CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
Production of Value-Added Isoprenoids by Expressing Pinus sabiniana
Methylbutenol (MBO) Synthase Gene in Escherichia coli and Nostoc punctiforme
A thesis submitted in partial fulfillment of the requirements
For the degree of Master of Science
in Biology
By
Dinesh Gupta
December 2014
The thesis of Dinesh Gupta is approved by:
__________________________________
Stan Metzenberg, PhD
___________________
Date
__________________________________
Michael L. Summers, PhD
___________________
Date
__________________________________
Chhandak Basu, PhD, Chair
___________________
Date
California State University, Northridge
ii
Dedication
My family and all my teachers and professors have always been a source of
knowledge and encouragement for me and they have played a very instrumental role in
succeeding me to this achievement. I would like to sincerely dedicate this work to all
of them.
iii
Acknowledgements
I would like to extend my huge gratitude to my mentor Dr. Chhandak Basu for
his invaluable supervision, unconditional help and endless support throughout this
study period. It was an honor to work with him. I am deeply indebted to Dr. Michael
L. Summers for providing me the opportunity to collaborate with his lab and for his
generous input and support. I owe special and sincere thanks to Dr. Stan Metzenberg
for his incredible support, guidance and encouragement.
I would like to specially thank Dr. Thomas Sharkey, Michigan State University
and Dr. Jay D. Keasling, University of California, Berkeley for providing us plasmids,
and Dr. Dennis Gray, Saginaw Valley State University for helpful information. It is my
pleasure to acknowledge Dr. Rheem Medh, Dr. Larry Baresi, Dr. Ernest Kwok, Dr. Ray
Hong, Dr. Sean Murray and Dr. Tim Karels, Department of Biology, CSUN and Dr.
Mike Kaiser, Department of Chemistry, CSUN. I would also like to thank all of my
fellow lab members and friends Homa, Jamsheer, Asada, Tigran, William, Raunaq,
Richa, Michel, Andrew, Emaan, Mujaheed, Jing and John. I am particularly thankful to
Anantha Peramuna, Jenevieve Polin and all the members from Dr. Summers lab.
I would like to acknowledge CSUN Graduate Equity Fellowship 2012 and 2013;
Research and Creative Activity Graduate Fellowship 2013 and Pamela M. Klein, M.D.
Scholarship in Biology 2014 for the partial financial support.
I am grateful to my loving parents Laxmi Narayan Rauniyar and Kaushilya
Devi Rauniyar; my dear brother Mukesh Gupta and sister Rukmini Rauniyar for their
unconditional love and support. A special thanks to Sujina Mali. Finally, I would also
like to thank all the people who had directly or indirectly helped me during the entire
study period.
iv
Table of Contents
Signature Page
ii
Dedication
iii
Acknowledgements
iv
List of Figures
vii
Abstract
ix
Chapter-I: Introduction
Value-added Isoprenoids
1
Isoprenoid Pathway
2
2-Methyl-3-buten-2-ol (MBO)
4
MBO Synthase
5
Microbial Host for MBO Production
6
Chapter-II: Engineering an Isoprenoid Pathway in Escherichia coli for Production of
2-Methyl-3-buten-2-ol: A Potential Biofuel
Background
8
Materials and Methods
11
Bacterial Strains and plasmids
11
Introduction and Analysis of Heterologous Pathways in E. coli
12
Dot Blot Analysis
13
MBO Production and Quantification by Gas Chromatography (GC)
14
MBO Toxicity Assay
15
Results and Discussion
15
Effect of MVA Pathway Induction on Growth
15
Confirmation of Heterologous MVA Pathways and MBO Synthase Genes in
Transformed E. coli
16
MBO Production Assay by Gas Chromatography
17
MBO Toxicity Assay on Cell Growth
20
Conclusion
21
Additional Information to Chapter-II
22
v
Chapter III: Expression of Pinus sabiniana methylbutenol (MBO) synthase gene leads
to enhance production of phytols in Nostoc punctiforme
Background
25
Materials and Methods
27
Strains and Growth Conditions
27
Vector Construction and Transformation
28
Chlorophyll Quantification
28
Reverse Transcription PCR (RT-PCR)
30
SDS-PAGE Analysis
31
Analysis of MBO Production
31
GC-MS Analysis of Total Lipid Extracted from Control vs. Transgenic
N. punctiforme
32
Localization and Quantification of Phytols Production in N. punctiforme 33
Determination of Extraction Efficiency
Results and Discussion
34
35
Analysis of MBO Synthase Transgene Expression by RT-PCR
35
Analysis of MBO Synthase Transgene Expression by SDS-PAGE
36
MBO Production in Control vs. Transgenic N. punctiforme
37
GC-MS Analysis of Total Lipid Extracted from Control vs. Transgenic
N. punctiforme
38
Explanation for Phytols Overproduction in Transgenic Strains SBG102
40
Localization and Quantification of Phytol Production in N. punctiforme
44
Estimation of More Accurate Phytol Production Using Extraction
Efficiency
46
Conclusion
47
Summary
49
References
51
Appendix
56
vi
List of figures
Figure 1: Cytosolic MVA and plastidic MEP pathway
4
Figure 2: A schematic representation of closed system bioreactor for harvesting
MBO from E. coli culture
7
Figure 3: MBO production via MVA pathway involving synthetic operons
11
Figure 4: Comparative study of growth characteristics of E. coli BG101, harboring
pMevT, and pMBI plasmids, with or without IPTG addition
16
Figure 5: Confirmation of recombinant E. coli BG102 by dot blot analysis
17
Figure 6: MBO production in E. coli BG104
19
Figure 7: Optimization of MBO production in BG104 strain using LB broth, Terrific
Broth, LB broth with O2, and Terrific Broth with O2
19
Figure 8: Study of MBO toxicity on the growth of E. coli BG104
20
Figure 9: Study of MBO toxicity in E. coli DH5α cell viability
24
Figure 10: Schematic diagram of pSUN4KK2-MBO vector construction
29
Figure 11: Confirmation of pSUN4KK2-MBO plasmid by restriction digestion
29
Figure 12: N. punctiforme culture in air tight containers
33
Figure 13: General flowchart to study phytols accumulation in N. punctiforme
34
Figure 14: Testing for MBO synthase transcription using RT-PCR
36
Figure 15: SDS-PAGE protein analysis of crude protein extracts from control and
transgenic strains of N. punctiforme
Figure 16: Differential production of phytols in transgenic strains
37
39
Figure 17: MS spectra match of the peak 1 (RT=10.500) produced in this study with
the nearest match from the MS data of the library
39
Figure 18: MS spectra match of the peak 2 (RT=10.667) produced in this study with
the nearest match from the MS data of the library
40
Figure 19: MS spectra match of the peak 3 (RT=10.792) produced in this study with
the nearest match from the MS data of the library
40
Figure 20: A plausible biosynthetic pathway leading to formation of phytol in
N. punctiforme
42
vii
Figure 21: Alternative model leading to overproduction of phytol through supply of
a metabolic intermediate created by MBO synthase to GDP synthase in
N. punctiforme
44
Figure 22: Quantification of phytol production in control SBG101 (white) verses
transgenic SBG102 strains (blue)
45
Figure 23: Quantification of phytols production in control SBG101 (white) verses
transgenic SBG102 strains (blue) considering an extraction efficiency of
63.7%
46
viii
Abstract
Production of Value-Added Isoprenoids by Expressing Pinus sabiniana
Methylbutenol (MBO) Synthase Gene in Escherichia coli and Nostoc punctiforme
By
Dinesh Gupta
Master of Science in Biology
Non-renewable fossil fuels are responsible to meet more than 80% of global
energy demand and its increasing price has ignited the interest in renewable biofuel
production. 2-Methyl-3-buten-2-ol (MBO) is a natural volatile 5-carbon alcoholic
compound produced by several pine species. MBO has the potential to be used as
biofuel. Higher energy content and less solubility in water make MBO superior than
bioethanol in terms of both energy output and cost effectiveness. In pine chloroplast,
methyl-erythritol-4-phosphate (MEP) isoprenoid pathway produce dimethylallyl
pyrophosphate (DMAPP) which is utilized by MBO synthase for production of MBO.
MBO production from its natural host is challenging due to its volatile nature. However,
the volatile nature of MBO makes its recovery easier when produced through bacterial
cultures in closed system bioreactor. The aim of this study is to produce MBO from
Escherichia coli and also from the photosynthetic microorganism Nostoc punctiforme.
MBO production in E. coli was attained by metabolic engineering with
mevalonate (MVA) dependent pathway to increase the intracellular supply of DMAPP
substrate and co-transformation with codon optimized Pinus sabiniana MBO synthase
gene. Production was characterized and quantified using gas chromatography (GC)
analysis. Further, MBO production was optimized using different culture media and
conditions. MBO toxicity to the host cells was also studied to estimate the maximum
amount of MBO that can be produced from E. coli culture.
N. punctiforme was used as another host to produce photosynthetically derived
MBO by expressing the MBO synthase gene from a plasmid under control of an
indigenous petE promoter. Reverse transcriptase (RT)-PCR and SDS PAGE were
performed to analyze the MBO synthase gene expression at mRNA and protein level.
Although, the transcription and translation of MBO synthase were confirmed,
detectable level of MBO production was not detected through gas chromatograph-mass
spectrometry (GC-MS). Instead, enhanced production of phytols in the transgenic strain
was observed and confirmed through a GC-MS analysis of total extracted lipids. To
explain the enhanced production of phytols in transgenic strains, two plausible
hypothesis were proposed; first, presence of an indigenous broad range substrate
specific prenyltrasferases and second, appropriation of a MBO synthase metabolic
intermediate by a native geranyl diphosphate synthase. To study the location of phytol
production and accumulation in N. punctiforme, cell fractionation was performed using
French pressure cell press and ultracentrifugation. Phytols were found to be present in
cytoplasmic fraction.
This study demonstrates feasibility of MBO production through bioengineering
of E. coli however further work is required to improve its production to an economically
efficient level. At the same time, this work also highlights the challenges of
bioengineering a non-native cyanobacterial host, N. punctiforme, for production of
useful compounds.
ix
Chapter-I
Introduction
Value-added Isoprenoids
Isoprenoids consist of a large family of organic compounds including cyclic and
acyclic hemiterpenes, monoterpenes, sesquiterpenes and different higher terpenoids,
carotenoids and their derivatives (Lichtenthaler 2010). Isoprenoids are product of
isoprenoid biosynthetic pathway which is essentially present in all forms of biological
system. Isoprenoids can be volatile or non-volatile and perform a diverse array of
biological functions in the host organisms. For instance, isoprenoids play important
roles in biosynthesis of vitamins, hormones, pigments (photosynthetic as well as nonphotosynthetic), components of cell wall, components of protein-targeting, molecules
involving in signaling, defense, virulence etc. and it also helps in protection against
oxidation, cell membranes stabilization and tRNA modification (Holstein and Hohl
2004; Lichtenthaler 2010; Lichtenthaler 2010; Vickers et al. 2014).
A number of isoprenoids have been used as an active ingredients in flavor,
fragrances and pharmaceuticals (Zwenger and Basu 2008; Kirby and Keasling 2009)
and these economically important isoprenoids are also known as value-added
isoprenoids. For instance, artemisinin and taxol are pharmaceutically important
isoprenoids which are respectively used as an antimalarial drug (Martin et al. 2003) and
as an anticancer compound (Ajikumar et al. 2010). Based upon its high market
demands, these isoprenoids have been synthesized through metabolic engineering of
isoprenoid pathway in microbial systems (Ajikumar et al. 2008; Van Noorden 2010;
Zhang et al. 2013).
1
Currently, efforts are underway in search of renewable and ecofriendly
alternative to non-renewable fossil fuels and this area of alternative energy research is
one of the most important subjects in biological science. Isoprenoids with physicochemical properties similar to biofuels (for example phytol) have been reported
(Muradov et al. 2014). Also, different isoprenoids like isoprene and beta-caryophyllene
have been proposed as potential renewable biofuel (Lindberg et al. 2010; Reinsvold et
al. 2011; Yang et al. 2012; Zurbriggen et al. 2012). Isoprene is also used as starting
material in production of synthetic rubber and a feedstock in the biosynthetic industry
(Lindberg et al. 2010). Similarly, 2-Methyl-3-buten-2-ol (MBO) is another compound
produced through isoprenoid pathway which has potential to be used as biofuel and also
as substrate biomaterial in synthetic biology.
Isoprenoid Pathway
Isoprenoids are produced from common five carbon precursors, isopentenyl
pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). IPP and DMAPP are
known to be produced either through methyl-erythritol-4-phosphate (MEP) pathway or
mevalonate (MVA) pathway (Figure 1). MEP pathway occurs in prokaryotes and
plant’s chloroplasts while MVA pathway occurs in archea and eukaryotic cytosol
(Lichtenthaler 2000).
The MEP pathway utilizes intermediates of glycolytic pathway for IPP and
DMAPP synthesis. First, DOXP synthase (DXS) catalyzes the condensation of
pyruvate and glyceraldehyde 3-phosphate into deoxy-xylulose-5-phosphate (DOXP).
In subsequent reactions DOXP reductase (DXR), 4-diphosphocytidyl-2-C-methyl-Derythritol
synthase
(CMS),
4-diphosphocytidyl-2-C-methyl-D-erythritol
kinase
(CMK), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS), (E)-4-
2
Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) synthase (HDS) and HMBPP reductase (HDR) respectively converts DOXP into IPP or DMAPP (Figure 1).
Then, basic isoprenoid units, IPP and DMAPP are able to inter-change by the enzyme
IPP isomerase (Barkley et al. 2004; Lindberg et al. 2010). In this pathway, DOXP
synthase is a rate limiting enzyme (Rodríguez-Concepción 2006) and it is controlled
through feedback regulation by both IPP and DMAPP (Banerjee et al. 2013).
In the MVA pathway, two molecules of acetyl CoA condense to produce
acetoacetyl CoA catalyzed by thiolase. Acetoacetyl CoA then combine with another
molecule of acetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) via
HMG-CoA synthase. Subsequent reactions are catalyzed by HMG-CoA reductase
(HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK) and
mevalonate-5-pyrophosphate decarboxylase (MVD) and finally convert HMG-CoA to
IPP or DMAPP. HMGR is the rate limiting enzyme in MVA pathway and it is under
post-translational feedback regulation by FPP (Gardner and Hampton 1999). Therefore,
to overcome this limitation, a truncated HMGR in which N-terminal transmembrane
domain has been removed is generally used in construction of a heterologous MVA
pathway (Polakowski et al. 1998; Martin et al. 2003).
3
Figure 1: Cytosolic MVA and plastidic MEP pathway (Roberts 2007).
DMAPP is the substrate for hemiterpene synthases such as isoprene synthase
and MBO synthase. Prenyltransferases also use DMAPP as an initial allylic unit and
catalyze the sequential condensation reaction with IPP for production of elongating
prenyl pyrophosphate precursors geranyl diphosphate (GPP, C10), farnesyl
diphosphate (FPP, C15) and geranylgeranyl diphosphate (GGPP, C20) (Kellogg and
Poulter 1997; Vickers et al. 2014). These prenyl diphosphates are then used in the
isoprenoid pathway for synthesis of different isoprenoids through respective
bioenzymatic reactions.
2-Methyl-3-buten-2-ol (MBO)
MBO (C5H10O) is a short chain volatile alcoholic hydrocarbon, naturally
released into atmosphere by some of the pine trees natively found in western North
America (Gray et al. 2011). Plants emit a diverse group of short volatile isoprenoids
and theirs production may be constitutive or stress-induced to protect plant against
different abiotic stresses (Sharkey and Singsaas 1995; Vickers et al. 2009; Loreto and
Schnitzler 2010) as well as biotic stresses (Fineschi and Loreto 2012). The role of
4
production of MBO in some of pine species is still unclear but has been shown to
increase upon exposure to high light and temperature and thought to play a similar role
as isoprene (a similar compound to MBO) in protecting plant against thermo-tolerance
(Gray et al. 2003).
Thomas D. Sharkey in 2009 pointed out MBO as a promising compound in
biosynthesis of useful products including biofuels (Sharkey 2009). Energy density of
MBO was found to be superior to ethanol and also MBO is less soluble in water than
ethanol
(http://www.wisys.org/uploads/media/WSTS_Guay_and_Singsaas_PP.ppt).
Ethanol is currently used as biofuel in fuel supplement (Lamsen and Atsumi 2012).
Cost effectiveness and higher energy content are some of the major requirements for
production of an ideal biofuel. MBO, with higher energy output and less solubility in
water, has potential to be developed into future biofuel. Different commercially
important compounds including flavor, fragrances and medicinally important
molecules can be produced using MBO as starting biomaterial. An isoforms of MBO,
3-methyl-2-butenol, can also be potentially used to synthesize vitamin A and vitamin E
(Chou and Keasling 2012).
MBO Synthase
MBO synthase is a soluble protein of molecular size of 70 kD which catalyzes
conversion of dimethylallyl pyrophosphate (DMAPP) into MBO (Gray et al. 2011).
MBO synthase gene from P. sabiniana has been sequenced and MBO synthase
recombinant protein produced in E. coli has been characterized (Gray et al. 2011).
Phylogenetically, MBO synthase and different monoterpene synthases genes belong to
terpene synthase (TPS) gene subfamily d1 (TPS-d1) (Gray et al. 2011). MBO synthase
has been shown to be dual in nature in vitro, producing both MBO and isoprene at a
5
ratio of 90:1 respectively but no isoprene production from its natural host plant has been
reported in vivo (Gray et al. 2011). Substrate DMAPP in plant chloroplast is produced
through MEP pathway where MBO synthase catalyzes its conversions into MBO.
Microbial Host for MBO Production
Although, several pine trees produce MBO naturally, but, one of the major
limitations in MBO production and collection from its natural host is its volatile nature.
Therefore in this study we sought to produce MBO from microbial hosts so that MBO
can be produced in a closed system and collected. Typically, biofuel produced through
bacterial cultures need to be phase separated from host cells and growth culture medium
through expensive and complicated procedures. However, the volatile nature of MBO
makes its recovery easier in this case if it could be produced from bacterial cultures. A
gaseous/aqueous two-phase photobioreactor for cyanobacterial culture and collection
of produced volatile isoprene has been shown before (Bentley and Melis 2012) and a
similar proposed bioreactor design for MBO production either from E. coli or N.
punctiforme is shown in figure 2.
Here, we hypothesize that (i) MBO can be produced from E. coli by engineering
it with a heterologous MVA pathway along with the expression of MBO synthase gene
from P. sabianiana, and (ii) photosynthetically derived MBO can be produced from a
cyanobacterium N. punctiforme by expressing MBO synthase gene, utilizing the native
MEP pathway. MBO production through two different host has its own advantages.
MBO production through a photosynthetic host, N. punctiforme, provides renewable
source while production through E. coli provide a host more compatible with existing
infrastructures. The production of advanced eco-friendly biofuels are required to
address the imminent problem of fuel crisis sustainably. In this scenario, our research
6
on isoprenoid-based biofuel production may be helpful in near future to address the
issue of global energy crisis.
Figure 2: A schematic representation of closed system bioreactor for harvesting MBO
from E. coli culture [Adapted from (Bentley and Melis 2012)]. Similar closed system
bioreactor for production of MBO from N. punctiforme is also possible with a little
variation.
7
Chapter-II
Engineering an Isoprenoid Pathway in Escherichia coli for Production of 2-Methyl-3buten-2-ol: A Potential Biofuel
Background
Both increasing demand for petroleum products and dependence on
nonrenewable fossil fuel has spurred research into efficient production of renewable,
ecofriendly alternative sources of biofuel. To enhance efficiency and integration into
existing infrastructure, new biofuels should have physicochemical properties
comparable with gasoline. Bioethanol has been used as gasoline supplement, however,
its low energy density combined with high purification cost inherently limits its optimal
use (Lamsen and Atsumi 2012). While attempts are being sought to increase efficiency
and reduce the overall cost of ethanol production (Lynd et al. 2008; Elshahed 2010),
identification and production of other potential renewable biofuels such as short chain
volatile alcohols and hydrocarbons are under investigation (Sharkey 2009; Lindberg et
al. 2010). The search for substances with enhanced chemical properties compared to
ethanol is driving explorations in this area (Fortman et al. 2008; Peralta-Yahya et al.
2011; Zhang et al. 2011; Chou and Keasling 2012). MBO (C5H10O) is a short chain
volatile alcohol naturally produced by several indigenous pine trees of western North
America (Harley et al. 1998; Schade et al. 2000; Lerdau and Gray 2003). The energy
content of MBO is calculated to be 106,000 BTU/gallon, and is more comparable with
petroleum (111,000–125,000 BTU/gallon) than ethanol (85,000 BTU/gallon)
(http://www.wisys.org/uploads/media/WSTS_Guay_and_Singsaas_PP.ppt).
Note: This chapter-II was published in journal of ‘Molecular Biotechnology’ in June
2014 in volume 56, issue 6, pp. 516-523. (Online published on 22 November 2013)
This chapter is written in the verbatim except for minor changes like figure number
and reference format.
8
MBO is also less soluble in water than ethanol, allowing capture of the volatile
MBO from the headspace of a culture without requirement of expensive post harvesting
steps. This volatile nature of MBO also increases production efficiency by preventing
excess accumulation of MBO in the growth media and subsequently its inhibitory effect
on microbial culture. In addition to being an ideal candidate for renewable biofuel,
MBO can also be used as starting biomaterial to produce number of value added
compounds. 3-Methyl-2-butenol, one of MBO isoform, can potentially be used in
synthesis of vitamin A, vitamin E, flavor or fragrances, and many other
pharmaceutically significant molecules (Chou and Keasling 2012).
The large family of organic compounds known as isoprenoids has been
recognized as important source of flavor, fragrances, and medicine (Zwenger and Basu
2008; Kirby and Keasling 2009). Many pharmaceutically important compounds have
been synthesized through metabolic engineering of the isoprenoid biosynthetic
pathway. For example, artemisinin, a potential antimalarial drug (Martin et al. 2003)
and taxol, an anticancer compound (Ajikumar et al. 2010), are commercially produced
in microbial systems. There are also recent reports on potentials of isoprenoid
molecules for use as renewable biofuels (Reinsvold et al. 2011; Yang et al. 2012;
Zurbriggen et al. 2012). Of relevance to this work is the observation that an intermediate
in isoprenoid synthesis is the substrate for MBO synthase.
Two isoprenoid biosynthetic pathways exist in plants: chloroplasts produce the
MBO precursor, dimethylallyl diphosphate (DMAPP), through methyl-erythritol-4phosphate (MEP) pathway, whereas in the cytosol, DMAPP is produced by the
mevalonate (MVA) pathway ( Rohmer 1999; Lange et al. 2000; Zurbriggen et al. 2012).
In the MEP pathway, pyruvate and glyceraldehyde 3-phosphate condense to produce
deoxy-xylulose-5-phosphate which is converted to DMAPP following six subsequent
9
enzymatic reactions (Lichtenthaler 2000). This pathway also exists in most of
prokaryotic bacteria, including Escherichia coli. In the MVA pathway, DMAPP is
produced from acetyl-CoA precursors using several enzymatic reactions as shown in
Figure 3. In P. sabiniana, DMAPP is directly converted to MBO, catalyzed by MBO
synthase (Gray et al. 2011). Although the gene for MBO synthase is encoded by nuclear
genome, a short N-terminal signal sequence targets this protein to chloroplast where the
DMAPP precursor is present due to its production via the MEP pathway (Lichtenthaler
1999; Gray et al. 2011). Recently, MBO has also been reported as one of the metabolites
in tert-Amyl alcohol (TAA) degradation. In A. tertiaricarbonis L108, MBO was
produced during TAA degradation in small amounts (less than 5%) (Schuster et al.
2012).
Several attempts have been made in the past to engineer native bacterial MEP
pathways to increase DMAPP precursor pools, however, limited success was reported
due to a ‘‘tight regulatory mechanism’’ (Martin et al. 2003; Chang and Keasling 2006;
Zurbriggen et al. 2012). To overcome this limitation, the MVA pathway in E. coli was
engineered so that the ‘‘upper half’’ of the mevalonate pathway genes were expressed
from plasmid pMevT, and ‘‘lower half’’ genes were expressed from plasmid pMBI
(Martin et al. 2003), which together constitute complete set of genes to efficiently
convert acetyl CoA to DMAPP (Figure 3). Following the addition of a codon optimized
P. sabiniana MBO synthase gene (Gray et al. 2011) to this genetic background, we
demonstrate the feasibility of MBO production in E. coli.
10
Figure 3: MBO production via MVA pathway involving synthetic operons. Shown in
orange are genes present in pMevT. atoB acetoacetyl-CoA thiolase, HMGS HMG-CoA
synthase, tHMGR: truncated HMG-CoA reductase; Shown in yellow are genes present
in pMBI. ERG12 mevalonate kinase, ERG8 phosphomevalonate kinase, MVD1
mevalonate pyrophosphate decarboxylase, idi IPP isomerase; Shown in blue is the gene
present in pMBO. MBOS MBO synthase from P. sabiniana. In the above MVA
pathway, atoB, and idi were isolated from E. coli, and all other genes were isolated
from Saccharomyces cerevisiae. The T5 and lac promoters are indicated by yellow and
blue triangles, respectively. Figure was adapted from: (Martin et al. 2003; Gray et al.
2011).
Materials and Methods
Bacterial Strains and Plasmids
Escherichia coli DH5α and SoluBL21 (AMS Biotechnology) strains were used
as expression hosts in Luria–Bertani (LB) or Terrific Broth growth media. The pMBO
plasmid (Gray et al. 2011) harboring P. sabiniana MBO synthase gene (GenBank No.
JF719039) optimized for expression in E. coli, was generously provided by Dr. Thomas
D. Sharkey, Michigan State University. For heterologous MVA pathway expression in
E. coli, two plasmids (pMevT and pMBI) encoding S. cerevisiae mevalonate pathway
11
genes were kindly provided by Dr. Jay D. Keasling, University of California, Berkeley.
All other important properties of bacterial strains and plasmids used in this study are
listed in Table 1.
Table 1: Bacterial strains, plasmids, and primers used in this study.
Relevant properties
Source
Strains
E. coli DH5α
Expression host
Lab stock
E.coli SoluB21
E. coli BG101
Expression host (DE3) with IPTG inducible T7 polymerase (Cms)
E. coli DH5α harboring pMevT and pMBI
Lab stock
This work
E. coli BG102
E. coli DH5α harboring pMBO, pMevT and pMBI
This work
E. coli BG103
E. coli SoluB21 harboring pMevT and pMBI
This work
E. coli BG104
Plasmids
pMevT
E. coli SoluB21 harboring pMBO, pMevT and pMBI
This work
Ori p15A, CmR, Plac, AtoB-HMGS-tHMGR
pMBI
Ori pBBR1, TetR, Plac, MK-PMK-PMD-Idi
pMBO
Ori pMB1, KanR , P. sabininana mbo synthase
Martin et al
2003
Martin et al
2003
Gray et al
2011
Primers
HMGS-f
5’GAATTAAGGAGGACAGCTAAATGAAACTCTCAACTAA
ACTTTG
5’AGTGTAATCCTCCTTATTTTTTAACATCGTAAG
idi-r
5’ATCCCGGGAGGAGGATTACTATATGCAAACGGAACAC
GTC
5’ATCCCGGGTTATTTAAGCTGGGTAAATG
MBOS-f
5’GCTTGTAGCGCGATTCAAACGGAA
Martin et al
2003
Martin et al
2003
Martin et al
2003
Martin et al
2003
This work
MBOS-r
5’AGACGCCTTTCATGTACTCTGGCA
This work
HMGS-r
idi-f
Introduction and Analysis of Heterologous Pathways in E. coli
Chemically competent E. coli cells were sequentially transformed with the
pMevT and pMBI plasmids using standard chemical transformation (Sambrook and
Russell 2001), resulting in E. coli strains BG101 (DH5α) and BG103 (SoluBL21).
Sequential plasmid transformations were performed; selected transformants were made
chemically competent before subsequent transformations. To test growth defects
caused by expression of the MVA pathway genes as mentioned previously (Martin et
12
al. 2003), the overnight culture of transformed E. coli BG101 was subcultured in LB
media supplemented with antibiotics at 37oC shaking at 200 rpm. After 2 h of culture
initiation, the culture was induced with 0.5 mM IPTG or water (for minus IPTG
controls) and incubated. To study the growth characteristics, optical density of 1 ml
cultures was measured at 600 nm before and after 3, 6, 9, and 24 h of IPTG induction
using a NanoDrop 2000c Spectrophotometer (Thermo Scientific, USA). After
confirming growth defects in strains containing the introduced mevalonate pathway
genes, pMBO was similarly transformed into BG101 and BG103, and selected using
triple antibiotic selection to produce strains BG102 and BG104, respectively.
Dot Blot Analysis
To confirm the compatibility and stability of three plasmids to coexist in an E.
coli expression host, dot blot analysis was performed on strain BG102. Plasmid DNA
from various strains of E. coli was extracted with Gene JET Plasmid Mini prep kit
(Fermentas Life Science, USA). An overnight culture of BG102 from a freezer stock
was used to inoculate 5 ml of LB broth with antibiotics and 0.5 mM IPTG in 4 tubes
for plasmid extraction for dot blot analysis. Plasmids were denatured by boiling, applied
to a positively charged nylon membrane, and UV cross-linked. Probes were PCR
amplified from purified plasmids, HMGS from pMevT, idi from pMBI, and MBOS
from pMBO (Table 1), using Dream Taq Master Mix (Fermentas, USA). All
oligonucleotide primers were purchased from Integrated DNA Technology, USA. PCR
condition included an initial denaturation at 95oC for 5 min, 30 PCR cycles with
denaturation at 95oC for 30 s, annealing for 30 s (HMGS: 53.5oC, idi: 57oC, MBOS:
55.2oC), and extension at 72oC for 1 min, with a final extension at 72oC for 5 min. The
amplified products were gel purified using NucleoSpin Gel and PCR clean-up kit
(Macherey–Nagel, Germany), and digoxigenin (DIG) labeled using DIG High Prime
13
Labeling Kit (Roche Applied Science, USA) following manufacturer’s instructions and
protocols described previously (Hart and Basu 2009). The pBIN-mGFP5-ER plasmid
(Haseloff et al. 1997) was used as negative control in each experiment. The
hybridization was performed at 42oC as reported before (Hart and Basu 2009). The
hybridized blot was washed as recommended by the manufacturer, and hybridization
detected by anti-DIG antibody conjugated with alkaline phosphatase, using NBT/BCIP
(nitroblue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl-phosphate in 67% DMSO)
for color detection.
MBO Production and Quantification by Gas Chromatography (GC)
To assay the amount of MBO produced by E. coli strains BG102 and BG104,
overnight cultures inoculated from freezer stocks were used to inoculate 3 ml of LB
broth containing antibiotics and 0.5 mM IPTG in sealable tubes. The culture tubes were
then capped with rubber stoppers, crimp sealed, and incubated for 48 h at 37oC shaking
at 200 rpm. After incubation, gas from the headspace was sampled and analyzed by
GC–FID using a Hewlett-Packard, HP6890 Gas Chromatograph equipped with a HP-5
column (crosslink 5 % PhMe siloxane 30 m X 0.25 μM film thickness) at 90oC with
helium as the carrier gas at a flow rate of 1.1 ml/min using a 1:20 split ratio. MBO was
identified by comparing retention time with a MBO standard (Sigma-Aldrich, USA).
The amount of MBO produced by bacterial cultures was calculated by comparing the
peak area of the chromatograph against MBO standard peak area prepared in identical
sealed vials containing an equal volume of water. The production of MBO was further
optimized using Terrific Broth and oxygenation using overnight cultures of E. coli
BG104 grown in the same media as used in the experiment. Oxygen enrichment in
culture tubes was performed by blowing pure oxygen into the headspace of culture
tubes and quickly capping the tubes in front of blowing oxygen. All sets of experimental
14
tests (cultures in LB, LB with O2, Terrific Broth, and Terrific Broth with O2) were
carried out in triplicates, and MBO production was analyzed by using GC–FID as
described above.
MBO Toxicity Assay
To test MBO toxicity to the cell growth, E. coli strain BG104 was grown at
37oC for 2 h in LB medium supplemented with antibiotics in a 125 ml flask. This stock
was used to aliquot 3 ml of bacterial culture to individual sterile vials with 20 mM
glucose. Various amounts of liquid MBO (Sigma-Aldrich, USA) ranging from 0.5 to
30 μl were added, and the vials were immediately caped and crimp sealed to minimize
the escape of volatile MBO. Culture density was measured spectrophotometrically at
600 nm after 10 h of incubation.
Results and Discussion
Effect of MVA Pathway Induction on Growth
The growth curve (Figure 4) showed that the exponential growth rate of E. coli
strain BG101 is similar with or without IPTG induction of the MVA pathway genes.
However, IPTG induction causes the culture to enter into stationary phase sooner and
reach a lower cell density than the uninduced control. Expression of the complete set
of MVA genes cloned under control of the lac promoter (in pMevT and pMBI plasmids)
can lead to accumulation of the end-product DMAPP to toxic concentrations (Martin et
al. 2003). Thus, the lower cell yield after IPTG induction, as compared to control
culture, is most likely due to production of excess precursor metabolites.
15
Optical Density (OD600)
1.2
1
0.8
0.6
With IPTG
0.4
Without IPTG
Point of Addition
0.2
0
0
10
20
30
Time, h
Figure 4: Comparative study of growth characteristics of E. coli BG101, harboring
pMevT, and pMBI plasmids, with or without IPTG addition. Induction of complete
mevalonate pathway genes cloned under Plac promoter accumulate inhibitory levels of
precursor metabolites such as DMAPP, a final product of mevalonate pathway, causing
observed growth retardation of E. coli culture induced with IPTG upon entry into
stationary phase.
Confirmation of Heterologous MVA Pathway and MBO Synthase Genes in
Transformed E. coli
Three genes from the respective plasmids (HMGS from pMevT, idi from pMBI,
and MBOS from pMBO) were used as specific probes after labeling with DIG. The
probes did not show nonspecific hybridization to a blotted control plasmid, and
hybridized to total plasmids isolated from the triple plasmid-bearing strain BG102. This
indicates that the three plasmids were compatible in the host strain as shown in duplicate
samples (Figure 5) as predicted by their three different origins of replication (Table 1).
The sampling of plasmids from strain BG102 was done in same condition as used in
MBO production, indicating that pMevT, pMBI, and pMBO were stable in the host cell
during experimental conditions. After establishing the presence of all three plasmids,
the strain was subjected to further tests for MBO production.
16
Figure 5: Confirmation of recombinant E. coli BG102 by dot blot analysis. Three
highlighted (yellow) genesHMGS (from pMevT), idi (from pMBI), and MBOS (from
pMBO) were PCR amplified, DIG labeled and used as labeled probe to confirm the
presence of plasmids pMevT, pMBI, and pMBO, respectively. Spot 1 pBIN-mGFP5ER (negative control); spot 2 respective plasmid itself (positive control); spot 3 total
plasmid isolate from E. coli BG102; spot 4 probe itself. Blots A1 and A2 (replicates)
pMevT confirmation; Blots B1 and B2 (replicates): pMBI confirmation; Blots C1 and
C2 (replicates): pMBO confirmation.
MBO Production Assay by Gas Chromatography
The negative controls, culture media with no cells, as well as samples containing
cultures of recombinant E. coli strains BG102 and BG104 without IPTG induction
showed only the presence of ethanol (ethanol was used to dissolve the chloramphenicol
and tetracycline antibiotics) in its headspace air as indicated by the peak at 2.31 min
retention time (Figure 6a, b). BG102 and BG104 cultures showed a new peak at 2.47
min specific to IPTG induction (Figure 6c, d). These peaks were determined to be MBO
by comparing its retention time on gas chromatograph against its standard (Figure 6e).
Based upon comparison to standard under identical conditions, MBO production by E.
coli strain BG102 was calculated in the range of 20 μl/l, whereas under same culture
conditions, 31.5 μl/l MBO was produced by E. coli SoluBL21 strain (BG104). The
increased production in the SoluBL21 (BG104) background may be due to the lack of
an OmpT protease, allowing higher amounts of a limiting enzyme to be present in the
cells. Further optimization for MBO production was carried out using higher MBO
17
producing SoluBL21 strain BG104. There was no difference in the amount of MBO
produced when cells were incubated in LB or Terrific Broth media without oxygen.
Interestingly, strain BG104 incubated without oxygen in Terrific Broth media exhibited
a new peak in addition to those of ethanol and MBO that appeared at 2.39 min retention
time. The identity of this compound is unknown, but did not migrate through the column
identical to known isoprene controls (data not shown). In case of LB media with excess
oxygen, production was increased to 50.7 μl/l (1.6 times more compared to LB without
oxygen), and in case of Terrific Broth media with oxygen, production increased to
103.3 μl/l (3 times more compare to Terrific Broth without oxygen) (Figure 7). This
result showed that E. coli produce MBO more efficiently in aerobic conditions with a
rich carbon source. Under laboratory conditions, the MBO synthase enzyme has been
shown to exhibit a dual activity, catalyzing DMAPP to both MBO as well as isoprene
(Gray et al. 2011). Under these in vitro conditions, MBO production is favored over
isoprene at a ratio of about 90:1 (Gray et al. 2011). In contrast, no in vivo isoprene
production from its natural pine tree host has been observed (Gray et al. 2011). Similar
to its natural host, we did not observe any detectable amount of isoprene production
from E. coli strains BG102 and BG104. The result confirmed feasibility of developing
E. coli host for production of volatile MBO through expression of codon optimized P.
sabiniana MBO synthase gene and supplying host cells with DMAPP precursor through
engineering of heterologous MVA genes.
18
Figure 6: MBO production in E. coli BG104. Gas chromatography profiles of control
cultures, a (media with all antibiotics, IPTG, but no cells) and b (culture not induced
with IPTG) showed only single peak of ethanol, while same cultures (in LB and LB +
O2) induced with 0.5 mM IPTG (c and d, respectively) showed two characteristic peaks
of ethanol and MBO. MBO and ethanol peaks were confirmed by comparison with a
pure MBO and ethanol standards (e). Times of elution (retention) from column are
indicated at the bottom of each figure in minutes. Also, retention time difference
between ethanol and MBO was found constant in each case (0.161 min).
140
103.37
120
MBO (µl/L)
100
80
50.71
60
40
31.54
30.95
20
0
LB
TB
LB with O2
TB with O2
Figure 7: Optimization of MBO production in BG104 strain using LB broth, Terrific
Broth, LB broth with O2, and Terrific Broth with O2. The highest MBO production
was 103 μl/l (0.985 mM) and was observed in Terrific Broth with O2.
19
MBO Toxicity Assay on Cell Growth
We used a closed system to study the toxicity of the product i.e., MBO on E.
coli BG104 (Figure 8), because MBO is volatile and would escape from the system if
not contained. To assure the E. coli growth, 20 mM of glucose was added to LB broth,
since in a closed system E. coli continues to grow by fermentation. The result showed
that up to 10 μl of MBO in 3 ml of culture (i.e., 31.6 mM MBO) is not inhibitory to
normal growth. According to this result, about 3.3 ml of MBO per liter of cell culture
can be produced without any cell growth inhibition. Furthermore, if a continuous
system in which the product is removed from headspace is used, accumulated MBO
would not inhibit production if kept below inhibitory concentrations in the culture. A
two-phase gaseous/aqueous bioreactor (Bentley and Melis 2012) could also be used to
harvest MBO, which may reduce the cost of production of MBO.
OD600 after 10 hrs after…
3
2.5
2
1.5
1
0.5
0
0
0.5
1
2
4
6
8
10
12
14
16
18
20
30
Figure 8: Study of MBO toxicity on the growth of E. coli BG104. MBO-induced
cytotoxicity was observed with the addition of 12 μl (38.3 mM) of MBO.
20
Conclusion
Engineered microbial platforms are convenient and cost effective approaches
for large scale production of recombinant proteins and their metabolic products. Several
pine trees produce MBO naturally, but one of the major limitations in MBO production
from its natural host is its volatile nature. The volatile nature of MBO, however, makes
its recovery easier when produced from bacterial cultures. Many other biofuel
compounds need to be phase separated from host cells and growth culture medium
through expensive and complicated procedures. Here, we report the production of 2Methyl-3-buten-2-ol, a naturally occurring volatile alcohol through engineering of
microbial metabolic pathways. In this work, two E. coli host strains, DH5α and
SoluBL21, were equipped with a complete set of MVA pathway genes to convert acetyl
CoA to the DMAPP precursor utilized by MBO synthase. The functionality of the
introduced MVA pathway was inferred to produce excess DMAPP upon IPTG
induction due to the lower growth rate during growth curve analysis and by subsequent
production experiments requiring IPTG induction for MBO expression. DMAPP was
then converted to MBO, catalyzed by a codon optimized P. sabiniana gene encoding
MBO synthase. MBO production by the SoluBL21 strain was found to be better than
in a DH5α strain, perhaps attributable to the absence of a protease in this strain allowing
increased enzyme activity. With this metabolic engineering strategy and experimentally
determined incubation conditions, MBO production in E. coli BG104 was optimized to
produce up to 103 μl/l (0.985 mM). This level of MBO production was far below that
found to inhibit cell growth. We propose that future strain development leading to MBO
production near the inhibitory concentration could be mitigated by harvesting MBO
using a condenser. Higher energy output and cost effectiveness are two major
requirements for an ideal biofuel. With higher energy content and less solubility in
21
water than ethanol, MBO is a promising candidate for future use. The report of MBO
production in this paper is novel, but not cost effective yet. However, MBO production
on an industrial scale must be significantly increased by media and strain optimization,
and by improved bioreactor design. Since the genes in pMevT and pMBI are eukaryotic
in nature, codon optimization suitable for expression in the production strain could be
undertaken to further boost MBO production.
Additional information to chapter-II unpublished data
Study of MBO Toxicity in terms of Cell Viability
Materials and Methods
The OD600 value does not represent dead or live cells. Therefore, to test MBO
toxicity upon the cell viability, E. coli DH5α was grown at 37oC for 2 h in LB medium
in a 125 ml flask. This stock was used to aliquot 3 ml of bacterial culture to individual
sterile vials with 20 mM glucose. Various amounts of liquid MBO (Sigma-Aldrich,
USA) ranging from 10 to 40 µl (31.6 to 126.9 mM) were added, and the vials were
immediately caped and crimp sealed to minimize the escape of volatile MBO. Culture
density was measured spectrophotometrically at 600 nm after overnight of incubation.
After noting down the OD600 value, the cultures were serially diluted from each vials
and 100 μl of diluted cultures were plated on LB agar plate from different dilutions
(10E-4, 10E-5 and 10E-6). Two spread plates were plated from each above dilution from
each vials. Colonies were counted after overnight incubation. Then the colony forming
unit (cfu) per ml of culture was compared with observed OD600 value to check whether
OD600 represents any dead cells. The cultures OD and cfu/ ml was compared
considering OD600 of 1 is equivalent to 5x10E8 cells/ ml.
22
Result
When expected cell concentration according to the OD600 was compared to
observed cell density (detail shown in Table 2), it was found that cell culture with 31.6
mM, to 44.6 mM of MBO has higher observed cell density than expected (Figure 9)
which shows all the cells are live and dividing. But, cell cultures with higher
concentration than 44.6 mM of MBO has lower observed cell density (less than half)
than expected (Figure 9) which shows with higher concentration of MBO the cultures
have more than half of the dead cells. This result showed that the higher concentration
of MBO than 44.6 mM is toxic and cause cell death. However, some cells that survive
at these higher MBO concentrations may have been adapted to deal with MBO toxicity
and become tolerant. These cells can be selected and used as host for better production
of MBO. Also, genetic study of these cells would be help to understand how these cells
has become more tolerant to MBO which would further be helpful in developing MBO
tolerant strain for better MBO production.
Table 2: Study of MBO toxicity in cell survival. Expected cell density per ml has been
calculated from OD600 value considering OD600 of 1 is equivalent to 5x10E8 cells/ ml.
Vol of
MBO
(µl)
MBO
concentration
(mM)
OD600
No. of colonies
(Mean cfu)
Expected cell
density
(cfu/ml)
0
10
12
14
16
18
20
24
30
40
0
31.6
38.3
44.6
50.9
57.3
63.6
76.3
95.3
126.9
2.96
2.75
2.77
2.55
2.18
2.07
1.92
1.41
0.99
0.00
238, 254 (246)
186, 174 (180)
251, 165 (208)
205, 231 (218)
70, 54 (62)
42, 49 (45)
38, 33 (35)
19, 27 (23)
201, 178 (189)
000
1.48 X 109
1.38 X 109
1.39 X 109
1.28 X 109
1.09 X 109
1.04 X 109
0.96 X 109
0.71 X 109
0.49 X 109
000
23
Observed cell
density
(cfu/ml)
2.46 X 109
1.80 X 109
2.08 X 109
2.18 X 109
0.62 X 109
0.45 X 109
0.35 X 109
0.23 X 109
0.19 X 109
000
3E+09
No of cells/ml
2.5E+09
2E+09
1.5E+09
1E+09
500000000
0
0
20
40
60
80
100
120
Concentration of MBO (mM)
Expected cfu/ml
Observed cfu/ml
Figure 9: Study of MBO toxicity in E. coli DH5α cell viability. Observed cell density
started to decrease than expected cell density after addition of ~50 mM MBO in the
culture medium.
24
Chapter - III
Expression of Pinus sabiniana methylbutenol (MBO) synthase gene leads to enhance
production of phytols in Nostoc punctiforme
Background
Cyanobacteria are the only group of bacteria capable of fixing carbon through
oxygenic photosynthesis similar to plants. For example, a quarter of total carbon
fixation is performed in the ocean alone by marine cyanobacteria (Flombaum et al.
2013). Cyanobacteria are a diverse group of prokaryotes which include both single
cellular and filamentous forms and found in a wide range of habitats. Nowadays,
cyanobacteria are one of the most important and maximally targeted microorganisms
for use in applied biotechnology (Abed et al. 2009). This is because of cyanobacteria’s
advantageous characteristics such as a shorter life cycle, simpler nutrients demand,
easier genetic manipulation (Frigaard et al. 2004; Heidorn et al. 2011; Ruffing 2011)
and higher solar energy conversion efficiency than crop plants (Melis 2009). For
example, the cyanobacterium Synechocystis sp, has been genetically engineered for
production of photosynthetic isoprene (Lindberg et al. 2010) and a sesquiterpene βcaryophyllene (Reinsvold et al. 2011). Furthermore, cyanobacteria are more
specifically preferred in biotech research for bio-energy production (Wang et al. 2012).
In this study, we have employed N. punctiforme which is a filamentous members of
cyanobacteria with ability to fix N2 through its heterocysts. Among five orders of
phylum cyanobacteria, N. punctiforme belongs to order Nostocales. N. punctiforme
adapted to deal with different abiotic stresses and can change their vegetative cells into
at least three different cellular forms depending upon the environmental conditions
namely, heterocyst (under nitrogen limitation); akinetes (under energy limitation for
example lack of light or phosphate) and motile hormogonia (to rescue from unfavorable
conditions) respectively (Argueta et al. 2004). Genome of N. punctiforme has been
25
sequenced and, with availability of advanced genetic techniques, this cyanobacterium
is an attractive host for various research (Meeks et al. 2001). In addition, N.
punctiforme, with the ability of N2 fixation, shows less nutrient demand that might be
helpful in cost effective production of various commercially important bio-synthetics
through this photosynthetic factory.
A photosynthetic host like N. punctiforme, has a direct advantage of producing
MBO by utilizing sunlight and environmental CO2 which makes this process
renewable, environmental friendly and without contributing in increase in global
warming. We reported production of MBO in E. coli using codon optimized P.
sabiniana MBO synthase gene along with an engineered MVA pathway, as described
in Chapter-II. In this Chapter, our aim was to produce photosynthetic MBO by
expressing MBO synthase gene in a cyanobacterial host utilizing the existing MEP
pathway for supply of DMAPP substrate. A report of production of isoprene upon
expression of isoprene synthase gene from cyanobacterium Synechocystis sp PPC 6803
(Lindberg et al. 2010) has indicated that the cyanobacterial MEP pathway could
produce and supply enough DMAPP substrate for isoprene, or here in this case for
MBO production.
In this work, MBO synthase gene was expressed through an artificial plasmid
vector containing an indigenous promoter for gene expression in N. punctiforme. Even
though, MBO synthase was successfully transcribed and translated, MBO production
was not detected. Instead, enhanced production of phytols in transgenic strains
containing MBO synthase was observed. To explain this result, we hypothesize two
possibilities, 1) presence of an indigenous prenyltransferase with broad range of
substrate specificity, or 2) appropriation of a catalytic intermediate of MBO synthase
enzyme to native GDP synthase enzyme which is ultimately channeled to phytols
26
biosynthesis. This work reveals information on metabolic channeling in cyanobacteria
and highlights the challenges of engineering a non-native host cells for biosynthesis of
economically important compounds.
Table 3: List of strains, plasmids and primers used in this study.
Strains
N. punctiforme
N. punctiforme
SBG101
N. punctiforme
SBG102
Plasmids
pSUN4KK2
Relevant properties
Source
Expression host
N. punctiforme harboring pSUNKK2
Lab stock
This work
N. punctiforme harboring pSUNKK2-MBO
This work
Ori pDC1 (cyano ori), ColE1 ori, NeoR, PetE, GFP
pSUN4KK2MBO
Primers
MBOS-f
Ori pDC1 (cyano ori), ColE1 ori, NeoR, PetE, MBOS,GFP
Ip and Summers,
unpublished data
This work
5’-GCTTGTAGCGCGATTCAAACGGAA
This work
MBOS-r
5’-AGACGCCTTTCATGTACTCTGGCA
This work
Materials and Methods
Strains and Growth Conditions
N. punctiforme ATCC 29133 was used as an expression host. Cultures were
grown in 50 ml of liquid Alan and Arnon growth media containing 5 mM MOPS buffer
(pH 7.5), 2.5 mM NH4Cl, 2.5 mM NaNO3 and 2.5 mM KNO3 in 125 ml Erlenmeyer
(EM) flasks or on solid media as previously described (Summers et al. 1995) using 10
µg ml-1 neomycin for plasmid selection. E. coli DH5α was grown in Luria-Bertani (LB)
broth and agar plates using 30 µg ml-1 kanamycin for plasmid selection cloning and
vector construction.
27
Vector Construction and Transformation
An expression vector pSUN4KK2 with an indigenous promoter for gene
expression in N. punctiforme was used to express MBO synthase gene (vector was
developed in Dr. Summers’ lab and generously provided by Dr. Michael L. Summers).
P. sabiniana MBO synthase gene optimized for E. coli expression contained in pMBO
(Gray et al. 2011) was excised using XbaI and NotI and sub-cloned under a copper
inducible promoter petE into a similarly digested pSUN4KK2 vector to develop
pSUN4KK2-MBO (Figure 10). A gel image showing MBO insert, pSUN4KK2
backbone and pSUN4KK2-MBO vector construct was shown in Figure 11. The correct
sequence and orientation of MBO synthase gene in the pSUN4KK2-MBO construct
was confirmed by sequencing (detail information was shown in appendix 1). The
pSUN4KK2 and pSUN4KK2-MBO plasmids were electroporated into N. punctiforme
using Gene PulserTM (BIO RAD, USA) following a method described before (Summers
et al. 1995) to generate control and transgenic strains SBG101 and SBG102
respectively. Information relevant on strains and plasmids are presented in Table 3.
Chlorophyll Quantification
For estimation of Chlorophyll concentration, 1 ml of culture for each sample
were used and extracted into 90% methanol (concentrated to 100 µl and 900 µl of 100%
methanol was added). The extracts were analyzed in quartz cuvette (Beckman Coulter
Inc., U.K.) at OD665 nm using DU 640 spectrophotometer (Beckman CoulterTM, USA)
and chlorophyll concentration was calculated as described before (Meeks and
Castenholz 1971).
28
Figure 10: Schematic diagram of pSUN4KK2-MBO vector construction.
Figure 11: Confirmation of pSUN4KK2-MBO plasmid by restriction digestion. L,
Ladder (bright bands are shown in kb); 1, MBO insert; 2, pSUN4KK2 backbone; 3,
pSUN4KK2-MBO vector construct digested with XbaI.
29
Reverse Transcription PCR (RT-PCR)
Total RNA was extracted from 50 ml of N. punctiforme SBG101 and SBG102
cultures grown to a chlorophyll concentration of about 6.5 µg Chla ml-1 using Qiagen
RNeasy Mini kit (QIAGEN, USA) following a protocol previously described
(http://microbiology.ucdavis.edu/meeks/xpro7a.htm). The extractions was performed
with two replications for each of control and transgenic strains. Quality and
concentration of RNA was assayed using a NanoDrop 2000c Spectrophotometer
(Thermo Scientific, USA). One microgram of RNA from all samples were used for
cDNA synthesis using iScript Reverse Transcriptase for RT-qPCR (BIO-RAD, USA).
Reverse transcription was performed in PCR tubes at 25oC for 5 min, 42oC for 30 min
and 85oC for 5 min. A negative control (NC) was run which contained the same amount
(1μg) of total RNAs from a transgenic strain SBG102 and iScript master mix (BIORAD, USA) but lacking reverse transcriptase. PCR was performed using equal volume
respective cDNAs as template, a set primers for MBO synthase detection (Table 3) and
master mix (Fermentas, USA). A positive control using pSUN4KK2-MBO plasmid and
2 negative controls, one with dH2O as template (N) and another (NN) using product of
NC from above reverse transcriptase steps were included. The product of NC contained
RNAs from transgenic strains and used as template in NN negative control to assure
that RNA samples were not contaminated with genomic DNA. PCR was performed at
an initial denaturation at 95oC for 5 min, 30 PCR cycles with denaturation at 95oC for
30 s, annealing at 55.2oC for 30 s and extension at 72oC for 1 min, with a final extension
at 72oC for 5 min.
30
SDS-PAGE Analysis
To study the expression of MBO synthase gene at protein level, cells from 1 ml
culture of strains SBG101 and SBG102 grown to approximately 6.5 µg Chla ml -1
chlorophyll (volume was adjusted to maintain same density of cells) were harvested by
centrifugation and the pellet suspended in a total volume of 300 µl with TBS (Tris-Base
saline). Cells were lysed by probe sonication (Ultrasonic Processor) using a microtip at
14% amplitude for 5 cycles of 30 s on ice. After centrifugation at 13,000 x g for 10 min
at 4oC, supernatants from both samples were collected and 15 µl of each sample used
for SDS-PAGE analysis.
Analysis of MBO Production
To assay MBO production from N. punctiforme, both control strain SBG101
and transgenic strain SBG102 were grown in closed vials and head space air was tested
through gas chromatograph (GC) and gas chromatograph-mass spectrophotometer
(GC-MS). Triplicates of 3 ml and 6 ml of liquid Alan and Arnon medium in sealable
tubes were inoculated with log phase SBG101 and SBG102 cultures respectively. The
cultures were then air-tight with rubber stoppers, crimp sealed and incubated for 7-10
days at 25oC with shaking under continuous photoperiod. Above experiment was also
repeated with addition of 25 mM NaCO3 in the media to overcome CO2 limitation in a
closed system and also with larger volume (59 ml) to be sure to have enough cells for
MBO production. Different cultures of N. punctiforme was shown in figure 12.
Following incubation, headspace air were sampled and analyzed both through a GC–
FID using a Hewlett-Packard,HP6890 Gas Chromatograph as previously described in
chapter-II and a QP2010S GC-MS (SHIMADZU, USA) equipped with a HP-5 column
(30m, 0.32mm, 0.25 µM) held at 65oC for 1 minutes, increased to 300oC at 15oC min-
31
1
, with a 300oC final temperature maintained for two minutes, with a split ratio of 1:75
and helium carrier gas at a flow rate of 1.2 ml min-1. MBO standard (Sigma-Aldrich)
was used as positive control.
GC-MS Analysis of Total Lipid Extracted from Control vs. Transgenic N.
punctiforme
N. punctiforme cultures grown to late log or early stationary phase were used
for lipid extraction. The cultures containing 175 μg Chla (volumes were adjusted
according to OD665 value) were concentrated to a final volume of 1 ml and total lipids
were extracted using Bligh and Dyer method (Bligh and Dyer 1959). Care was taken in
handling samples to minimize any biasness in the result. Equal volume of extracted
lipids were dried out under N2 gas and then dissolved in equal volume of hexane
(Sigma-Aldrich, USA) before injected into a QP2010S GC-MS (SHIMADZU, USA).
The settings used for GC-MS was the same as described above. Peaks produced through
were analyzed and compared with NIST/EPA/NIH Mass Spectral Library (Data
version: NIST 11, Software version 2.0).
32
Figure 12: N. punctiforme culture in air tight containers. (A) Small vials for 6 ml
culture; (B) Large flask for 59 ml cultures; (C) N. punctiforme cultures for lipid
extraction; (D) Lipid extracts.
Localization and Quantification of Phytols Production in N. punctiforme
Cell pellets harvested by centrifugation from an early stationary phase cultures
containing 360 μg Chla, were resuspended in 25 mM Tris-HCl (pH 7.5), 250 mM
sucrose and lysed by using two passes through a French pressure cell press set at 16000
psi. The lysate was placed in an ultracentrifuge tube and 3 ml of deionized water was
gently added on the top of the lysate. Ultracentrifugation was performed for 150 min at
26000 rpm at 23oC. Following centrifugation, a top layer containing lipid droplets,
middle layer containing cytoplasmic contents of the cells, and pellet containing cell
membranes and cell debris were collected in separate tubes. Total lipids were extracted
from each of the fractions separately and analyzed through GC-MS as above (Figure
13). Phytol production was quantified by comparing peak areas with that of a phytol
standard (Sigma-Aldrich, USA).
33
Figure 13: General flowchart to study phytols accumulation in N. punctiforme. CM, cell
membranes containing fraction; CP, cytoplasm containing fraction; LD, lipid droplet.
Determination of Extraction Efficiency
To calculate efficiency of lipid extraction protocol a nonvolatile internal control
eicosane, a C20 straight chain hydrocarbon which was not produced indigenously in N.
punctiforme, was used. Preliminary experiments were conducted to determine an
amount of eicosane that could produce peak in a physiologically comparable range
using 1μl of different concentration of eicosane in hexane through GC-MS (calculation
is shown in appendix-2). This resulted in the addition of 1.5 μl of 3mg/ml eicosane
solution to the samples prior to extraction and total lipid extraction was conducted. The
34
extracts were then dried out under N2 gas and dissolved in 50 μl hexane before 1 μl of
it was run through GC-MS.
Results and Discussion
Analysis of MBO Synthase Transgene Expression by RT-PCR
The P. sabiniana MBO synthase gene was cloned in the pSUN4KK2 vector to
form pSUN4KK2-MBO (Figure 11) in the correct orientation for transcription from the
copper inducible cyanobacterial petE promoter. N. punctiforme strains SBG101
containing the empty vector and SBG102 containing pSUN4KK2-MBO were
constructed using electroporation and subsequent antibiotic selection.
To test if
transcription of MBO synthase gene occurred, RT-PCR was performed. RT-PCR
results showed the presence of MBO synthase cDNA in both biological replications of
transgenic strains SBG102 (Figure 14). The size of the PCR product for replicate
SBG102 samples exactly matched the size of the positive control but neither of the
replicates for the empty vector control strains SBG101 contained MBO synthase
cDNA. The negative controls N, using water as the template, and NN, using same
amount of template from a reaction lacking reverse transcriptase confirms that the
bands produced in the RT-PCR for SBG102 originally came from cDNA derived from
mRNA and not from DNA contamination in the RNA samples. These results indicate
that transgene MBO synthase is present and expressed in the transgenic strain of N.
punctiforme SBG102 at the mRNA level.
35
Figure 14: Testing for MBO synthase transcription using RT-PCR. Lanes L is ladder
(100 bp plus), N is negative control for PCR, NN is negative control using product from
negative control of RT step, P is positive control using pSUN4KK2-MBO, K1 and K2
are biological replication of control strain SBG101 and KM1 and KM2 are biological
replication of transgenic strain SBG102.
Analysis of MBO Synthase Transgene Expression by SDS-PAGE
To study MBO synthase transgene expression at the protein level, SDS-PAGE
was performed. Total soluble protein extracts were used for SDS-PAGE. Analysis of
coomassie stained gels indicated the presence a protein of approximately 70 kD in the
transgenic strain SBG102 sample that was not present in the control strain SBG101
sample (Figure 15). This observation supports the contention that MBO synthase
enzyme is produced in the host N. punctiforme strain SBG102.
In this study MBO synthase is expressed under a copper inducible petE
promoter which is indigenous to N. punctiforme where it transcribes the photosynthetic
electron carrier plastocyanin. The petE promoter exhibits a basal level of expression in
cultures grown with ammonia in copper-containing media such as used in our
experiments and this basal expression is only about 19% of the averaged expression of
the 20 most highly transcribed genes based upon microarray analysis (Campbell et al.
2007). MBO toxicity in E. coli at above 38.3 mM was observed in our previous work
(presented in chapter-II) and this is one reason we preferred a weak promoter to express
MBO synthase in this study. However, a band is clearly observed at 70 kD in the
transgenic strain which exactly matches the expected size of MBO synthase (Gray et
36
al. 2011). This result indicates the expression of MBO synthase transgene at protein
level. Only crude soluble proteins were used in SDS-PAGE since MBO synthase is a
soluble protein (Gray et al. 2011).
Figure 15: SDS-PAGE protein analysis of crude protein extracts from control and
transgenic strains of N. punctiforme. Lanes, 1 is total soluble protein from control strain
SBG101; 2 is total soluble protein from transgenic strain SBG102; L is standard protein
ladder in kilodaltons (kD). A faint band similar in size to MBO synthase (comparable
to 70 kD and present in lane 2 but absent in lane 1) indicates expression of MBO
synthase at protein level.
MBO Production in Control vs. Transgenic N. punctiforme
Headspace air samples were used to analyze the production of volatile MBO
from control SBG101 and transgenic SBG102 cultures through a GC-FID and GC-MS.
None of the samples from control as well as transgenic strains showed any detectable
peak comparable to MBO standard (data not shown). We further repeated this
experiment with addition of NaCO3 in media to supplement the cyanobacteria with CO2
as well as with larger volumes of culture to ensure an adequate amount of host cells
were present. However, none of the headspace air samples from appropriately incubated
cultures were found to contain detectable level of MBO. Assuming the translated MBO
synthase as an active enzyme, this result could be possible only if intracellularly
37
produced MBO was incorporated into cell material or somehow utilized in vivo to
produce non-volatile larger isoprenoid compounds.
GC-MS Analysis of Total Lipid Extracted from Control vs. Transgenic N.
punctiforme
It was hypothesized that MBO was polymerized into higher molecular weight
non-volatile isoprenoids which are hydrophobic in nature, therefore analysis of total
lipids was conducted. GC-MS analysis of lipid samples from strains SBG101 and
SBG102 indicated differential expression of three peaks at retention times (RT) of
10.500, 10.667 and 10.792 min. These peaks were respectively found to be phytol
acetate and two phytol sterioisomers (here we termed them all together as phytol) with
91, 93 and 93% similarity match to NIST/EPA/NIH Mass Spectral Library (Data
version: NIST 11, Software version 2.0). These were found to be produced in higher
amount in lipid extracts from SBG102 than in SBG101 (Figure 16). There are 29
different sterioisomers of phytol available through the NCBI PubChem. Phytols
produced herein from N. punctiforme matched to a phytol [InChI=1S/C20H40O/c117(2)9-6-10-18(3)11-7-12-19(4)13-8-14-20(5)15-16-21/h15,17-19,21H,6-14,16H2,15H3](http://webbook.nist.gov/cgi/cbook.cgi?ID=C102608537&Units=SI&Mask=200)
Our purchased phytol standard (Sigma-Aldrich, USA)
matched to a phytol
[InChI=1S/C20H40O/c1-17(2)9-6-10-18(3)11-7-12-19(4)13-8-14-20(5)15-1621/h15,17-19,21H,6-14,16H2,1-5H3/b20-15+/t18-,19-/m0/s1]
(http://webbook.nist.gov/cgi/cbook.cgi?ID=C150867&Units=SI&Mask=200).
We
were unable to find the exact commercially available standards for phytols identified
from N. punctiforme in this study, however, fragmentation pattern of mass spectra
produced for these peaks match to authentic NIST/EPA/NIH Mass Spectral Library
38
with more than 90% similarity (Figures 17-19). These results indicate MBO was
incorporated into a 20-carbon branched chain alcohol molecule identical to an isomer
of phytol that was also present in an acetylated form.
Figure 16: Differential production of phytols in transgenic strains SBG102 (pink) verses
control strains SBG101 (black) where peak 1, 2 and 3 represent phytol acetate and 2
phytols (likely sterioisomers), respectively based upon the nearest match to GC-MS
NIST/EPA/NIH Mass Spectral Library, Data version: NIST 11, Software version 2.0.
Numbers on the bottom represent retention time (RT) in minutes.
Figure 17: MS spectra match of the peak 1 (RT=10.500) produced in this study with
the nearest match from the MS data of the library. In the figure upper panel represents
MS spectra of sample while lower panel shows the MS spectra of nearest match
compound from the library.
39
Figure 18: MS spectra match of the peak 2 (RT=10.667) produced in this study with
the nearest match from the MS data of the library. In the figure upper panel represents
MS spectra of sample while lower panel shows the MS spectra of nearest match
compound from the library.
Figure 19: MS spectra match of the peak 3 (RT=10.792) produced in this study with
the nearest match from the MS data of the library. In the figure upper panel represents
MS spectra of sample while lower panel shows the MS spectra of nearest match
compound from the library.
Explanation for Phytol Overproduction in Transgenic Strains SBG102
GC-MS analysis of total lipids extracts showed that an enhanced production of
phytols in transgenic strains SBG102. To explain this result we hypothesized one of
the two plausible pathways namely, model-I (Figure 20) and an alternative model
(Figure 21) that is supported by previously reported information.
In model-I, we propose that intracellularly produced MBO in N. punctiforme is
utilized immediately by a prenyltransferase (C10-C20) with a broad range of substrate
specificity to over produce geraniol (GOH), farnesol (FOH) and then to geranylgeraniol
(GGOH) required substrates for subsequent phytol over-production (Figure 20).
Schuller et al. (2012) shown that a cyclic dipeptide N-prenyltransferase (CdpNPT) can
use a diverse range of substrates, and Koeduka et al. (2011) has expressed a
40
Streptomyces prenyltransferase HypSc (SCO7190) possessing broad-range substrate
specificity in tomato and shown production of prenylated flavonoids, 3'-dimethylallyl
naringenin (C20H20O5) (Koeduka et al. 2011; Schuller et al. 2012). These studies
indicate that prenyltransferases can utilize a broad range of substrates including those
lacking a diphosphate group, supporting our hypothesis that a broad range
prenyltransferase exists in N. punctiforme.
Isopreonoids consist of a large family of organic compounds and broadly
classified based upon its carbon numbers into hemiterpenes (C5), monoterpenes (C10),
sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40), longerchain poly-isoprenoids and their derivatives that are produced from IPP and DMAPP
precursors. DMAPP is the substrate for hemiterpene synthases like isoprene synthase
and MBO synthase. Prenyltransferases also use DMAPP as an initial allylic unit and
catalyze the sequential condensation reaction with IPP for production of elongated
prenyl pyrophosphate precursors geranyl diphosphate (GDP; a 10-carbon C10
compound), farnesyl diphosphate (FDP; C15) and geranylgeranyl diphosphate (GGDP;
C20) (Kellogg and Poulter 1997; Vickers et al. 2014). These prenyl diphosphates are
used in synthesis of different isoprenoids through respective bioenzymatic reactions
in the isoprenoid pathway. Class I bacterial prenyltransferase contains a short-chain
(C15-C20) prenyltransferases (Koike-Takeshita et al. 1995; Ogura and Koyama 1998).
Ohto et al. 1999, reported three different Class I prenyltransferase from a
cyanobacterium Synechococcus elongatus and those prenyltransferases were able to
accept DMAPP as the initial allylic substrate (Ohto et al. 1999). A KEGG database
search for the terpene backbone biosynthetic pathway in N. punctiforme has shown the
presence of enzymes for the MEP pathway as well as for steps 1, 2, 4 and 6 in model-I
(Figure 20) (http://www.genome.jp/kegg-bin/show_pathway?npu00900).
41
A pblast
with protein sequence of enzymes for steps 5 and 7 in model-I (Figure 20) into N.
punctiforme have shown the presence of different potential genes.
Figure 20: A plausible biosynthetic pathway leading to formation of phytol in N.
punctiforme. In the above diagram enzymes I, 1, 2, 4, 5, 6 and 7 shown in green are
potential native enzymes: IPP isomerase (enzyme I) geranyl diphosphate (GDP)
synthase (enzyme 1); farnesyl diphosphate (FDP) synthase (enzyme 2); geranylgeranyl
diphosphate (GGDP) synthase (enzyme 4); pyrophosphatases (enzyme 5);
geranylgeranyl reductase (GGR) (enzyme 6) and geranylgeraniol (GGOH) reductase
(enzyme 7). Enzyme 3 is the foreign MBO synthase. GDP synthase, FDP synthase and
GGDP synthase shown in gray as 1*, 2* and 4* respectively, are bacterial class I
prenyltransferases with proposed broad range substrates specificity. Solid black arrow
represents already known or proposed pathways while gray arrows show plausible
reactions proposed in this model. Structures are adopted from NCBI pubchem
compound library (http://pubchem.ncbi.nlm.nih.gov).
42
Although broad spectrum prenyltransferases have been reported, we do not find
one that uses a linear substrate similar to MBO, GOH or FOH. Therefore, over
production of phytol in the transgenic strain may occur through an alternative model
(Figure 21). MBO synthase catalyzes the conversion of DMAPP to MBO through a
transient carbocation molecule (Silver and Fall 1995). Similarly, as GDP synthase
catalyzes condensation of DMAPP and IPP, it first dephosphorylates DMAPP into the
same transient carbocation (Burke et al. 1999). It is plausible that recombinant MBO
synthase produced here in N. punctiforme may have lost activity resulting in the final
hydrolysis but retain activity resulting in intermediate formation, perhaps due to miss
folding or any number of other factors. As such, it might be able to accept its DMAPP
substrate, dephosphorylate it into carbocation intermediate but lose it before its
hydrolysis into MBO. Leaked carbocation intermediate in this way might be used
instantly by GDP synthase to produce GDP which can be utilized in the following steps
to produce phytols as shown in model-I. Either model could explain why production of
volatile MBO was not observed.
Cyanobacteria have been shown to utilize the MEP pathway for production of
phytol (Disch et al. 1998). The first reaction of the MEP pathway catalyzed by DOXP
synthase is controlled through feedback inhibition by both IPP and DMAPP (Banerjee
et al. 2013; Rodríguez-Concepción 2006). If a similar control mechanism exists in
cyanobacteria, this would explain the limited production of phytols in control strain
SBG101. In the transgenic strain however, recombinant MBO synthase could
continuously convert DMAPP substrate to either MBO or a carbocation intermediate
and in this way lower the concentration of DMAPP below threshold requirement for
feedback inhibition. As a consequence, DMAPP would be expected to be produced at
43
a higher rate in the transgenic strain which in turn would lead to more MBO for
incorporation into phytols using either of the proposed models.
Figure 21: Alternative model leading to overproduction of phytol through supply of a
metabolic intermediate created by MBO synthase to GDP synthase in N. punctiforme.
MBO synthase (enzyme 3); GDP synthase (enzyme 1); transient carbocation common
to both reactions is shown in brackets. Top and bottom panel is adapted from (Silver
and Fall 1995) and (Burke et al. 1999), respectively. OPP denotes pyrophosphate (PPi)
attachment.
Localization and Quantification of Phytol Production in N. punctiforme
To find out the location where phytols accumulated, cells were fractionated into
lipid droplets, cytoplasm and membranes containing fractions, using ultracentrifugation. GC-MS analysis was performed for total lipid extracts from each
fractions. The results indicated that the peaks for phytols with retention times of 10.500,
10.667 and 10.792 min were appeared only from the fraction containing cytoplasm but
not in the fraction containing lipid droplets or membranes for all three biological
replicates of transgenic and control strains. We also quantified phytols production in
44
transgenic compared to the control by comparing the peak area with those of a phytol
isomer standard. The phytol standard produced two peaks at a RT of 12.125 and 12.275
min. Phytols production, based upon the comparison under identical conditions, in
transgenic strains SBG102 were calculated to be 1.575 μg, 0.245 μg and 0.411 μg per
mg of Chla respectively for phytol acetate (RT=10.500), phytol (RT=10.677) and
phytol (RT=10.792) (Figure 22). Similarly phytols production in control strain SBG101
found to be 0.715 μg, 0.107 μg and 0.193 μg per mg of Chla respectively (Figure 22).
Thus, the production of these phytols in the transgenic strain was more than twice to
that of the control strain (Figure 22) (data was presented in appendix table A).
1.8
1.575
Control
Transgenic
1.6
1.4
μg/mg Chla
1.2
1
0.715
0.8
0.6
0.411
0.4
0.245
0.2
0.193
0.107
0
Phytol acetate (RT=10.50)
Phytol (RT=10.66)
Phytol (RT=10.79)
Figure 22: Quantification of phytol production in control SBG101 (white) verses
transgenic SBG102 strains (blue). The y-axis indicates amount of phytols in µg mg-1
Chlorophyll a and x-axis shows differentially expressed phytols identified in this study
(n=3). Error bars denote standard error.
45
Estimation of More Accurate Phytols Production Using Extraction Efficiency
An internal control, eicosane (peak at RT of 4.325), was used to calculate
efficiency of the lipid extraction protocol (data not shown). Peak area of eicosane
produced from standard solution (expected peak area) were compared with the peak
area produced from the lipid extracts (observed peak area) to calculate the extraction
efficiency. The extraction efficiency was found to be only 63.7% (calculation is shown
below in appendix-2). This extraction efficiency was used to more accurately calculate
the amount of phytols produced by these cells indigenously and found to be 2.473 μg ,
0.384 μg and 0.646 μg for transgenic and 1.122 μg , 0.168 μg and 0.303 μg per mg of
Chla for control respectively for phytol acetate (RT=10.500), phytol (RT= 10.677) and
phytol (RT= 10.792) (Figure 23) (data was presented in appendix table B).
Control
Transgenic
3
2.5
μg/mg Chla
2
1.5
1
0.5
0
Phytol acetate (RT= 10.50)
Phytol (RT=10.66)
Phytol (RT=10.79)
Figure 23: Quantification of phytols production in control SBG101 (white) verses
transgenic SBG102 strains (blue) considering an extraction efficiency of 63.7%. The yaxis indicates the amount of phytols in µg mg-1 Chlorophyll a and x-axis shows
differentially expressed phytols identified in this study (n=3). Error bars denote
standard error.
46
Conclusion
Use of photosynthetic microbial hosts for renewable production of useful
compounds of interest especially for biofuel, is one of the best choice. It allows us to
produce economically important compounds directly from sunlight through utilization
of environmental CO2. A shorter life cycle, simple nutrient demand and genetic
simplicity make cyanobacteria one of the most attractive target microbial host for use
in applied biotechnology (Abed et al. 2009). Different isoprenoids such as isoprene and
β-caryophyllene
have
been
successfully
produced
through
cyanobacterium
Synechocystis sp. PCC 6803 (Lindberg et al. 2010; Reinsvold et al. 2011; Zhang et al.
2011). In this work, the photosynthetic host N. punctiforme was transformed with a P.
sabiniana MBO synthase gene for production of photosynthetically derived MBO, a
hemiterpene alcohol. MBO synthase was expressed under a native petE promoter by
the help of a vector construct pSUN4KK2-MBO.
Although, we confirmed MBO synthase gene transcription (Figure 14) and
translation (Figure 15) by performing a RT-PCR and SDS-PAGE, respectively, MBO
production could not be confirmed at a detectable level. However, we found production
of phytols in higher amount in the transgenic strain expressing the MBO synthase gene
(Figure 16). Over production of phytols with MBO transgene expression in N.
punctiforme was then explained with the two plausible pathways, model-I (Figure 20)
and alternative model (Figure 21).
Phytol production from GGDP or GGOH in these models, is in accordance with
a previous study which showed that reduction of carbon labeled GGDP into carbon
labeled phytol when incubated with purified spinach chloroplasts (Soll and Schultz
1981). From the above study we can confer the presence of both reductase and
47
phosphatase activity in the chloroplast. Since, cyanobacteria are evolutionarily related
to chloroplasts, we assume similar properties exist in our host and over production of
GDP or GOH, FDP or FOH and GGDP or GGOH could lead to enhanced production
of phytols as observed in our results.
Phytol, a diterpene alcohol, is an economically useful isoprenoids. Phytol has
been reported to be used in commercial production of synthetic vitamin K (Daines et
al. 2003), vitamin E (Netscher 2007) and also used as an active ingredient in production
of different fragrances, cosmetics and non-cosmetics (McGinty et al. 2010). Phytols
have been demonstrated to have a number of pharmaceutically important properties
including: antinociceptive and antioxidant (Santos et al. 2013); anti-inflamatory and
antiallergic (Ryu et al. 2011); antimicrobial activity against Mycobacterium
tuberculosis (Rajab et al. 1998; Saikia et al. 2010) and Staphylococcus aureus (Inoue
et al. 2005); antischistosomal activity against Schistosoma mansoni (de Moraes et al.
2014) and antidibetic property in rats (Elmazar et al. 2013). Moreover, phytol’s
physico-chemical properties are comparable with diesel biofuel (Muradov et al. 2014).
We conclude that synthesis of this value added isoprenoids, phytol compounds,
can be enhanced in cyanobacteria by intracellular supply of the foreign metabolite
MBO that is integrated into existing metabolism. Further work will be required to find
the mechanism of incorporation and understand the metabolic complexities limiting
expression of compounds of potential interest to medicine and energy.
Note: Chapter-III has been accepted for publication in the journal ‘Bioengineered’.
48
Summary
Current efforts are underway in search of sustainable and environmentally
friendly alternative biofuels which have physico-chemical properties similar to fossil
fuels. Methylbutenol is a short chain volatile isoprenoid compound and have potential
to be used as biofuel. Several pine trees emit MBO, however, it is not feasible to harvest
MBO from its natural source. To this end, metabolic engineering and MBO production
in microbial platform would be more convenient and cost effective approach. In this
study, we employed two microbial host for MBO production because each host have
its own unique advantage. E. coli is more compatible with existing infrastructures while
a photosynthetic host, N. punctiforme, provides a renewable source for MBO
production.
In this study, we successfully showed the feasibility of MBO production
through bioengineering of E. coli host and proposed that MBO production can be
improved to an economical level with further optimizations of culture media, strains
and bioreactor design.
Although, expression of MBO synthase was attained
successfully in N. punctiforme, our result did not indicate production of volatile MBO.
Instead of MBO production, enhancement in phytols production in N. punctiforme
SBG102 strain was observed. This result could be explained based upon two plausible
models which propose either presence of an indigenous prenyltransferase with broad
range substrate specificity or appropriation of MBO carbocation intermediate by native
GDP synthase. These plausible models are also supported by previously reported
information.
49
We conclude that MBO can be produced in bioengineered E. coli expressing
MBO synthase along with MVA pathway to supply DMAPP substrate and with further
optimization work its production can be significantly improved. We also conclude that
intracellular supply of MBO or its intermediate could lead to over produce phytols in
N. punctiforme. Further research work will be required to find the mechanism of
incorporation of MBO or its intermediate into phytol production and to understand the
metabolic complexities of the cyanobacterial host N. punctiforme which limits the
production of compound of interest through this host.
50
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Appendix
1. Cloning of P. sabiniana MBO synthase gene in pSUN4KK2 expression vector to
develop pSUN4KK2-MBO for N. punctiforme (This sequence analysis was done by
Dr. Michael L. Summers)
MCS region of pSUN4KK2 with GFP highlighted in green. pSUN4KK2 is 10,037 bp
(795 lost between XbaI / SacI)
GFP F and R, are bold and underlined
(114 bp)
GFP F2/R2 are italicized and underlined (261 bp)
M13F is strikethrough
CCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACG
CAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACG
ACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTG
AGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCT
CGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG
CTATGACCATGATTACGCCAAGCTTGCATGCGCTGCTGCCACCGCTGAGC
AATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTT
TGCTGAAAGGAGGAACTATATCCGGATATCCACAGGACGGGTGTGGTCGC
CATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGG
GCGGCGGCCAAAGCGGTCGGACAGTGCTCCGAGAACGGGTGCGCATAGA
AATTGCATCAACGCATATAGCGCTAGAGTCGACCTGCAGATAGGGCATT
GGGCATTGGGAAGTATCCATTCCCCCTTTCCCCTTCCCCATTCCCTACTGA
TTTCACCGATTTTTGCAATAAATTGCTCCCCATTTATTAAAAAAAAATTGT
ATCTATGACAGATTGTCATATTTGGTGTTGATTTTATTTAAAATGAGAAAG
GAAAAAAAATTGTTTGTTAGGCTGCAGCCAAGCTTATCGATTTCGAACCC
56
GGGGTACCGGGCCCTCTAGACCGGTGCGGCCGCGGTACCGAATTCCTCGA
GTAATTTACCAACACTACTACGTTTTAACTGAAACAAACTGGAGACTCAT
AATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAA
TTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGA
AGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGG
AAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGT
TCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAA
GAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAG
ATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACC
CTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAA
CATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACA
TCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGC
CACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAA
TACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTC
GACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGG
TCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGC
TCTACAAATACGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAA
AACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCC
AGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTT
GCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCA
TCTGTGCGGTATTTCACACCGCATAAATTCTTTTGTTATATCGGCGGAAAG
CTTTGAGCGTAAACCAGGACGTTGCATCCAGGTTTATCAAACATACGATG
AAAGTCTAAAAGAAGAATCTAGCGAATCAGAATAGGAAAATTAGACCCG
CAAGGTAACTTACAACTTCAACTTCCAGTTAGAGTAGCTAGTTGAAGTAA
GTGGAAGTCGGAAGCTGGAAGCTGGTGAGTGATGCAGTGCGTGGCTGCAT
57
CAAGATTTGCAATAAAACACTTTGCAAATTTTGATGCACCTGGGGCGATC
GCCTTTCACGGTGTTTCGTCACTGGATGGGGGCGAGTGCGCGAGTGTTCTA
GCTTGCTGATTATTTATTCTGCTAGAACATTGCAGCAACTCCAAGGCTTGA
GCGAGTGCAACTTGGGCAACGTTGATCTGAGTCCTGGCTTGAGTCAAGGC
GTTTCGGCGTTCGATGCCCAGCCGCGCCTCTGAGTCGCGGGGGTCGCCGTC
GTGGCTGAGGTGGATGACTCGGACTTTATCGCTTTTCTCGCTCGGCTCAAA
AATTGCCTCGGCAGCGGTCA
MBO (lowercase underlined) cloned as an XbaI/NotI fragment into pSUN4KK2.
This would give a Cu-inducible expression that we could monitor by the cotranscribed GFP protein. 5bp lost and 1818 gained for a total plasmid size of 11.850
kb.
TTAAAAAAAAATTGTATCTATGACAGATTGTCATATTTGGTGTTGATTTTA
TTTAAAATGAGAAAGGAAAAAAAATTGTTTGTTAGGCTGCAGCCAAGCTT
ATCGATTTCGAACCCGGGGTACCGGGCCCtctagaaataattttgtttaactttaagaggaggtaa
aacatatgcatcatcatcatcaccatctggtcccgcgtggcagccatatgatcactaccgaatcgggcgaaggcgtgcag
cgtcgcattgccaaccaccacagcaatctgtgggacgataactttattcagtctttgagcaccccgtatggtgcgatcagcta
tcatgagagcgcccagaaactgattggcgaggttaaggagatgatcaactcgattagcctgaaagatggtgaactgattac
cccaagcaacgacctgctgatgcgtctgagcatcgttgacagcattgagcgtctgggtatcgatcgtcatttcaagtctgaga
tcaaatccgcgctggactatgtttatagctattggaatgagaagggtatcggctggggtcgtgacagcgttgtcgcggacct
gaacagcacggcgttgggtctgcgcacgctgcgtctgcacggttacccggtcagcagcgacgttttgcagcacttcaaag
aacagaaaggtcaatttgcttgtagcgcgattcaaacggaaggtgagatccgctcggttctgaacctgttccgtgcgagcca
aattgcgttcccgggtgagaaagtcatggaggaagctgaagttttctccacgatctacctgaaagaggcgattttgaaactg
ccggtgtgtggtctgagccgcgagattagctacgtgctggagtacggctggcacatcaatctgccacgcctggaggcgcg
58
taactacatcgacgtgtttggtgaggacccgatctacttgacgccgaatatgaaaacgcagaagctgctggagctggcgaa
actggaattcaacatgtttcactcgctgcaacaacaagagctgaaattgctgagccgctggtggaaagacagcggcttcag
ccagatgaccttcccgcgtcatcgtcacgtggagtactacaccctggcgagctgcatcgacagcgagccgcagcatagct
cttttcgtttgggttttgctaaaatctttcacctggcgacggtgctggacgatatctacgataccttcggcacgatggacgagtt
ggagttgtttaccgcggcagttaagcgttggcacccgagcgcgaccgagtggctgccagagtacatgaaaggcgtctaca
tggttctgtatgaaaccgtcaacgaaatggcgggtgaagcggagaaaagccaaggccgtgatacgctgaactacggccg
taatgcgttggaggcttatatcgatgcgagcatggaagaagcgaaatggatctttagcggttttctgccgacctttgaggagt
atctggacaacggcaaagtaagctttggctatggcatcggtacgctgcaaccgatcctgaccttgggcatccctttcccgca
ccacattctgcaagagatcgacttcccgtcccgtctgaacgacgttgccagcagcatcctgcgcctgaaaggtgacatcca
cacctatcaggcagagcgcagccgcggtgagaagagcagctgcatcagctgctatatggaggagaatccggaaagcac
cgaagaggatgctattaaccacatcaatagcatggtagataagctgctgaaggagctgaactgggagtacctgcgcccag
atagcaatgtcccgattaccagcaagaagcatgcgttcgacatcctgcgtgccttctatcacctgtacaaataccgtgatggc
ttctcggtggcgaattacgagatcaaaaatctggttatgacgaccgtgatcgaaccggtcccgctggacgacgacgacaag
gagctcgtcgacgcggccgcGGTACCGAATTCCTCGAGTAATTTACCAACACTACTA
CGTTTTAACTGAAACAAACTGGAGACTCATAATGAGTAAAGGAGAAGAA
CTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAAT
GGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGG
AAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATG
GCCAACACTTGTC
59
This translates into:
Histidine tagged MBO synthase protein (underlined) with 27 additional amino acids
(indicated in Bold) at the C-terminal end of the protein before a stop codon is reached.
MHHHHHHLVPRGSHMITTESGEGVQRRIANHHSNLWDDNFIQSLSTPYGAIS
YHESAQKLIGEVKEMINSISLKDGELITPSNDLLMRLSIVDSIERLGIDRHFKSEI
KSALDYVYSYWNEKGIGWGRDSVVADLNSTALGLRTLRLHGYPVSSDVLQH
FKEQKGQFACSAIQTEGEIRSVLNLFRASQIAFPGEKVMEEAEVFSTIYLKEAIL
KLPVCGLSREISYVLEYGWHINLPRLEARNYIDVFGEDPIYLTPNMKTQKLLE
LAKLEFNMFHSLQQQELKLLSRWWKDSGFSQMTFPRHRHVEYYTLASCIDSE
PQHSSFRLGFAKIFHLATVLDDIYDTFGTMDELELFTAAVKRWHPSATEWLPE
YMKGVYMVLYETVNEMAGEAEKSQGRDTLNYGRNALEAYIDASMEEAKWI
FSGFLPTFEEYLDNGKVSFGYGIGTLQPILTLGIPFPHHILQEIDFPSRLNDVASS
ILRLKGDIHTYQAERSRGEKSSCISCYMEENPESTEEDAINHINSMVDKLLKEL
NWEYLRPDSNVPITSKKHAFDILRAFYHLYKYRDGFSVANYEIKNLVMTTVIE
PVPLDDDDKELVDAAAVPNSSSNLPTLLRFN*NKLETHNE*RRRTFHWSCPN
SC*IRW*C*WAQIFCQWRG*R*CNIRKTYP*IYLHYWKTTCSMANTC
60
2.
Calculation for Extraction efficiency
A stock solution of 3 mg/1000 μl of eicosane was made in hexane.
3 mg/1000 μl
= 3 μg/ μl
Therefore, 1 μl of this stock solution contains 3 μg of eicosane.
5 μl of this stock was diluted to 1000 μl by adding 995 μl of hexane to make standard
solution.
Or, 15 μg/1000 μl = 15 ng/ μl
Therefore, 1 μl of this standard solution contains 15 ng of eicosane.
1.5 μl of stock solution was added to the sample during lipid extraction and the N2
dried extracts were finely dissolved in 50 μl of hexane.
Therefore,
1.5 X 3 μg of eicosane in 50 μl of hexane.
Or, 4.5 μg/50 μl = 90 μg/ 1000 μl = 90 ng/ μl
So, 1 μl of this extracts contain an amount equivalent to 90 ng of eicosane.
Then, 1 μl of each standard solution and extracts were run through GCMS and
produced peak area of eicosane were noted. The step was repeated one more time.
Average area were calculated and compared to find out the lipid extraction efficiency
of the protocol used in this study.
61
For Standard,
Peak area for eicosane standard in sample 1 (PA1) = 1562132
Peak area for eicosane standard in sample 2 (PA1) = 1510317
Average peak area = 1536224.5
(1 μl of standard i.e. 15 ng of eicosane produced average peak area of 1536224.5)
For Lipid Extracts,
Peak area for lipid extract in sample 1 (kPA1) = 6457122
Peak area for lipid extract in sample 2 (kmPA1) = 5284690
Average peak area = 5870906
(1 μl of lipid extract i.e. 90 ng of eicosane produced average peak area of 5870906)
From above,
15 ng of eicosane produce peak area = 1536224.5
Or, 1 ng of eicosane produce peak area = 1536224.5/15
Or, 90 ng of eicosane produce peak area = 1536224.5 X 90/15
Therefore, expected peak area for 90 ng of eicosane = 9217347
Observed peak area for 90 ng of eicosane after extraction = 5870906
Therefore,
Extraction efficiency = observed peak area/expected peak area X 100%
= 5870906/9217347 X 100% = 63.7%
62
1.
Table showing amounts phytols calculated based on peaks area comparison to an
isomeric phytol standard. Table A and B, represent data calculated for phytols
production from control and transgenic strains without and with considering
correction using extraction efficiency of 63.7% respectively.
Table A.
Amount of phytols calculated based upon peaks area comparisons with standard.
Sample-1
Sample-2
Sample-3
Mean
St. deviation
St. error
Control strains SBG101
Phytol
Phytol
acetate (P1)
(P2)
0.2592
0.0354
1.0614
0.1544
0.8236
0.1312
0.7147
0.1070
0.4120
0.0631
0.237887
0.036421
Phytol
(P3)
0.0783
0.2838
0.2178
0.1933
0.1049
0.060574
Transgenic strains SBG102
Phytol
Phytol
acetate (P1)
(P2)
1.5213
0.2173
1.7098
0.2825
1.4941
0.2348
1.5751
0.2449
0.1175
0.0337
0.067823
0.019483
Phytol (P3)
0.3829
0.4632
0.3874
0.4112
0.0451
0.026049
Table B.
Amount of phytols calculated based upon peaks area comparisons with standard.
Sample-1
Sample-2
Sample-3
Mean
St. deviation
St. error
Control strains SBG101
Phytol
Phytol
acetate (P1)
(P2)
0.4069
0.0556
1.6662
0.2424
1.2929
0.2060
1.122
0.1680
0.64681
0.0990
0.37344
0.0572
Phytol (P3)
0.1229
0.4455
0.3419
0.3034
0.1647
0.0951
63
Transgenic strains SBG102
Phytol
Phytol
Phytol (P3)
acetate (P1)
(P2)
2.3882
0.3411
0.6011
2.6841
0.4435
0.7272
2.3455
0.3686
0.6082
2.4726
0.3844
0.6455
0.18440
0.0530
0.0708
0.10647
0.0306
0.0409