catching up with the times: development of advanced...
TRANSCRIPT
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
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2
Trace minerals CO2
(Organic carbon) H2O
H2
O2
Pigments
Oils (TAG/PUFA)
Starch
Inputs Outputs
Biomass Fermentation Feed Biogass
Recombinant products
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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
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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
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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)
0
10
20
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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
50
100
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250
Rela
tive
pro
du
cti
vit
y (
%)
+
25
i
ii
iii
iv
v
vi
26 0
200
400
600
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1000
1200
1400
1600
1800
Pat
cho
ulo
l µg
/g
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250
300
350
i i+ii iii iv v vi
Re
lati
ve
pro
du
cti
vit
y (
%)
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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
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+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
15
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
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0
5
10
15
20
25
30
35
UVM4+N
TesA+N
UVM4-N
TesA-N
lipid
co
nte
nt
of
bio
mas
s [%
]
lipid content of UVM4 and TesA under +N and -N
TL
NL
PL
31
Polar lipids +N -N
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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
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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
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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
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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
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45
Control gLucLpIBP WT
Secreted ice binding protein in ice cream
-15 ˚C 49% sucrose
Andreas Leiter, Dr. Volker Gaukel
1 mm
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47
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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
5.00
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
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
mg
/ g
dry
bio
mas
s
Polar lipid FAMEs under -N
UVM4- TesA-62
0.00
0.50
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
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0.5
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0.7
0.8
0.9
Hyd
rate
par
titi
on
ind
ex
GFPLpIBP gLucLpIBP WT BSA E. coli Algae
CEP
Algae CEP
Application