcohol, in the presence of water, specifically at a thiol group

This work is about a thioesterase: tesA

thiol bond

61

0.00

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10.00

15.00

20.00

25.00

30.00m

g /

g d

ry b

iom

ass

Polar lipid FAMEs under +N

UVM4+ TesA+

0.00

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20.00

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45.00

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/ g

dry

bio

mas

s

Polar lipid FAMEs under -N

UVM4- TesA-62

0.00

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1.00

1.50

2.00

2.50

mg

/ g

dry

bio

mas

s

Neutral lipid FAMEs (also free fatty acids) under +N

UVM4+ TesA+

0.00

10.00

20.00

30.00

40.00

50.00

60.00

mg

/ g

dry

bio

mas

s

Neutral lipid FAMEs (also free fatty acids) under -N

UVM4- TesA-63

MEP pathway

MVA pathway

Cytosol Chloroplast

G3P C3

+ Pyruvate C3

DXP C5

MEP

CDP-ME

CDP-MEP

ME-cPP

HMBPP

IPP C5

DMAPP C5

GGPP C20

Carotenoids Chlorophylls

C45 (PQ pool)

DXS

DXR

MCT

CMK

MDS

HDS

HDR

IDI

GGPPs

PSY

GGR

SPPs

3 Acetyl-CoA

HMG-CoA

MVA

MVAP

MVAPP

IPP C5

GPP C10

DMAPP C5

HMGS

IDI

CO2

FPP C15

GGPP C20

Monoterpenes

Sesquiterpenes Sterols

Diterpenes

HMGR

MK

PMK

MDC

GPPs

GGPPs FPPs

Plants and heterokont algae (diatoms)

Prof. Dr. Thomas Noll Angelika Schemel Sonja Siwiora

Julian Wichmann Thomas Baier

Prof. Dr. Dirk Lütkemeyer Prof. Dr. Frank Gudermann Dr. Anke Rattenholl Benjamin Pütz Viktor Kartushin

Prof. Dr. Virginia K Walker Kristy Moniz Tara Vanderveer

Prof. Dr. Karsten Niehaus Hanna Bednarz

Junior Prof. Dr. Torsten Seidel Martina Lummer

Dr. Volker Gaukel Andreas Leiter Prof. Dr. Clemens Posten Lena von Riesen Christian Steinweg

Prof. Dr. Olaf Kruse The entire Kruse Group

Prof. Dr. Jörn Kalinowski Vera Ortseifen

65

Acknowledgements

Prof. Dr. Volker Wendisch

Stern D, Witman GB, Harris EH (2008) The Chlamydomonas Sourcebook, Vol. 1-3, 2nd ed. Academic Press, Elsevier. 2032 pages

66

C. reinhardtii cell wall

67

Issue: cell wall proteins hinder chromatography

68

Concen

tra

te

Flo

w-t

hro

ugh

Elu

tion 1

Elu

tion 2

Intracellular vs. secreted protein production – different issues to overcome

Strep-tactin chromatography

Wash

Stern D, Witman GB, Harris EH (2008) The Chlamydomonas Sourcebook, Vol. 1-3, 2nd ed. Academic Press, Elsevier. 2032 pages

69

Nitrate reductase complementation restores nitrate growth

70

0

2

4

6

8

10

12

14

16

18

0 24 48 72 96

Ce

ll D

en

sity

(x1

0^

6 c

ells

/mL)

Time (h)

NUVM3

NUVM2

CC1883

UVM4

Growth on NO3 restored in ‘NUVM’ strains

71

A good system has standards

Lauersen, K. J., Kruse, O., and J. H. Mussgnug. 2015. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology and Biotechnology. 99(8), 3491-3503

72

Stefan Schulze, Prof. Dr. Michael Hippler

GOI Selectable marker

2A peptide

APVKQTLNFDLLKLAGDVESNPG ^ P

Bleomycin R

73

Rasala et al 2012

EU-HORIZON 2020

Collaborative Research Project

“PHOTOFUEL”

12 groups with Volkswagen as the leading participant

This project has received

funding from the European

Union’s Horizon 2020

research and innovation

programme under grant

agreement No 640720

75

0

0.2

0.4

0.6

0.8

1

1.2

0 24 48 72 96 120

Op

tica

l de

nsi

ty (

75

0 n

m)

Time (h)

UVM4

UVM4 + pJRgLucLpAFP

UVM4 + pJRCAgLucLpAFP

Results - cultivations

ATG NdeI EcoRI

H R i 3’ i2 gLuc LpIBP

StrepII-TAA

mVenus

T. versicolor laccase

T. versicolor laccase

B. pumilis laccase

T. versicolor laccase

B. pumilis laccase

Flavobacterium sp. OPH

Flavobacterium sp. OPH

EcoRV cCA

BglII

CfAFP

Chloromonas sp. AFP

76

A own diagram, B. modified from Middleton et al. (2012), C. modified from Lauersen et al (2011), D. modified from Hakim et al (2013) with author permission.

77

Ice recrystallization is a common cause of frozen food spoilage

0 h

20 h

-15 ˚C

78

Background

0.05 µM LpIBP (~1 mg L-1)

0 h

20 h

-15 ˚C 79

Background

80

2 HexNAc (GlcNAc), 6-7 Hexose (Man) and 2 Pentose (Xyl) Stefan Schulze, Prof. Dr. Michael Hippler

Results

~0.25% total soluble

intracellular protein

Secreted to 0.7-1 mg L-1

5 µg protein

load

Lauersen, K. J., Kruse, O., and J. H. Mussgnug. 2015. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology and Biotechnology. 99(8), 3491-3503 81

Intracellular YFP

Secreted FPs

82

CEP partitioned into crystalline tetrahydrofuran gas hydrate. Possible ‘green’ hydrate inhibitor

CEP

gLucLpIBP

CEP

WT

Lauersen et al. (2013) Ice recrystallization inhibition mediated by a nuclear-expressed and secreted recombinant ice binding protein in the microalga Chlamydomonas reinhardtii.

Applied Microbiology and Biotechnology 97(22), 9763-9772.

0

0.1

0.2

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0.4

0.5

0.6

0.7

0.8

0.9

Hyd

rate

par

titi

on

ind

ex

GFPLpIBP gLucLpIBP WT BSA E. coli Algae

CEP

Algae CEP

Application