The Catabolism of Complex Carbohydrates in
Four Representative Examples of the
Geobacillus Genus
Submitted by George Mackford Taylor, to the University of Exeter
as a thesis for the degree of Masters of Science by Research in
Biosciences, August 2014.
This thesis is available for Library use on the understanding that it is copyright material
and that no quotation from the thesis may be published without proper
acknowledgement.
I certify that all material in this thesis which is not my own work has been identified and
that no material has previously been submitted and approved for the award of a degree
by this or any other University.
Signature…………………………………………………………................................
ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
I would like to acknowledge my supervisor Prof. John Love and colleague Dr.
Tom Howard for their valuable insights and guidance, which have been vital to this
project. I would also like to thank Dr. Thomas Lux for his bioinformatic analysis of the
Geobacillus genome, which was essential in starting this research and offer my thanks
to Shell Research Ltd. who financially supported this project.
2
ABSTRACT
ABSTRACT
The ever-increasing demand for transportation fuels, the decrease in global
petroleum reserves, and the negative impact of greenhouse gases make renewable
and sustainable biofuels an imperative for the future.
First-generation biofuels
produced from food crops are limited by cost and competition with food supply.
Despite considerable effort to produce fuels from lignocellulosic biomass, chemical and
enzymatic pretreatment to solubilise the biomass prior to microbial bioconversion
remains a major economic barrier to the development of an industrial process. Here
we report the complete genomic surveys of complex carbohydrate catabolism in four
representative examples of the Geobacillus genus: G. thermodenitrificans, G.
thermoglucosidans, G. stearothermophilus and G. kaustophilus. In concordance with
the genomic survey the empirical confirmation of mono-, di- and poly- saccharide
degradation in each species was investigated. The data collected indicates that all four
species were able to utilise a wide range of C5 and C6 monosaccharides and the
disaccharide cellobiose, which are all catabolic products of lignocellulosic biomass. G.
thermodenitrificans was also able to catabolise one of the major carbohydrates of
lignocellulose, xylan.
G. thermoglucosidans, G. stearothermophilus and G.
kaustophilus however were unable to utilise xylan for growth. Comparison of the G.
thermodenitrificans and G. thermoglucosidans xylan degradation genes highlighted a
xylan 1,4-β-xylosidase predicted in G. thermodenitrificans but not predicted in G.
thermoglucosidans, which suggested an incomplete pathway.
We created several
constructs with Gtn xynB to test sub-cellular localisation of this enzyme and whether
introduction allows xylan utilisation in G. thermoglucosidans.
3
LIST OF CONTENTS
LIST OF CONTENTS
LIST OF FIGURES
6
LIST OF TABLES
8
1. INTRODUCTION
9
1.1 Cellulose
11
1.2 Hemicellulose
11
1.3 Lignin
13
1.4 Synthetic catabolism of lignocellulose
15
2. MATERIALS AND METHODS
19
2.1 Bacteria
19
2.2 Media
19
2.3 Plasmids
19
2.4 Transformations
19
2.5 Genome analysis
21
2.6 Data analysis
22
3. RESULTS
23
3.1 In silico analysis of carbohydrate catabolic potential in Geobacillus
23
3.1.1 Phylogeny of the Geobacillus genus and selection of representative species 23
3.1.2 Identification and mapping of carbohydrate catabolic enzymes
3.2 Formulation of minimal growth media for Geobacillus
3.2.1 Geobacillus growth is sustained by tryptone and peptone
23
27
27
3.2.2 Geobacillus growth in ASM medium and formulation of a new minimal medium
31
3.3 In vivo analysis of carbohydrate metabolism in representative Geobacillus
37
3.3.1 Controls: Geobacillus growth without carbohydrate addition or in glycerol
37
3.3.2. Geobacillus growth with C6 saccharides
41
3.3.3. Geobacillus growth with C5 saccharides
41
3.3.4. Geobacillus growth in hydrotreated lignocellulose (RAPT)
46
3.4 Engineering xylan catabolism in Geobacillus thermoglucosidans
46
3.4.1 Plasmid constructs and transformation
48
4
LIST OF CONTENTS
3.4.2 Growth of Geobacillus thermoglucosidans engineered with xylanase
51
3.4.3 Gtn xynB contains an active extracellular export signal
51
4. DISCUSSION
55
4.1.1 Catabolism of carbohydrates in the Geobacillus genus
55
4.2.1 Transgene expression
56
4.3.1 Wider aspect
57
5. CONCLUSION
60
6. BIBLIOGRAPHY
61
5
LIST OF FIGURES
LIST OF FIGURES
Figure 1-1. Bioconversion strategies for converting lignocellulose into biofuels
10
Figure 1-2. Catabolic pathway for the degradation of cellulose to glucose
12
Figure 1-3. Catabolic pathway for the degradation of xylan to xylose
14
Figure 2-1. Plasmid map of pS797
20
Figure 3-1. Phylogenetic tree based on the predicted pangenome of all selected
Geobacilli
24
Figure 3-2. Glycolysis pathway map
25
Figure 3-3. Growth of G. thermodenitrificans in TGP media
32
Figure 3-4. Growth of G. thermodenitrificans in variations of TGP media
33
Figure 3-5. Growth of G. thermodenitrificans in a range of tryptone and peptone
concentrations
Figure 3-6.
34
G. thermodenitrificans growth in a minimal tryptone or peptone
concentration supplemented with glucose or glycerol
35
Figure 3-7. Growth of G. thermodenitrificans in TGP and ASM media
36
Figure 3-8. Growth of G. thermodenitrificans in ASM buffered with a range of
MOPS concentrations
38
Figure 3-9. pH and bacterial growth of G. thermodenitrificans in buffered ASM
39
Figure 3-10. Growth of four representative Geobacillus species in MASM without
carbohydrate or in the presence of glycerol
40
Figure 3-11. Growth of four representative Geobacillus species investigated in the
presence of C6 saccharides
42
Figure 3-12. Growth of Geobacillus in MASM supplemented with microcrystalline
cellulose (avicel)
44
Figure 3-13. Growth of Geobacillus in the presence of C5 saccharides
45
Figure 3-14. Growth of G. thermodenitrificans in the presence of hydrotreated
lignocellulose “RAPT”
47
Figure 3-15. Plasmid constructs for G. thermoglucosidans xylan catabolism
49
Figure 3-16. Plasmid construct for Gtn xynB cellular localization
50
LIST OF FIGURES
Figure 3-17. Growth of G. thermoglucosidans engineered with pXYNB
52
Figure 3-18. Growth of G. thermoglucosidans engineered with pPldhA::XYNB
53
Figure 3-19. Expression of sGFP in G. thermoglucosidans engineered with
pXYNB70::sGFP
54
7
LIST OF TABLES
LIST OF TABLES
Table 1-1. Literature review of Geobacillus species carbohydrate utilisation
17
Table 3-1. Enzymes involved in the catabolism of glycerol to glyceraldehyde-3phosphate in Geobacillus
26
Table 3-2. Enzymes involved in the catabolism of cellulose to glucose in
Geobacillus
28
Table 3-3. Enzymes involved in the catabolism of xylan to glyeraldehyde-3phosphate in Geobacillus
29
Table 3-4. Enzymes involved in the catabolism of arabinose to glyeraldehyde-3phosphate in Geobacillus
Table 3-5.
30
Maximal specific growth rates of the four Geobacillus species
investigated on a range of carbon sources
43
INTRODUCTION
1. INTRODUCTION
The increasing demand for fuels, limited secure accessible global petroleum
reserves, and the impact of greenhouse gases on climate change have increased the
need for renewable and sustainable biofuels (1, 2, 3, 4). First generation biofuels have
been derived from food crops such as sugar cane, corn, wheat, and sugar beets (5, 6,
7). However, producing a high volume of first generation biofuels from food crops
creates direct competition with the food supply driving higher food prices (5). For next
generation biofuels to be economically and environmentally sustainable, they must be
produced from renewable resources that do not compete with food supply and have
mitigation potential for greenhouse gas emissions.
Biofuels produced from more
abundant and underused resources such as plant biomass (second generation), algal
biomass (third generation) and greenhouse gases such as carbon monoxide and
carbon dioxide (fourth generation) (5) could overcome and prevent the link between
food and fuel price. Plant biomass (lignocellulose) is the most abundant biomass on
Earth and consists of about 70% sugars (5, 8) and for this reason it is a leading
candidate for a renewable substrate.
Plant biomass presents a major challenge for downstream processing as the
majority of carbon is derived from the plant cell wall in the form of recalcitrant structural
biopolymers rather than residing in the more easily accessible sugars, starch, and oil
fractions (9, 10). The composition of cell walls varies widely among plant species (11),
but is primarily composed of five components: cellulose (~35-60%), hemicellulose
(~20-35%), lignin (~10-25%), pectin, and minerals that are collectively referred to as
lignocellulose (12).
Lignocellulose has drawn intense interest as a potentially
sustainable carbon source because degradation of cellulose and hemicellulose directly
yields fermentable hexose (C6) and pentose (C5) sugars.
Consolidated bioprocessing (CBP) is an approach for second-generation biofuel
(bioethanol) production from lignocellulosic feedstocks. The process has generated
much interest because it utilizes microorganisms to perform the direct hydrolysis and
the fermentation of biomass to biofuel a single process without the need for added
enzymes. Current strategies for second-generation biofuel production require three
major operational steps: physiochemical pretreatment, enzymatic saccharification and
fermentation (Fig 1-1) (5, 13).
Pretreament and enzymatic hydrolysis represent
substantial cost. It is predicted that the use of cellulolytic microbes for consolidated
bioprocessing and eliminating pretreatment would reduce bioprocessing costs by 40%
(2).
However, few industrially tractable microbes possess the inherent ability to
catabolise these complex sugars.
9
INTRODUCTION
A
B
Conven onal Strategy
1st genera on CBP
(current strategy)
Plant Biomass
Plant Biomass
C
2nd genera on CBP
Plant Biomass
Physiochemical pretreatment
(High temperature,
acid or alkali treatment)
Pretreated
Biomass
Pretreated
Biomass
Enzyma c
hydrolysis
Fermentable
Sugars
CBP; hydrolysis
and fermenta on
by a single organism
Single Step
BioProcessing:
hydrolysis and
fermenta on
by a single organism
without
Pretreatment
Fermenta on
Biofuel Recovery
Biofuel Recovery
Biofuel Recovery
Figure 1-1. Bioconversion strategies for converting lignocellulose into biofuels.
(A) typical current bioprocess, (B) consolidated bioprocessing (CBP) of pretreated plant
biomass involving saccharolytic fermentative thermophile, and (C) CBP of untreated
plant biomass involving saccharolytic fermentative thermophile. Shaded boxes
highlight stages performed by microbes.
10
INTRODUCTION
1.1 Cellulose
Cellulose is the major load-bearing component of plant cell walls and is thought to be
the most abundant biopolymer on Earth (14).
Cellulose microfibrils are insoluble,
cable-like structures composed of approximately 24 hydrogen-bonded chains
containing β(1,4)-linked glucose molecules (15, 16). The glucan chains are parallel,
and successive glucose residues are rotated 180 degrees to form a repeating
disaccharide unit called cellobiose. This allows glucan chains to form a flat, relatively
inflexible, ribbon-like crystalline structure held together by hydrogen bonds and Van der
Waals forces to form microfibrils (17).
Cellulases break down cellulose. Both bacterial and fungal species have been
reported to produce thermostable cellulases.
Thermophilic and mesophilic fungal
genera such as Aspergillus, Rhizopus, Trichoderma, Sclerotium, Sporotrichum
thermophile, Thermoascus thermophile var. coprophile, Chaetomium thermophile and
Coniochaeta ligniara
belonging
to
the
(18, 19) produced cellulases.
genera
Bacillus,
Geobacillus,
Several thermophilic bacteria,
Caldibacillus,
Acidothermus,
Caldocellum, and Clostridium also produce thermostable cellulase (20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31).
The depolymerisation of cellulose is catalysed by cellulases (Fig. 1-2). Three
cellulases have been reported, endoglucanase (1,4- β-D-glucan glucohydrolase [EC
3.2.1.4]), exoglucanase (1,4-β-D-glucan cellobiohydrolase [EC 3.2.1.91]), and βglucosidase (β-D-glucoside glucohydrolase [EC 3.2.1.21]) work synergistically to
hydrolyze cellulose (32, 33).
Endoglucanase hydrolyses the (1→4)-β-D-glucosidic
linkages in cellulose randomly, producing oligosaccharides, cellobiose and glucose.
Exoglucanase hydrolyses (1→4)-β-D-glucosidic linkages in cellulose, releasing
cellobiose from either the non-reducing or reducing termini and β-glucosidase
hydrolyses cellobiose to glucose (34). Alternatively, cellulose can undergo hydrolysis
of the non-reducing terminal releasing α-D-glucose, which is catalysed by αglucosidase ([EC 3.2.1.20]).
1.2 Hemicellulose
Hemicellulose is a general designation for a diverse class of linear and branched
polysaccharides that differ widely in composition among plant development (12). Most
hemicelluloses have a β(1,2)-linked glucan, xylan, galactan, mannan, or glucomannan
backbone that is branched with single or longer glycosyl residues (35).
Although
hemicellulose represents a potentially large fermentable pool of sugar, it presents
challenges to biofuel production.
11
INTRODUCTION
Cellulose Catabolism
Cellulose
Endoglucanase
(EC 3.2.1.4)
Exoglucanase
(EC 3.2.1.91)
α-glucosidase
(EC 3.2.1.20)
Cellobiose
β-glucosidase
(EC 3.2.1.21)
2x Glucose
Figure 1-2. Catabolic pathway for the degradation of cellulose to glucose. The
degradation of cellulose requires the synergistic action of multiple enzymes to degrade
the polysaccharide to glucose. The mechanism involves endoglucanase randomly
hydrolysing cellulose into oligosaccharides, which in turn are subject to either
exoglucanase, which catalyses the release of cellobiose, or α-glucosidase, which
catalyses the successive release of glucose from the non-reducing terminal.
Subsequently β-glucosidase hydrolyses cellobiose to generate 2 glucose units.
12
INTRODUCTION
Xylan is the most abundant hemicellulose in grasses such as Panicum virgatum
(switchgrass) and Miscanthus (rhizomatous grass) and angiosperm hardwoods, such
as Eucalyptus globulus (eucalyptus), Salix (willow) and Populus (aspen) (36, 37). The
backbone of xylan consists of a linear polymer of β-(1,4)-linked xylosyl units, which is
most commonly decorated with arabinosyl, xylosyl, or glucuronic acid substituents (37,
38). However, the structure of xylan is highly variable among different species largely
due to the differing pattern of substituents (12).
Xylan degrading enzymes are produced by various organisms like fungi, bacteria,
yeast, marine algae, protozoans, snails, crutaceans, insects, and seeds (39, 40).
Bacteria and fungi are large producers of thermostable xylanases (40, 41).
Thermostable xylanases are produced by fungi including Laetiporus sulphureus (42),
Talaromyces thermophiles (43), Thermomyces lanuginosus (44), Nonomuraea
flexuosa (45) and Thermoascus aurantiacus (45).
Thermophilic bacteria such as
Bacillus (46, 47, 48), Geobacillus (49, 50), Thermotoga (51, 52), Acidothermus (53),
Paenibacillus
(54,
55),
Thermoanaerobacterium
(56),
Actinomadura
(57),
Alicyclobacillus (58), Anoxybacillus (59), Nesterenkonia (60), and Enterobacter (61)
have been reported to produce thermostable xylanases.
Complete enzymatic hydrolysis of hemicellulose requires the synergistic action of
numerous enzymes including endo-β-1,4-xylanases ([EC 3.2.1.8]), xylan 1,4-βxylosidases ([EC 3.2.1.37]), α-L-arabinofuranosidase ([EC 3.2.1.55]), α-glucuronidases
([EC 3.2.1.139]), acetylxylan esterases ([EC 3.1.1.72]), feruloyl esterases ([EC
3.1.1.73]), mannan endo-1,4-β-mannanases ([EC 3.2.1.78]), β-1,4-mannosidases ([EC
3.2.1.25]), and arabinan endo 1,5-α-L-arabinosidases ([EC 3.2.1.99]) (34, 41, 62, 63).
In the case of xylan, endo-1,4-β-xylanase and xylan 1,4-β-xylosidase hydrolyse
the xylan backbone (Fig. 1-3).
Endo-1,4-β-xylanase hydrolyses internal (1,4)-β-D-
xylosidic linkages in xylan backbone resulting in varied length xylooligosaccharides
(xylobiose, xylotriose, xylotetrose, etc.).
Xylan 1,4-β-xylosidases catalyses the
hydrolysis of xylooligosaccharides from the non-reducing termini into individual xylose
units (64).
1.3 Lignin
Lignin is a highly complex aromatic heteropolymer whose biological role in plants
is to increase cell wall integrity and resistance to attack by pathogens (65). It is mainly
composed of the monolignols ρ-courmaryl, coniferyl, and sinapyl alcohols, which give
rise to the ρ-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units (66). This
rigid structure can comprise up to 30% plant biomass and encloses both cellulosic and
13
INTRODUCTION
Xylan Catabolism
Xylan
α-L-Arabinofuranosidase (EC 3.2.1.55),
α-Glucuronidases (EC 3.2.1.139),
Acetylxylan esterases (EC 3.1.1.72),
Feruloyl esterases (EC 3.1.1.73)
Xylan
Arabinose
Endo-β-1,4-xylanase
(EC 3.2.1.8)
Xylan 1,4-βxylosidase
(EC 3.2.1.8)
Xylose
Figure 1-3.
Catabolic pathway for the degradation of xylan to xylose.
The
degradation of xylan requires the synergistic action of multiple enzymes to debranch
the hemicellulose and to depolyermise the backbone. Initially debranching is catalysed
by α-L-arabinofuranosidase, α-glucuronidase, acetylxylan esterase and feruloyl
esterase. The subsequent backbone is depolymerised by endo-1,4-β-xylanase, which
catalyses the random hydrolysis generating xylooligosaccharides, which in turn are
subject to xylan 1,4-β-xylosidase that catalyses the release of xylose units from the
non-reducing terminal.
14
INTRODUCTION
hemicellulosic fractions making their microbial degradation impossible without the
depolymerisation of lignin (7, 67, 68, 69).
Despite the naturally evolved recalcitrance of lignin, some microbes have evolved
enzymatic approaches to lignin degradation.
The most active lignin degraders
identified to date are fungi, such as those belonging to the white-rot or brown-rot
families that decompose wood (70, 71, 72). The current model for microbial lignin
degradation invokes the oxidative combustion of lignin mediated by a broad range of
small molecule oxidants produced by metalloenzymes, such as vetratryl alcohol cation
radical (73) and various Mn (III) coordination complexes (74, 75). These diffusible
mediators, rather than the enzymes are thought to react direcly with lignin to generate
radical sites with the substrates and initiate a cascade of bond scission reactions that
ultimately leads to its decomposition to smaller aromatic compounds, CO2, and water
(65).
1.4 Synthetic catabolism of lignocellulose
Considerable effort has been made to develop a single microbe capable of both
saccharification (hydrolysis of polysaccharides) and fermentation to avoid the
substantial expense of using saccharolytic enzyme mixtures (76). The heterologous
expression of saccharolytic enzymes has been demonstrated in a number of
organisms, including Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia
coli (76, 77, 78). Xylanases were engineered into E. coli to make fatty-acid-derived
fuels (FA-fuels) directly from hemicellulose; fatty acid ethyl esters (FAEEs) were
produced at 11.6 mg l-1 from a glucose and hemicellulose mix (79). Cellulases are
complex enzymes and difficult to express heterologously (80). However, E. coli strains
have also been engineered to grow on either cellulose or hemicellulose and,
separately, produce pinene, butanol or FAEEs.
Co-cultures of two such strains
-1
produced 71 mg l FAEEs from pretreated switchgrass (77). E. coli expressing a betaglucosidase was able to produce isopropanol from cellobiose at titers up to 4 g l-1 (81).
B. subtilis has been engineered to overexpress an endoglucanase and express
minicellulosomes on its surface (82, 83). Although these approaches have resulted in
progress in cellulose and hemicellulose utilisation, the overall enzyme activity is still
very low compared with that of naturally cellulolytic organisms and the rates of
hydrolysis are not sufficient for an industrial process that produces a low value product
such as automobile fuel (76).
Thermophilic microorganisms have a number of potential advantages over
mesophiles as a microbial chassis for the conversion of lignocellulosic biomass to
liquid transport fuels. Growing at temperatures of 50-80 ˚C thermophiles are sources
of highly active and thermostable enzymes (18, 20, 21, 84, 85, 86, 87, 88). These
15
INTRODUCTION
thermostable enzymes offer several potential advantages in the hydrolysis of
lignocellulosic materials such as the increased solubility of reactants and products,
resulting in higher reaction velocities thus decreasing the amount of enzyme needed
(87, 89); shorter hydrolysis times; decreased risk of contamination, and thus, increased
productivity; facilitated recovery of volatile products e.g. ethanol (90); and decreased
cost of energy for cooling after thermal pretreatment. Moreover thermophiles have
been widely accepted as the most efficient microbial group as producers of
thermostable cellulases and xylanases for the degradation of lignocellulosic biomass
(47, 85, 91).
For example, thermophile Thermoanaerobacterium species were
engineered with a thermostable mannanase and three cellulolytic enzymes in aim of
improving the bacteria as a model system for lignocellulosic degradation (92).
The Geobacillus genus is one promising candidate for lignocellulose to biofuel
conversion; its thermophilic nature is desirable for biomass breakdown and fuel
isolation (93).
Members of the genus are capable of aerobic and facultatively
anaerobic growth at temperatures between 40 and 70 ˚C (94).
Geobacillus can
ferment both hexose and pentose sugars (95) and, moreover, extracellular endo/
exoglucanase activity has been demonstrated in Geobacillus species, such as
Geobacillus sp. R7 (84) and Geobacillus sp. T1 (17).
Geobacillus sp. R7 and sp. T1 have not demonstrated growth on cellulose, only
to produce cellulase in its presence (17, 84). In addition, literature concerning cellulase
activity in Geobacillus reports levels with an activity too low to support growth. For
example, under optimal conditions Geobacillus spp. have produced endoglucanase
activities of 0.0113 U ml-1 (30), 0.058 U ml-1 (21) and 0.064 U ml-1 (96). Moreover,
literature shows fermentation of all C5 and C6 sugars to be in the presence of an
alternative carbon source (Table 1-1), such as tryptone or yeast extract that could be a
potential substrate for growth. Therefore this project investigates the input pathway of
Geobacillus by analysing their growth on a variety of carbon sources that compose
lignocellulose.
We report here the presence of catabolic pathways for mono-, oligo- and polysaccharides, demonstrating the ability of four representative species from Geobacillus
genus to ferment specific carbohydrates. This thesis presents a genomic survey of the
catabolic pathways of specific sugars in G. thermodenitrificans, G. thermoglucosidans,
G. stearothermophilus and G. kaustophilus.
Subsequently, the growth of G.
thermodenitrificans, G. thermoglucosidans, G. stearothermophilus and G. kaustophilus
was monitored on these carbohydrates to confirm the catabolic pathways indicated by
genomic mapping. Having established sugars that cannot be natively fermented by the
four Geobacillus species a synthetic pathway was designed and engineered for xylan
16
INTRODUCTION
Carbon Source
L-Arabinose
L-Arabinose
Geobacillus Species
G.kaustophilus
G. thermoglucosidans
Tryptone/ Yeast Extract
10 g/l Tryp
Tryp
Reference
(97)
(98)
Cellobiose
G. thermoglucosidans
10g/l Tryp
(99)
Cellobiose
G. thermoglucosidans
1% YE
(95)
Cellobiose
G.kaustophilus
10 g/l Tryp
(97)
Cellobiose
G. Strain C5
0.5g/l YE
(100)
Glucose
G. thermoglucosidans
Tryp
(98)
Glucose
G. thermoglucosidans
10g/l Tryp
(99)
Glucose
G. Strain C5
0.5g/l YE
(100)
Glucose
G. caldoproteolyticus
0.5g/l YE
(101)
Glycerol
G. Strain C5
0.5g/l YE
(100)
Glycerol
G. thermoglucosidans
1% YE
(95)
Glycerol
G. caldoproteolyticus
0.5g/l YE
(101)
Maltose
G.kaustophilus
10 g/l Tryp
(97)
Soluble Starch
Starch
Sucrose
G.kaustophilus
G. caldoproteolyticus
G. thermoglucosidans
10 g/l Tryp
0.5g/l YE
Tryp
(97)
(101)
(98)
Sucrose
G.kaustophilus
10 g/l Tryp
(97)
Sucrose
Sucrose
Trehalose
G. Strain C5
G. caldoproteolyticus
G. Strain C5
0.5g/l YE
0.5g/l YE
0.5g/l YE
(100)
(101)
(100)
Xylan
G. thermodenitrificans
3g/l YE
(102)
D-Xylose
G. thermoglucosidans
10g/l Tryp
(99)
D-Xylose
G.kaustophilus
10 g/l Tryp
(97)
D-Xylose
G. thermoglucosidans
1% YE
(95)
D-Xylose
G. thermoglucosidans
Tryp
(98)
D-Xylose
G. Strain C5
0.5g/l YE
(100)
Xylooligosaccharide
G.kaustophilus
10 g/l Tryp
(97)
Table 1-1. Literature review of Geobacillus species carbohydrate utilisation.
Geobacillus is capable of growing on a wide range of carbohydrates, however,
reported media contained carbon in the form of tryptone or yeast extract.
17
INTRODUCTION
catabolism in G. thermoglucosidans. Several constructs were designed to test subcellular localisation of the engineered enzyme and whether its introduction allows xylan
utilisation in G. thermoglucosidans.
18
MATERIALS AND METHODS
2. MATERIALS AND METHODS
2.1 Bacteria
Bacteria
species
were
Geobacillus
thermodenitrificans
K1041,
G.
thermoglucosidans DSM 2542 (formerly G. thermoglucosidasius DSM 2542), G.
stearothermophilus DSM 22, G. kaustophilus DSM 7263 and Escherichia coli S17-1.
G. thermodenitrificans and E. coli strains were gifts from Biosyntha, Welwyn Garden
City, Hertfordshire, UK.
G. thermoglucosidans, G. stearothermophilus and G.
kaustophilus were purchased from the DSMZ culture collection.
2.2 Media
To determine Geobacillus carbohydrate catabolism in vivo, bacteria were grown
on a 90mm petri dish (Thermo Fisher Scientific, Waltham, MA, USA) containing TGPagar (tryptone 17 g l-1, peptone 3 g l-1, NaCl 5 g l-1, K2HPO4 2.5 g l-1, sodium pyruvate 4
g l-1, 0.4% (w/v) glycerol and 16 g l-1 agar). Single colonies were selected and grown
overnight in 5 ml TGP-broth at 55 ˚C, with orbital shaking at 220 r.p.m. 1 ml of these
cultures, which were grown at 55 ˚C, 220 r.p.m for 24-32 h, were used to inoculate 50
ml of MASM (8 mM citric acid, 5 mM MgSO4, 20 mM NaH2PO4, 10 mM K2SO4, 25 mM
(NH4)2SO4, 80 μM CaCl2, 1.65 μM Na2MoO4, 1 g l-1 yeast extract, 5 ml-1 trace element
solution and 160 mM MOPS). The trace element solution contained: 1.44 g l-1
ZnSO4.7H2O, 0.56 g l-1 CoSO4.6H2O, 0.25 g l-1 CuSO4.5H2O, 5.56 g l-1 FeSO4.6H2O,
0.89 g l-1 NiSO4.6H2O, 1.60 g l-1 MnSO4, 0.08 g l-1 N3BO3, 60 mM H2SO4. The OD600
was used as a surrogate for bacterial growth and was measured using a
BioPhotometer (Eppendorf).
For the conjugal transformation E. coli and Geobacillus were cultured in LB
(tryptone 10 g l-1, yeast extract 5 g l-1 and NaCl, 5 g l-1) and mLB (tryptone 10 g l-1,
yeast extract 5 g l-1 NaCl 5 g l-1, MgSO4.7H2O 0.145 g l-1, CaCl2.2H2O 0.134 g l-1,
FeSO4. 7H2O 0.011 g l-1 and nitrilotriacetic acid trisodium salt 0.288 g l-1).
2.3 Plasmids
For this study pS797 was used as the mobilised plasmid designed for conjugal
transfer (Fig. 2-1).
The plasmid was a gift from Biosyntha, Welwyn Garden City,
Hertfordshire, UK.
2.4 Transformations
2.4.1 E. coli heat shock transformation
E. coli S17-1 was engineered with plasmids for conjugal transfer with
Geobacillus. E. coli transformation was performed using the heat shock method. 1 μl
19
MATERIALS AND METHODS
Figure 2-1. Plasmid map of pS797. pS797 is a mobilisable plasmid designed for
conjugal transfer, courtesy of Biosyntha Technology Ltd. The plasmid contains the Nic
region and traJ gene for conjugal transfer. The plasmid also carries the repBST1 origin
of replication (Geobacillus), the ColE origin of replication (E. coli), ampicillin resistance
(E. coli), kanamycin resistance (Geobacillus) and the Biobrick MCS.
20
MATERIALS AND METHODS
of plasmid DNA was added to the thawed E. coli competent cells. After 30 min of
incubation on ice, the suspension was incubated for 30 s at 42 ˚C and returned to the
ice for 2 min. 1 ml of LB was added and the culture incubated for 1 h at 37 ˚C at 220
r.p.m. 100 μl of the culture was spread on LB-agar plates containing ampicillin, 100 µg
ml-1 and grown overnight at 37 ˚C.
2.4.2 Geobacillus conjugal transformation
The E. coli donors were grown on LB plates containing ampicillin, 100 μg ml-1 at
37 ˚C overnight. 15 μg of a colony was collected, washed once with 1 ml of LB
medium and resuspended in antibiotic-free LB. Concurrently, the recipient Geobacillus
was grown in mLB-agar at 55 ˚C overnight.
resuspended in the conjugal mix.
3 μg’s of Geobacillus colonies was
The conjugal mix was dispensed onto LB-agar
plates in 10 µl aliquots, which were allowed to dry before incubation at 37 ˚C for 6-7 h.
Following incubation colonies were resuspended in 1 ml LB. 10-1 and 10-2 dilutions
were made in LB and 200 µl aliquots of each were spread on mLB-agar plates
containing kanamycin, 12µg ml-1. Plates were incubated at 55 ˚C for 48 h isolating
kanamycin-resistant Geobacillus transformants.
2.5 Genome analysis
The genome assemblies were performed by Thomas Lux, the bioinformatics
specialist
in
the
Love
Laboratory.
The genomes
of
the
strains
of
G.
thermodenitrificans, G. thermoglucosidans, G. stearothermophilus and G.kaustophilus
used in this project were sequenced at the Exeter University Next Generation
Sequencing Facility, using the Illumina HiSEQ or MiSEQ platforms and resulted in
theoretical genome coverage over 500-fold for each genome. The ensuing sequences
were assembled de novo and annotated with reference to the previously sequenced
Geobacillus genomes present at that time in the NCBI Genbank database: G.
thermodenitrificans NG80-2 (NC_009328), G. thermoglucosidasius C56 YS93
(NC_015660), G. kaustophilus HTA426 (NC_006510), G. thermoleovorans CCB US3
UF5
(NC_016593),
Geobacillus
C56
T3
(NC_014206),
Geobacillus
HH01
(NC_020210), Geobacillus JF8 (NC_022080), Geobacillus WCH70 (NC_012793),
Geobacillus
Y412MC52
(NC_014915),
Geobacillus
Y412MC61
(NC_013411),
Geobacillus Y4 1MC1 (NC_014650).
To construct the Geobacillus phylogenetic tree, the Geobacillus pangenome (i.e.
all the genes in all of the genomes) was determined using the sequence clustering
algorithms: OrthoMCL (OMCL), COGtriangles (COG), and the bidirectional best hit
(BDBH).
Phylogenetic alignments were performed using MUSCLE software.
21
MATERIALS AND METHODS
Conserved positions were located with Gblocks software and PhyML software running
an approximate likelihood-ratio test (aLRT) algorithm (103).
2.6 Data analysis
Data was processed using Graphpad Prism v6.0 for mac (GraphPad Software,
San Diego, USA). Nonlinear regression was used to generate bacterial growth curves
using the sigmoidal model, namely a “one-site, specific binding with Hill slope”
equation. This is the simplest model that most accurately fits the data.
22
RESULTS
3. RESULTS
3.1 In silico analysis of carbohydrate catabolic potential in Geobacillus
3.1.1
Phylogeny of the Geobacillus genus and selection of representative
species
A phylogenetic tree of the Geobacillus genus was constructed using the available
genomes of all the fully sequenced Geobacillus retrieved from the National Center for
Biotechnology Information (NCBI) database. The resulting tree (Fig. 3-1) shows the
predominance of 3 clades containing between 3 and 5 different species, and 4
offshoots from the main branch, each containing only 1 species.
We therefore
selected, as representative examples of the genus, four Geobacillus species: G.
kaustophilus
(Gkt;
representative
of
clade
A),
G.
thermodenitrificans
(Gtn;
representative of clade B), G. thermoglucosidans (Gth; representative of clade C) and
G. stereothermophilus (Gst; representative of a single offshoot).
3.1.2 Identification and mapping of carbohydrate catabolic enzymes
The NCBI and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases
were screened for the enzymes for carbohydrate catabolism in the four selected
Geobacillus (Gtn, Gth, Gst and Gkt) by comparing protein sequences. Sequences
assigned putative and hypothetic functions were inspected manually through searches
against PFAM and PROSITE databases. Putative metabolic pathways were analysed
by Metacyc and KEGG databases. In the four selected Geobacillus, as in bacteria
generally, carbohydrate catabolism feeds into the glycolytic pathway (Fig 3-2) that
results in the production of pyruvate, which is ultimately catabolised to lactic acid,
acetic acid, acetylaldehyde and ethanol, depending on the O2 status of the medium.
Although glycerol is not a carbohydrate, it is the carbon source for Geobacillus
growth that is most referred to in the literature and was therefore deemed an
appropriate substrate with which to start this investigation.
Glycerol catabolism
followed the phosphorylation pathway, which involved glycerol phosphorylation by
glycerol kinase (EC 2.7.1.30) to generate glycerol-3-phosphate, which in turn is
dehydrogenated by glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), yielding NADH
and
dihydroxyacetone
phosphate,
which
is
finally
converted
by
triose
phosphoisomerase (EC 5.3.1.1) to glyceraldehyde-3-phosphate and enters the
glycolytic cycle (Table. 3-1).
Genes encoding the enzymes for glycerol catabolism to
glyceraldehyde-3-phosphate are predicted in the genomes of the four Geobacillus
species investigated.
23
RESULTS
Figure 3-1. Phylogenetic tree based on the predicted pangenome of all selected
Geobacilli. The tree, courtesy of Dr. Thomas Lux, highlights three clades and 4
offshoots from the main branch. To provide a full sample of the Geobacillus genus four
representative
examples
thermodenitrificans
(clade
were
B),
selected;
G.
G.
kaustophilus
thermoglucosidans
(clade
(clade
C)
A),
G.
and
G.
stearothermophilus (offshoot D).
24
RESULTS
Figure 3-2. Glycolysis pathway map. Green rectangles: Enzymes of the glycolytic
pathway that are predicted in the genome of the four Geobacillus species investigated;
White rectangle: other proteins not predicted in Geobacillus genome.
The
carbohydrate catabolism of Geobacillus feeds into glycolysis generating lacate,
acetate, acetaldehyde and ethanol. Illustration of complete molecular pathway was
generated using the KEGG PATHWAY software and are used with permission
(http://www.genome.jp/kegg/kegg1.html).
25
RESULTS
Genbank Accession Number
Enzyme
EC
Number
Glycerol kinase
2.7.1.30
YP_001125333.1 YP_004588513.1 EZP78188.1 YP_147213.1
N
Glycerol-3phosphate
dehydrogenase
1.1.1.94
YP_001126250.1 YP_004587515.1 EZP75923.1 YP_148073.1
N
Triose
phosphoisomerase
5.3.1.1
YP_001127095.1 YP_004586562.1 EZP74972.1 YP_148909.1
N
Gtn
Gth
Gst
SignalP
Gkt
Table 3-1. Enzymes involved in the catabolism of glycerol to glyceraldehyde-3phosphate in Geobacillus.
The enzymes predicted in the genomes of G.
thermodenitrificans (Gtn), G. thermoglucosidans (Gth), G. stearothermophilus (Gst)
and G. kaustophilus (Gkt), which are involved in the catabolism of glycerol to
glyceraldehyde-3-phosphate.
26
RESULTS
Lignocellulose is a composite material containing both the C6 and C5
polysaccharides cellulose and xylan (Fig. 1-2, Fig. 1-3).
During saccharification,
cellulose is degraded to the monosaccharide glucose via the disaccharide cellobiose,
and xylan is degraded to the monosaccharides arabinose and xylose.
Cellulose degradation can be catalysed via two pathways both of which require
the synergy of multiple enzymes, (Fig. 1-2).
Genes coding for the catabolism of
cellulose to glucose are predicted in the genomes of all four Geobacillus species
investigated (Table 3-2). Cellulose is catabolised by endoglucanase (EC 3.2.1.4) to
randomly generate cellulose oligosaccharides, cellobiose and glucose. The cellulose
oligosaccharides then undergo continuous hydrolysis catalysed by α-glucosidase (EC
3.2.1.20), which releases glucose units until the oligomer has been catabolised to
cellobiose, which in turn is hydrolysed by β-glucosidase to 2 glucose units.
Subsequently, glucose feeds into the glycolytic pathway.
The genomes of all four Geobacillus species contain multiple genes coding for
xylanase degrading enzymes capable of catabolising xylan to xylose (Table 3-3).
Xylan is catabolised into varied length xylooligosaccharides by endo-1,4-β-xylanase
(EC 3.2.1.8), which is consecutively degraded into xylose units by xylan 1,4-βxylosidase (EC 3.2.1.37). Xylose catabolism followed the isomerase pathway where
xylose is converted by xylose isomerase (EC 5.3.1.5) to D-xylulose that is successively
phosphorylated by xylose kinase (EC 2.7.1.17) to generate D-xylulose-5-phosphate.
D-xylulose-5-phosphate, an intermediate of the pentose phosphate pathway, is then
metabolised by transketolase (EC 2.2.1.1) yielding glyceraldehyde-3-phosphate, which
can feed into glycolysis.
The backbone of xylan is commonly decorated with arabinose substituents.
Arabinose is catabolised via the isomerisation of L-arabinose to L-ribulose, which is
catalysed by L-arabinose isomerase (EC 5.3.1.4). L-ribulose is then phosphorylated by
ribulokinase (EC 2.7.1.16) generating L-ribulose-5-phosphate, which in turn is
catabolised by L-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4) producing D-xylulose5-phosphate, which is then metabolised by transketolase (EC 2.2.1.1) yielding
glyceraldehyde-3-phosphate, which can feed into glycolysis. Genes encoding the
enzymes for arabinose catabolism to glyceraldehyde-3-phosphate are predicted in the
genomes of the four Geobacillus species investigated (Table 3-4).
3.2 Formulation of minimal growth media for Geobacillus.
3.2.1 Geobacillus growth is sustained by tryptone and peptone
To investigate Geobacillus carbohydrate catabolism in vivo, we initially cultured
G. thermodenitrificans in tryptone-peptone-pyruvate-glycerol (TGP) medium, as
27
RESULTS
Genbank Accession Number
Enzyme
EC
Number
Endoglucanase
3.2.1.4
Gtn
Gth
Gst
SignalP
Gkt
YP_001126727.1 YP_004587002.1 EZP76424.1 YP_148566.1
N
α-glucosidase
3.2.1.20 YP_001124655.1 YP_004589171.1 EZP78655.1 YP_146468.1
N
β-glucosidase
3.2.1.21 YP_001126360.1
YP_148190.1
N
β-glucosidase
3.2.1.21
YP_001125856
YP_004588242
YP_147709.1
N
β-glucosidase
3.2.1.21
YP_001127225
YP_004586372
EZP75177.1 YP_149067.1
N
Table 3-2. Enzymes involved in the catabolism of cellulose to glucose in
Geobacillus. The enzymes predicted in the genomes of G. thermodenitrificans (Gtn),
G. thermoglucosidans (Gth), G. stearothermophilus (Gst) and G. kaustophilus (Gkt),
which are involved in the catabolic pathway for the degrade cellulose to glucose.
28
RESULTS
Enzyme
EC
Number
Endo-1,4-βxylanase
3.2.1.8
Endo-1,4-βxylanase
Genbank Accession Number
Gtn
Gth
SignalP
Gst
Gkt
YP_001125870.2 YP_004588260.1
ABI49951.1
WP_020279794
Y
3.2.1.8
YP_001125883.1 YP_004588273.1
2Q8X_A
WP_020279011
N
Xylan 1,4-βxylosidase
3.2.1.37
YP_001125867.1
ABI49959.1
WP_020279302
N
Xylan 1,4-βxylosidase
3.2.1.37
YP_001125878.1 YP_004588268.1
ABI49941.1
WP_020279006
N
Xylan 1,4-βxylosidase
3.2.1.37
YP_001125884.1 YP_004588274.1
ABI49956.1
WP_020279012
N
Acetylxylan
esterase
3.2.1.72
YP_001125885.1 YP_004588275.1
ABI49936.1
GAD13923
N
Xylan α-1,23.2.1.131
glucuronosidase
Q09LY5
N
Xylose
isomerase
Xylose kinase
5.3.1.5
YP_001125866.1 YP_004588257.1
ABI49955.1
YP_147728.1
N
2.7.1.17
YP_001125865.1 YP_004588256.1
ABI49954.1
YP_147727.1
N
Transketolase
2.2.1.1
YP_001125304.1 YP_004588539.1 EZP78159.1/
YP_147185.1
N
Table 3-3. Enzymes involved in the catabolism of xylan to glyeraldehyde-3phosphate in Geobacillus.
The enzymes predicted in the genomes of G.
thermodenitrificans (Gtn), G. thermoglucosidans (Gth), G. stearothermophilus (Gst)
and G. kaustophilus (Gkt) that function to degrade xylan to glyceroaldehyde-3phophate.
29
RESULTS
Enzyme
EC
Number
Genbank Accession Number
SignalP
Gtn
Gth
Gst
Gkt
YP_001125900.1
GAJ44956.1
Q9S467.2
YP_147757.1
N
Ribulokinase 2.7.1.16 YP_001125901.1
GAJ44957.1
Q9S468.1
YP_147758.1
L-ribulose-5phosphate 4- 5.1.3.4. YP_001125902.1
GAJ44958.1
Q9S469.1
YP_147759.1
epimerase
Transketolase 2.2.1.1 YP_001125304.1 YP_004588539.1 EZP78159.1 YP_147185.1
N
L-arabinose
isomerase
5.3.1.4
Table 3-4. Enzymes involved in the catabolism of arabinose to glyeraldehyde-3phosphate in Geobacillus.
The enzymes predicted in the genomes of G.
thermodenitrificans (Gtn), G. thermoglucosidans (Gth), G. stearothermophilus (Gst)
and G. kaustophilus (Gkt), which have the potential to degrade arabinose to
glyceroaldehyde-3-phophate.
30
N
N
RESULTS
recommended by our industrial partner Biosyntha. Growth was monitored by optical
density at 600 nm (OD600).
Following inoculation and a lag phase of approximately 2
h, G. thermodenitrificans grew exponentially for 6 h to an OD600 of 2.5, when a drastic
reduction in OD600 was observed. In the absence of glycerol, the bacteria grew as
observed previously with glycerol for 6 h, but no decrease in OD600 was observed, and
the culture attained a final OD600 of 5 after 24 h (Fig 3-3). To determine which of the
remaining medium constituents was able to sustain microbial growth, a series of
growth experiments in media containing all combinations of tryptone, peptone or
pyruvate was performed (Fig. 3-4). G. thermodenitrificans did not grow at all in media
containing only 4 g l-1 of sodium pyruvate. G. thermodenitrificans grew poorly (Maximal
OD600 ≈ 0.5) in media containing peptone and pyruvate, but well in media containing
tryptone and pyruvate (Maximal OD600 ≈ 5). It therefore appeared that pyruvate does
not sustain Geobacillus growth, but that tryptone, and to a lesser extent, peptone do.
However, these experiments were performed according to the original TGP formulation
that contains 17 g l-1 of tryptone compared to only 3 g l-1 of peptone.
When equal concentrations of tryptone or peptone were used in the medium, G.
thermodenitrificans grew equally well (Fig. 3-5). As the concentration of tryptone or
peptone was reduced, bacterial growth decreased. At 0.75 g l-1 tryptone or peptone,
the maximum OD600 was approximately 0.5. Using the OD600 0.5 as a basal level of
growth, G. thermodenitrficans was grown in 0.75 g l-1 of tryptone and/ or peptone
supplemented with either 4 g l-1 of glycerol or glucose (Fig 3-6). In each case the
addition of glycerol or glucose to tryptone, peptone or tryptone and peptone did not
allow G. thermodenitrificans to grow to an OD600 greater than basal level of the no
carbon control (≈ 0.5), therefore preventing analysis of the bacterial growth on these
carbon sources. While these experiments were being performed, Biosyntha contacted
us with a new “minimal medium” formulation, labelled ASM. As we were interested in
carbohydrate metabolism, not in formulating a new minimal medium for Geobacillus,
we transferred to ASM.
3.2.2 Geobacillus growth in ASM medium and formulation of a new minimal
medium
ASM medium contains (8 mM citric acid, 5 mM MgSO4, 20 mM NaH2PO4, 10 mM
K2SO4, 25 mM (NH4)2SO4, 80 μM CaCl2, 1.65 μM Na2MoO4, 1 g l-1 yeast extract, 5 ml-1
trace element solution. The Trace element solution contained: 1.44 g l-1 ZnSO4.7H2O,
0.56 g l-1 CoSO4.6H2O, 0.25 g l-1 CuSO4.5H2O, 5.56 g l-1 FeSO4.6H2O, 0.89 g l-1
NiSO4.6H2O, 1.60 g l-1 MnSO4, 0.08 g l-1 N3BO3, and 60 mM H2SO4). To compare base
line growth of G. thermodenitrificans in TGP and ASM the OD600 was monitored over
24 h without supplementation of any additional carbon substrate (Fig. 3-7). Following
31
RESULTS
Figure 3-3. Growth of G. thermodenitrificans in TGP media. G. thermodenitrificans
was grown in TGP (black circles), which contained the full complement of components,
and TGP (No C.S) (white circles), which did not contain the added carbon source
glycerol, therefore considered a no carbon source control.
G. thermodenitrificans
grown in the presence of glycerol showed lower levels of growth compared to cultures
grown in the absence of glycerol. Data from the growth of G. thermodenitrificans in
TGP media was fitted using the one site – specific binding with Hill slope equation
(solid line), while the data collected from point of aggregation was fitted using a
connecting line (dashed line). The error bars shown represent the standard deviation
at 95% confidence intervals using 4 degrees of freedom.
32
Bacterial Growth (OD600)
RESULTS
7
TPPy*
T*Py*
*PPy*
T***
*P**
**Py*
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
Time (h)
Figure 3-4. Growth of G. thermodenitrificans in variations of TGP media.
G.
thermodenitrificans was grown in media containing variations of tryptone, peptone and
sodium pyruvate; TPPy* (tryptone, peptone and sodium pyruvate), T*Py* (tryptone and
sodium pyruvate), *PPy* (peptone and sodium pyruvate), T*** (tryptone), *P**
(peptone) and **Py* (sodium pyruvate); to determine which of the constituents was
able to support growth. The data demonstrated sodium pyruvate was not able to
sustain G. thermodenitrificans growth, however, the inclusion of tryptone was sufficient
for growth, and to a lesser extent peptone too.
Data from the growth of G.
thermodenitrificans was fitted using the one site – specific binding with Hill slope
equation. The error bars shown represent the standard deviation at 95% confidence
intervals using 4 degrees of freedom.
33
RESULTS
Bacterial Growth (OD600)
A
7
17 gl-1
13.4 gl-1
10.2 gl-1
6.8 gl-1
3.4 gl-1
1.7 gl-1
0.75 gl-1
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
20
24
28
Time (h)
Bacterial Growth (OD600)
B
7
6
5
4
3
2
1
0
0
4
8
12
16
Time (h)
Figure 3-5. Growth of G. thermodenitrificans in a range of tryptone and peptone
concentrations. G. thermodenitrificans was grown in 17, 13.4, 10.2, 6.8, 3.4, 1.7 and
0.75 gl-1 of tryptone (A) and peptone (B). G. thermodenitrificans grew equally well in
both tryptone (A) and peptone (B) with the final OD600 being directly related to their
concentration. Data from the growth of G. thermodenitrificans was fitted to the one site
– specific binding with Hill slope equation.
The error bars shown represent the
standard deviation at 95% confidence intervals using 4 degrees of freedom.
34
RESULTS
Figure. 3-6.
G. thermodenitrificans growth in a minimal tryptone or peptone
concentration supplemented with glucose or glycerol.
-1
G. thermodenitrificans
-1
grown in (A) 0.75 g l tryptone supplemented with 4 g l glucose or glycerol grew to a
similar OD600 as the no carbon source base line ~ 0.5, where no carbon source is taken
as no added glucose or glycerol to the minimal tryptone concentration. When cultured
in (B) 0. 75 g l-1 peptone supplemented with 4 g l-1 glucose or glycerol G.
thermodenitrificans grew to a similar final OD600 as the no carbon basal level of growth,
where no carbon source is taken as no added glucose or glycerol to the minimal
peptone concentration. Culturing G. thermodenitrificans in (C) 0.75 g l-1 both tryptone
and peptone supplemented with 4 g l-1 glucose or glycerol bacterial growth entered
stationary phase at a lower OD600 than the no carbon control, where no carbon source
is taken as no added glucose or glycerol to the minimal tryptone and peptone. Data
from the growth of G. thermodenitrificans was fitted to the one site – specific binding
with Hill slope equation. The error bars shown represent the standard deviation at 95%
confidence intervals using 4 degrees of freedom.
35
RESULTS
A
Bacterial Growth (OD600)
7
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
Time (h)
Bacterial Growth (OD600)
B
8
7
6
7
5
4
6 pH
3
2
5
1
0
0
4
8
12
16
20
24
28
4
Time (h)
Figure 3-7. Growth of G. thermodenitrificans in TGP and ASM media. (A) G.
thermodenitrificans grew to a higher OD600 (≈ 5) in TGP (black circles) compared to
ASM (white circles) (≈ 1) in the absence of any additional carbon source. (B) G.
thermodenitrificans grown in ASM supplemented with glucose (black circles, grey line)
demonstrated an initial fast growth rate, but a final OD600 similar to the baseline of the
bacterial growth in the absence of glucose (white circles, grey line). The pH (diamond)
of G. thermodenitrificians grown in the presence of glucose (black solid line) shows a
rapid drop that coincides with the drop in OD600. The pH of G. thermodenitrificans
grown in the absence of glucose (black dotted line) is sustained at ≈ 7 over 24 h. Data
from the growth of G. thermodenitrificans was fitted using the one site – specific
binding with Hill slope equation (grey solid line), while the data collected from point of
aggregation was fitted using a connecting line (grey dashed line). The error bars
shown represent the standard deviation at 95% confidence intervals using 4 degrees of
freedom.
36
RESULTS
inoculation and a lag phase of 2 h G. thermodenitrificans grew in both media. In TGP,
G. thermodenitrificans grew exponentially for 14 h reaching a maximal OD600 ≈ 5
whereas in ASM the exponential phase lasted 4 h and the culture reached a maximal
OD600 ≈ 1, therefore providing a suitably low baseline from which to monitor growth in
subsequent experiments.
Biosyntha routinely added 10 g l-1 of glucose to the ASM-base formulation. When
G. thermodenitrificans was cultured in this richer medium, a rapid exponential growth
was observed (to an OD600 ≈ 2 in 4 h), followed by a reduction in OD600 to
approximately 1 after a further 2 h. This reduction in OD600 coincided with a decrease
in medium pH, from 7 to 5.4; this acidification was not observed in the absence of
glucose (Fig 3-7). To mitigate the acidification of the media, G. thermodenitrificans
was grown in buffered ASM with 1% glucose, (Fig 3-8). The results indicate that
increasing the buffering alleviate pH changes in the media (Fig. 3-9).
In addition
buffering slows the growth of G. thermodenitrificans and delays aggregation of the
culture. The buffering of ASM-1%Gluc results in a higher cell density. However, in all
instances aggregation still occurred by 12 h.
Consequently to determine carbon utilisation we used a re-formulated ASM. This
medium, termed MASM, was ASM containing 160 mM MOPS added as a buffering
system.
3.3 In vivo analysis of carbohydrate metabolism in representative Geobacillus
The carbohydrate catabolism of G. thermodenitrificans, G. thermoglucosidans, G.
stearothermophilus and G. kaustophilus was investigated by monitoring growth in
MASM supplemented with glycerol (C3H8O3), glucose (C6H12O6), cellobiose (C6H12O11),
cellulose ([C6H12O6]n), xylose (C5H10O5), arabinose (C5H10O5) and xylan ([C5H8O4R]n).
Glycerol was chosen because it is the main carbon source for Geobacillus growth in
the literature.
Moreover, glycerol is the principal by-product of triacylglyceride
hydrolysis (i.e. the production biodiesel) and therefore plentiful in the waste stream of
the first generation biodiesel industry. The other compounds were selected because
they represent the main products of lignocellulose acid hydrolysis.
3.3.1 Controls: Geobacillus growth without carbohydrate addition or in glycerol.
All four Geobacillus species grew very little when incubated in MASM without any
addition of the carbohydrate glycerol, thereby providing a low baseline (maximal OD600
≈ 1) from which to monitor growth in subsequent experiments (Fig 3-10). When MASM
was
supplemented
with
10
g
l-1
of
glycerol,
G.
thermodenitrificans,
G.
thermoglucosidans and G. stearothermophilus grew to a final OD600 of approximately
4.5, 3.5, and 3.5, respectively. The growth kinetic between these three species was
37
Bacterial Growth (OD600)
RESULTS
7
320 mM
240 mM
160 mM
80 mM
60 mM
40 mM
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
Time (h)
Figure 3-8. Growth of G. thermodenitrificans in ASM buffered with a range of
MOPS concentrations. Increasing the concentration of MOPS slows the growth rate
of G. thermodenitrificans, delays aggregation and allows growth to a higher OD600.
Data from the growth of G. thermodenitrificans media was fit to the one site – specific
binding with Hill slope equation (solid line), while the data collected from the point of
aggregation was fitted using a connecting line (dashed line). The error bars shown
represent the standard deviation at 95% confidence intervals using 4 degrees of
freedom.
38
RESULTS
7
pH
6
4
6
2
5
32
0
24
0
16
0
80
60
40
0
0
Bacterial Growth (OD600)
OD.600 at Time of Aggregation
pH at end point (24h)
pH at Aggregation
[mM MOPS]
Figure 3-9. pH and bacterial growth of G. thermodenitrificans in buffered ASM.
Increasing the concentration of MOPS mitigates the pH changes after 24 h (black
columns) and at the point of bacterial aggregation (light grey column). Increasing the
buffering allows G. thermodenitrificans to grow to a higher OD600 before aggregation
(dark grey column). The error bars shown represent the standard deviation at 95%
confidence intervals using 4 degrees of freedom.
39
RESULTS
Bacterial Growth (OD600)
A
7
No Carbon Source
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
Time (h)
Bacterial Growth (OD600)
B
7
Glycerol
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
32
Time (h)
Figure 3-10. Growth of four representative Geobacillus species in MASM without
carbohydrate or in the presence of glycerol. G. thermodenitrificans (circles), G.
thermoglucosidans (squares), G. stearothermophilus (triangle) and G. kaustophilus
(inverted triangle) grown in (A) MASM without carbohydrate supplementation showed a
low baseline level of growth, OD600 ≈ 1. The four species growth in (B) MASM
supplemented with glycerol showed G. thermodenitrificans, G. thermoglucosidans and
G. stearothermophilus growing to a higher OD600 than their respective baseline in (A),
while G. kaustophilus did not reach an OD600 greater than its no carbon baseline. G.
thermodenitrificans OD600 decreased at 16 h (dashed line), which was ascribed to
aggregation. Data from the growth of Geobacillus species was fitted to the one site –
specific binding with Hill slope equation (solid line), while the data collected from the
point of aggregation was fitted using a connecting line (dashed line). The error bars
shown represent the standard deviation at 95% confidence intervals using 4 degrees of
freedom.
40
RESULTS
different:
G. thermodenitrificans had a lag-phase of 2 h, followed by exponential
growth to 16 h after inoculation. At 16 h, however, the OD600 decreases by 50% from
its peak of 5 to 2.5 (dashed line on the figure); this is ascribed to aggregation or
sporulation that is documented in Geobacillus.
Samples were taken from these
cultures at 24 h, and prepared for Scanning Electron Microscopy, to ascertain whether
aggregation or sporulation had occurred. However, fixation was not optimal and the
subsequent images were of insufficient quality to illustrate either possibility.
G.
thermoglucosidans had a long lag-phase of approximately 12 h, followed by
exponential growth to 24 h, before entering stationary phase. G. stearothermophilus
had a 6 h lag phase, an exponential phase to 16 h, followed by a typical stationary
phase. G. kaustophilus did not grow beyond the baseline of OD600 ≈ 1, indicating that
it was unable to metabolise the glycerol in the time available (24 h).
3.3.2. Geobacillus growth with C6 saccharides
G. thermodenitrificans, G. thermoglucosidans, G. stearothermophilus and G.
kaustophilus all grew to an
OD600 between 5 and 6.5 when cultured in MASM
-1
supplemented with 10 g l of glucose or cellobiose (Fig. 3-11). The growth kinetics of
each bacterial species (Table 3-5) were similar in either medium containing the mono
and di-saccharide, indicating that the bacteria had the capacity to catabolise the
carbohydrate
provided.
G.
thermodenitrificans,
G.
thermoglucosidans,
G.
stearothermophilus did not enter stationary phase, as is usually understood by the
terminology, but underwent a drastic reduction in OD600, which we ascribe to
aggregation.
Unlike the other species investigated, G. kaustophilus showed no
decrease in OD600 after exponential phase, but neither did it enter stationary phase,
which may have been due to its longer lag-phase. Geobacillus were cultured in MASM
supplemented with cellulose.
However, because cellulose is insoluble, it was not
feasible to monitor growth using OD600. Instead, aliquots were harvested from the
medium and plated onto TGP-agar. The number of colony forming units (cfu) on plates
indicated bacterial growth in the MASM-cellulose suspension. No Geobacillus species
grew with cellulose as the additional substrate (Fig. 3-12).
3.3.3. Geobacillus growth with C5 saccharides
All four Geobacillus species investigated grew to an OD600 between 4.5 and 6
when cultured in MASM supplemented with 10 g l-1 of xylose or arabinose except in the
case of G. kaustophilus that did not grow on arabinose (Fig. 3-13). The growth kinetics
of G. thermodenitrificans, G. thermoglucosidans and G. stearothermophilus (Table 3-5)
were similar in either medium supplemented with the C5 monosaccharides, excluding
G. stearothermophilus, which did not enter stationary phase in the presence of xylose,
but rather experienced a decrease in OD600 that was ascribed to aggregation.
41
RESULTS
A
Bacterial Growth (OD600)
7
Glucose
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
32
24
28
32
Time (h)
Bacterial Growth (OD600)
B
7
Cellobiose
6
5
4
3
2
1
0
0
4
8
12
16
20
Time (h)
Figure 3-11. Growth of four representative Geobacillus species investigated in
the
presence
of
C6
saccharides.
G.
thermodenitrificans
(circles),
G.
thermoglucosidans (squares), G. stearothermophilus (triangle) and G. kaustophilus
(inverted triangle) grown in MASM supplemented with glucose (A) showed growth to a
higher OD600 than the MASM baseline (OD600 ≈ 1), however a decrease in OD600
(dahsed line) was observed in G. thermodenitrificans, G. thermoglucosidans and G.
stearothermophilus. The four species grown in MASM supplemented with cellobiose
(B) grew to OD600 between 5 and 6.5.
A decrease in OD600 was observed in G.
thermodenitrificans, G. thermoglucosidans and G. stearothermophilus (dashed line).
The decrease in OD600 was ascribed to aggregation.
Data from the growth of
Geobacillus species was fitted to one site – specific binding with Hill slope equation
(solid line), while the data collected from the point of aggregation was fitted using a
connecting line (dashed line). The error bars shown represent the standard deviation
at 95% confidence intervals using 4 degrees of freedom.
42
RESULTS
μmax (h-1) on Range of Carbon Sources
Geobacillus
Spp.
No
C.S
Gtn
0.85
Gth
0.48
Gst
0.68
Gkt
0.81
Range
of
relative
error
0.860.84
0.470.49
0.670.69
0.790.82
Glucose
0.99
0.78
0.65
0.55
Range
of
relative
error
0.981.0
0.770.80
0.650.65
0.530.56
Cellobiose
1.02
0.82
0.58
0.62
Range
of
relative
error
1.021.03
0.760.89
0.550.62
0.610.62
Glycerol
0.94
0.66
0.64
0.59
Range
of
relative
error
0.940.95
0.650.68
0.63064
0.580.60
Arabinose
0.77
0.70
0.57
0.37
Range
of
relative
error
0.750.80
0.670.73
0.560.57
0.350.39
Xylose
0.77
0.71
0.77
0.33
Range
of
relative
error
0.730.84
0.680.74
0.750.79
0.310.35
Xylan
0.84
0.76
0.50
0.59
Range
of
relative
error
0.760.94
0.750.77
0.420.62
0.520.63
MASMRAPT
1.05
Range
of
relative
error
0.931.1
RAPT
0.56
Range
of
relative
error
0.510.72
Table 3-5. Maximal specific growth rates of the four Geobacillus species investigated on a range of carbon sources. Specific growth rates
(μ) were calculated using μ = ln (x2 – x1)/ (t2 – t1) for each time point, where the specific growth rate is the slope of the line, maximal specific growth
rate (μmax) was then selected. Error of μmax was calculated using standard deviation, which was then applied to the specific growth rate equation to
create ± values for the μmax. G. thermodenitrificans (Gtn) displayed the fastest growth rate of the four species when grown on C6 saccharides. When
grown on C5 saccharides G. thermodenitrificans (Gtn), G. thermoglucosidans (Gth) and G. stearothermophilus (Gst) shared a much similar maximal
specific growth rates while G. kaustophilus (Gkt) was considerably lower.
43
RESULTS
A
B
5×106
5×106
G. thermoglucoidans
G. thermodenitrificans
4×106
CFU per ml
CFU per ml
4×106
3×106
2×106
1
2
3
4
5
Time (days)
6
0
7
C
2
3
4
5
6
7
6
7
D
G. stearothermophilus
CFU per ml
4×106
CFU per ml
1
Time (days)
5×106
3×106
2×106
1×106
0
2×106
1×106
1×106
0
3×106
1
2
3
4
5
6
7
Time (days)
2.0×107
1.8×107
1.6×107
1.4×107
1.2×107
1.0×107
2.0×106
1.5×106
1.0×106
5.0×105
0.0
G. kaustophilus
1
2
3
4
5
Time (days)
Figure 3-12. Growth of Geobacillus in MASM supplemented with microcrystalline
cellulose (avicel). No Geobacillus species investigated grew to a higher cell density
in the presence of cellulose (grey column) compared to its absence (black column)
over 7 days.
The error bars shown represent the standard deviation at 95%
confidence intervals using 4 degrees of freedom.
44
RESULTS
B
7
Bacterial Growth (OD600)
Bacterial Growth (OD600)
A
Xylose
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
7
Arabinose
6
5
4
3
2
1
0
0
32
4
8
12
16
20
24
Time (h)
Time (h)
Bacterial Growth (OD600)
C
7
Xylan
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
Time (h)
Figure 3-13.
Growth of Geobacillus in the presence of C5 saccharides.
G.
thermodenitrificans (circles), G. thermoglucosidans (squares), G. stearothermophilus
(triangle) and G. kaustophilus (inverted triangle) cultured in MASM supplemented with
xylose (A) grew to OD600 between 4.5 and 6.5, however, a decrease in OD600 was
observed in G. thermodenitrificans (dashed line), which was ascribed to aggregation.
(B) G. thermodenitrificans, G. thermoglucosidans and G. stearothermophilus grew in
MASM supplemented with arabinose, however G. kaustophilus grew to a similar OD600
as the baseline for MASM (OD600 ≈ 1) indicating it cannot catabolise the C5
monosaccharide. (C) G. thermodenitrificans grew in the presence of xylan, while G.
thermoglucosidans, G. stearothermophilus and G. kaustophilus grew to a similar OD600
as the baseline for MASM (OD600 ≈ 1) suggesting they cannot catabolise the
polysaccharide. Data from the growth of Geobacillus species was fitted to the one site
– specific binding with Hill slope equation (solid line), while the data collected from the
point of aggregation was fitted using a connecting line (dashed line). The error bars
shown represent the standard deviation at 95% confidence intervals using 4 degrees of
freedom.
45
28
RESULTS
G. kaustophilus had a lag-phase of 14 h, followed by exponential growth to 28 h before
entering stationary phase when grown in the presence of xylose.
These results
indicate xylose can be catabolised by all four species investigated and arabinose by G.
thermodenitrificans, G. thermoglucosidans and G. stearothermophilus. G. kaustophilus
did not grow beyond the baseline of OD600 ≈ 1 in the presence of arabinose, indicating
that it was unable to metabolise the C5 carbohydrate in the time available (24 h).
Geobacillus were cultured in MASM supplemented with 10 g l-1 of xylan.
G.
thermodenitrificans grew to a final OD600 ≈ 5.5. G. thermodenitrificans had a lag-phase
of 2 h, followed by exponential growth to 16 h after inoculation, before entering
stationary phase. G. thermoglucosidans, G. stearothermophilus and G. kaustophilus
did not grow beyond the baseline of OD600 ≈ 1 when cultured in xylan, indicating they
were unable to metabolise the polysaccharide in the time available (24 h).
3.3.4. Geobacillus growth in hydrotreated lignocellulose (RAPT)
Hydrotreated lignocellulose is a complex mix of C5 and C6 saccharides with
varying levels of polymerization.
To determine whether Geobacillus could grow in
hydrotreated lignocellulose, G. thermodenitrificans was cultured in MASM, water or 160
mM MOPS, each supplemented with 10 ml l-1 of an industrial hydrotreated
lignocellulose termed “RAPT” (Fig 3-14). RAPT is produced by Shell and the method
of production is proprietary. G. thermodenitrificans grew in MASM supplemented with
10 ml l-1 of RAPT. After inoculation and a 4 h lag phase Geobacillus grew to an OD600
of ≈ 3.5. Following this, water supplemented with 10 ml l-1 of RAPT was used as a
medium, however, incubating RAPT at 55 ˚C increased the rate of acid hydrolysis in
the
pre-treated
RAPT
and
therefore
acidified
the
media
preventing
G.
thermodenitrificans growth. To overcome acidification, 160 mM MOPS was added as a
buffering system. In the buffered media G. thermodenitrificans grew to an OD600 of ≈ 7.
The growth kinetics of G. thermodenitrficans (Table 3-5) showed a lag phase of 6 h
and an exponential phase of 10 h before reaching a typical stationary phase 16 h after
inoculation, indicating that the bacteria had the capacity to grow solely on pre-treated
lignocellulosic biomass.
3.4 Engineering xylan catabolism in Geobacillus thermoglucosidans
G. thermoglucosidans, G. stearothermophilus and G. kaustophilus displayed no
growth in the presence of cellulose or xylan as carbon sources, but were able to grow
on the simpler saccharides, cellobiose, glucose, xylose and arabinose. Conversely, G.
thermodenitrificans grew well on xylan, but not on cellulose.
A genomic comparison between the four Geobacillus species revealed that G.
thermodenitrificans, G. stearothermophilus and G. kaustophilus possess all the genes
46
RESULTS
Bacterial Growth (OD600)
8
MASM-RAPT
RAPT
RAPT-MOPS
7
6
5
4
3
2
1
0
0
4
8
12
16
20
24
28
Time (h)
Figure 3-14. Growth of G. thermodenitrificans in the presence of hydrotreated
lignocellulose “RAPT”.
G. thermodenitrificans was capable of growing in MASM
supplemented with RAPT (black line), but not RAPT alone (dark grey line) unless it
had been buffered with MOPS (light grey line) where the bacteria reached an OD 600 of
≈ 7. Data from the growth of G. thermodenitrificans was fitted using the one site –
specific binding with Hill slope equation. The error bars shown represent the standard
deviation at 95% confidence intervals using 4 degrees of freedom.
47
RESULTS
to encode the enzymatic pathway for xylan degradation; it therefore appears that the
absence of xylan catabolism in G. stearothermophilus and G. kaustophilus is not due to
the lack of pathway competence, but possibly due to the enzymes not being
expressed.
However, time constraints did not allow the investigation of that
hypothesis.
In contrast, the molecular pathway for xylan catabolism in G.
thermoglucosidans lacks a gene encoding the xylan 1-4-β-xylosidase enzyme. In G.
thermodenitrificans, that enzyme is encoded by the xynB open reading frame
(accession number YP_001125867.1). We therefore proposed that the incomplete
xylan catabolic pathway in G. thermoglucosidans might be complemented by the G.
thermodenitrificans xynB coding sequence (Gtn xynB) and confer xylan catabolic
activity to the engineered G. thermoglucosidans.
3.4.1 Plasmid constructs and transformation
To transform Gtn xynB into G. thermoglucosidans, three plasmid constructs were
generated. The plasmid backbone was from pS797 and the DNA fragments were
synthesized by DNA2.0.
For all constructs, the gene terminator was the pS718
fragment, which is a synthetic stem-loop terminator derived from the plasmids pS797.
pXYNB (Fig. 3-15) contains the coding sequence of Gtn xynB with 100 base-pairs
upstream of the ATG start codon, which we assumed represented the native promoter.
In pPldhA::XYNB (Fig. 3-15), the 100 base-pairs upstream of the Gtn xynB coding
sequence (PxynB) was replaced by the promoter sequence of the constitutively
expressed ldhA gene from G. thermodenitrificans.
Due to its function as a xylanase, and given the fact that xylan can probably not
diffuse into bacterial cells, we hypothesized that Gtn xynB may be an extracellular
enzyme. We therefore performed a SignalP 4.1 (CBS) scan on the primary amino-acid
(AA) sequence (535 AA) of Gtn xynB. This scan revealed no obvious putative export
signal. Typically, peptide export signals are 20-40 AA in length (99). We therefore
fused the coding sequence for the 70 AA from the amino-terminus of Gtn xynB to the
5´ terminus of the SUPERFOLDER GREEN FLUORESCENT PROTEIN (sGFP; 104)
coding sequence. sGFP is a heat tolerant variety of GFP and used as a fluorescent
reporter in thermophilic bacteria. The fusion construct was located downstream of
PxynB, and the resulting plasmid termed pXYNB70::sGFP (Fig. 3-16).
Following conjugation with E. coli transformant strains, each carrying one of the
three plasmids, and plating onto selective agar medium (TGP-agar with 12.5 µg ml-1
kanamycin), 12 G. thermoglucosidans colonies were retrieved for each construct.
These colonies were then streaked onto selective plates, twice. A single colony out of
the 12 was selected, transferred to 5 ml liquid TGP and incubated overnight. 1 ml of
the bacterial suspensions from these cultures were transferred to 50 ml MASM
48
RESULTS
Biobrick prefix
Biobrick suffix
PxynB
promoter
xynB
pS718
terminator
pXYNB
Biobrick prefix
Biobrick suffix
ldhA
promoter
xynB
pPldhA::XYNB
pS718
terminator
Figure 3-15. Plasmid constructs for G. thermoglucosidans xylan catabolism.
Both plasmid constructs designed use the pS797 plasmid backbone and contain the
coding sequence of Gtn xynB, and the pS718 gene terminator. pXYNB contains the
100 base-pair upstream coding sequence of xynB, which we assumed represented the
native promoter, while pPldhA::XYNB contains the promoter sequence for the
constitutively expressed ldhA gene from G. thermodenitrificans.
49
RESULTS
Biobrick prefix
Biobrick suffix
PxynB
promoter
xynB
70
pXYNB70::sGFP
sGFP
pS718
terminator
Figure 3-16. Plasmid construct for Gtn xynB cellular localization. The construct
pXYNB70::sGFP contains the native promoter PxynB which drives expression of the
70 AA amino-terminus of Gtn xynB fused to 5´ terminus of the SUPERFOLDER
GREEN FLUORESCENT PROTEIN (sGFP) coding sequence. The pS718 fragment is
used as a gene terminator and the construct uses the pS797 backbone.
50
RESULTS
supplemented with 10 g l-1 xylan and growth monitored.
3.4.2 Growth of Geobacillus thermoglucosidans engineered with xylanase
Growth of the engineered G. thermoglucosidans was monitored by OD600. G.
thermoglucosidans engineered with pXYNB grew no better than the wild-type
bacterium in the presence or absence of xylan (Fig. 3-17), indicating that this strain
was unable to catabolise xylan. G. thermoglucosidans engineered with pPldhA::XYNB
appeared to have a shorter lag-phase than the wild-type, but the final OD600 and µmax
(0.38 h-1) for the transformant was no greater than of the wild-type (Fig. 3-18, Table. 35). These data are therefore ambiguous, but it appears that xylan cannot sustain the
growth of the engineered G. thermoglucosidans.
However, due to time constraints, we were unable to perform important aspects
of this series of experiments, notably transformation with the actual Gtn xynB
sequences, as opposed to the presence of a functioning selectable marker, was not
confirmed, and only one transformant colony per transformation was screened for
growth in the presence of xylan.
3.4.3 Gtn xynB contains an active extracellular export signal
G. thermoglucosidans engineered with pXYNB70::sGFP was grown in MASM
supplemented with 10 g l-1 glucose, xylose or xylan. Bacteria grew in the presence of
glucose and xylose but not in the presence of xylan. Following centrifugation, sGFP
fluorescence was observed in the supernatant medium, but not in the cell pellet of
cultures grown in the presence of glucose but not in the presence of xylose or xylan
(Fig. 3-19). Consequently, it appears that the 70 AA N-terminus of Gtn xynB does
indeed contain a signal sequence for the export of the protein from the bacterial
cytosol.
51
Bacterial Growth (OD600)
RESULTS
3
Gth pXYNB
Gth WT
Gth WT (No Xylan)
2
1
0
0
4
8
12
16
20
24
28
Time (h)
Figure 3-17. Growth of G. thermoglucosidans engineered with pXYNB. G.
thermoglucosidans (Gth) engineered with pXYNB and grown in MASM supplemented
with xylan (black line) grew to no greater OD600 than the WT G. thermoglucosidans
grown in MASM (dotted line) or MASM supplemented with xylan (grey line). Data from
the growth of G. thermoglucosidans was fitted to one site – specific binding with Hill
slope equation.
The error bars shown represent the standard deviation at 95%
confidence intervals using 4 degrees of freedom.
52
Bacterial Growth (OD600)
RESULTS
3
Gth pPldhA::XYNB
Gth WT
Gth WT (No Xylan)
2
1
0
0
4
8
12
16
20
24
28
Time (h)
Figure 3-18. Growth of G. thermoglucosidans engineered with pPldhA::XYNB.
G. thermoglucosidans (Gth) engineered with pPldhA::XYNB and grown in MASM
supplemented with xylan (black line) grew to no greater OD600 than the WT G.
thermoglucosidans grown in MASM (dotted line) or MASM supplemented with xylan
(grey line). Data from the growth of G. thermoglucosidans was fitted to one site –
specific binding with Hill slope equation. The error bars shown represent the standard
deviation at 95% confidence intervals using 4 degrees of freedom.
53
RESULTS
Figure 3-19.
Expression of sGFP in G. thermoglucosidans engineered with
pXYNB70::sGFP.
G. thermoglucosidans was grown for 24 h in MASM without
carbohydrate supplementation (No C.S.) or in the presence of either glucose, xylose or
xylan. After 24 h the cultures were pelleted by centrifugation and sGFP fluorescence
was observed under UV light.
The expression of sGFP was observed in the
supernatant of cultures as opposed to the pellet therefore suggesting the presence of a
signal sequence. When G. thermoglucosidans was grown in the presence of glucose a
high level of GFP expression was observed, but not in the presence of xylose or xylan.
However, this is qualitative data as cultures were only observed under UV light and
further analysis is therefore required for quantifiable data.
54
DISCUSSION
4. DISCUSSION
This investigation examined the input pathways for a range of mono-, di- and
poly- saccharides in four representative examples of the Geobacillus genus: G.
thermodenitrificans, G. thermoglucosidans, G. stearothermophilus and G. kaustophilus.
4.1.1 Catabolism of carbohydrates in the Geobacillus genus
In previous reports, species of the genus Geobacillus have attracted a lot of
attention with their ability to grow on a wide range of C5 and C6 saccharides at high
temperatures (105).
Having developed MASM to mitigate acidification and delay
aggregation of Geobacillus we were able to monitor bacterial growth on a range of
these carbohydrates.
Our studies of the four Geobacillus species support these
findings having demonstrated growth on glycerol (excluding G. kaustophilus), glucose,
cellobiose, xylose and arabinose (excluding G. kaustophilus), which typically compose
the pre-treated lignocellulosic hydrolysate “RAPT”. G. thermodenitrificans was capable
of using “RAPT” as the sole source of carbon for its growth.
The catabolism of cellulose and xylan, the carbohydrate components of
lignocellulose, were also investigated. In agreement with recent studies we found that
the four Geobacillus species investigated were predicted to contain a number of
cellulose and xylan degrading genes (17, 84, 106), however when cultured in xylan,
only G. thermodenitrificans could grow.
Moreover, none of the four species was
capable of growing on cellulose. These findings add to current reports of cellulase and
xylanase activity by demonstrating that natively produced cellulases and xylanases are
largely incapable of supporting growth. The lack of growth on these polysaccharides
could be linked to reports of low lignocellulosic enzyme activity in Geobacillus species
(21, 30, 96). It was therefore hypothesised that cellulases and xylanases were being
produced but expressing a very low activity that is not capable of supporting bacterial
growth, hence, not sufficient for the industrial application of bioprocessing
lignocellulose to biofuel (106).
Certain species of the genus Geobacillus are able to ferment mono- and disaccharides, making it a strong candidate for a microbial chassis that can produce
biofuels from glycerol and pre-treated lignocellulosic biomass. However, the direct
conversion of untreated lignocellulose to biofuel is hypothesised to be a more
economical and sustainable bioprocess.
Currently Geobacillus does not have the
catabolic potential to degrade cellulose and xylan at a rate to support its own growth,
therefore additional engineering will be required if its to be used as a chassis for onestep consolidated bioprocessing.
55
DISCUSSION
4.2.1 Transgene expression
To improve the input pathway of the Geobacillus genus we attempted to engineer
G. thermoglucosidans to grow on xylan. G. thermoglucosidans lacked a xylan 1-4-βxylosidase predicted in G. thermodenitrificans genome, which suggested an incomplete
xylan catabolic pathway. It was hypothesised that engineering the enzyme Gtn xynB
would complete the pathway and allow G. thermoglucosidans to catabolise xylan.
Engineering this gene into G. thermoglucosidans required successful transfection of
plasmid DNA.
(105).
Limited strains of Geobacillus have successfully been transformed
In most cases highly specialised and time consuming methods were used,
thereby making this problem an urgent area for individual and detailed study.
However, our findings contradict the literature as G. thermoglucosidans proved
amenable to genetic manipulation. All three constructs (pXYNB, pPldhA::XYNB and
pXYNB70::sGFP) were successfully transformed.
G. thermoglucosidans engineered with pXYNB and pPldhA::XYNB showed no
difference in bacterial growth compared to the wild-type bacteria in the presence or
absence of xylan. However, more research is necessary before obtaining a definitive
answer about whether Gtn xynB is about to catabolise xylan. Further research into the
number of screened colonies is necessary since only 1 transformant for both plasmids
was grown on xylan.
Moreover, since the gene expression of Gtn xynB was not
investigated further research is required for its validation (107, 108). In addition to the
gene expression the enzyme could be purified (109) and assayed for activity using pnitrophenyl
β-d-xylopyranoside
(p-NPX)
(109,
110).
Additional
biochemical
characterisation of xynB such as the optimal temperature for growth, thermostability
and the effect of pH on activity could also be investigated.
Studies of G. thermoglucosidans engineered with pXYNB70::sGFP provided
evidence that the 70 AA N-terminus of Gtn xynB contains a signal sequence, which
was not predicted by SignalP 4.1 (CBS). In future work the identified 70 AA sequence
can be used to improve secretion of engineered cellulases and xylanases. In addition
to this novel signal sequence, a previously reported signal peptide from G.
thermoglucosidans C56-YS93 endo-β-1,4-xylanase has been identified (99). Evidence
from B. subtilis highlights the importance of using a signal peptide that works well in
concert with the protein to be expressed (111, 112).
With two identified signal
sequences and a number of genes encoding strongly predicted signal peptides in G.
thermoglucosidans NCIMB 11955, SignalP 4.1 (CBS), (99) there is potential to further
investigate the improvements that would be gained by the refinement of secretion of
lignocellulose degrading enzymes.
56
DISCUSSION
When
growing
G.
thermoglucosidans
engineered
with
pXYNB70::sGFP
expression of sGFP was not observed in the presence of xylose or xylan. Expression
of sGFP was observed in the presence of glucose. However, this experiment was
initially carried out to identify whether the sequence of Gtn xynB contained a signal
sequence and therefore the fluorescence of bacterial cultures was only observed under
UV light. Therefore our analysis of the GFP expression when grown on alternative
carbohydrates is completely qualitative and further research needs to be carried out to
provide quantifiable data. These improvements could be achieved by analysing GFP
expression using flow cytometry, which would provide a per cell intensity for GFP
expression.
None the less, considering the qualitative data, G. thermoglucosidans does not
grow in the presence of xylan, but does in the presence of xylose. Therefore the
concept of a growth dependent promoter can be excluded.
It appears the native
promoter may require glucose to induce the system. This result is important because
Gtn xynB catalyses the degradation of xylan (108) yet our results suggest it’s induced
by glucose, which is neither a substrate nor product of the enzyme. While there is no
evidence the result could be explained by Geobacillus growing in lignocellulosic
environments where cellulose and xylan would both be present.
Glucose is the
product of cellulose degradation and may therefore be in plentiful supply. Hence the
native promoter, PxynB, could be primed by the glucose and therefore express Gtn
xynB, which in turn produces the xylan 1-4-β-xylosidase and degrades xylan. While
the finding was unexpected it raises a number of questions for future research.
Carrying this concept forward and the hypothesis that a glucose and xylan mix
will give a higher level of bacterial growth compared to glucose on its own, it would be
interesting to titre the concentration of glucose to establish a minimal concentration that
induces fluorescence of sGFP. Using this minimal concentration of glucose and a
range of xylan concentrations the growth of G. thermoglucosidans engineered with
pXYNB could then be monitored.
If the catabolism of xylan was regulated by glucose it would be interesting to
understand the global metabolism of Geobacillus by combining metabolic analysis with
transcriptome analysis. Growing the bacteria on “RAPT” would provide a complex mix
of C5 and C6 saccharides allowing the investigation into whether or not genes under
the control of one metabolic pathway interfere with another.
4.3.1 Wider aspect
In order to develop Geobacillus as an efficient producer of biofuel from
lignocellulose, a concerted effort to engineer the genome needs to be made. There
are currently two barriers preventing the use of lignocellulose as a renewable
57
DISCUSSION
substrate. These include overcoming the recalcitrance of lignin to allow access to the
carbohydrates (cellulose and xylan) and also engineering the catabolism of cellulose
and xylan. We investigated the later.
To successfully degrade cellulose Geobacillus needs to be engineered to
increase expression of a free enzyme system of endo- and exoglucanases and other
glycosyl hydrolases to optimal levels therefore mitigating the low expression levels
shown by native bacteria. G. thermoglucosidasius NCIMB 11955 has been previously
engineered to grow on cellulose by overexpressing and secreting Cel5A from
Thermotoga maritima (99). Bacillus subtilis growth on amorphous cellulose has also
been previously achieved by overexpressing the native BsCel5 (82). Engineering E.
coli with the endocellulase Cel from Bacillus sp. D04 and the β-glucosidase Cel3A from
Cellvibrio japonicus (77) has also been shown to provide a cellulolytic system capable
of catabolising cellulose and allowing E. coli to grow on ionic- liquid-pretreated
switchgrass. These examples show that cellulose catabolism can be attained with as
few as two enzymes although the reported efficiencies are too low for an industrial
bioprocess.
While the bioconversion of xylan is possible in G. thermodenitrificans, it is
important to explore engineering its catabolism in the other three species as they may
possess potential downstream metabolic advantages for the production of biofuels.
Successful engineering of xylan catabolism in E. coli used two xylanases: an
endoxylanase catalytic domain (Xyn10B) from Clostridium stercorarium and a xylanase
(Xsa) from Bacteroides ovatus. Xylan catabolism occurred when both genes were
fused to OsmY, an efficient excretion fusion partner. The report showed the sole
expression of Xyn10B was not sufficient for growth on xylan; both enzymes were
required (79). In another report, E. coli was engineered with the endoxylanase Xyn10B
from Clostridium stercorarium and the xylobiosidase gly43F from C. japonics (77).
These examples prove xylan catabolism can be achieved with as few as two enzymes,
however, the reported efficiencies were too low for an industrial bioprocess.
Enzymes are also needed to debranch the substituents of xylan such as acetyl,
arabinosyl, xylosyl, glucuronic acid, 4-O-methylglucuronic acid and ferulic acid
substituents (38). Our results demonstrate the catabolism of arabinose and xylose,
however the catabolism of acetyl, glucuronic acid, 4-O-methylglucuronic acid and
ferulic acid were not investigated. If Geobacillus cannot catabolise these substituents
the xylan backbone could be blocked from the active site of xylanases therefore
preventing degradation. This could be overcome with the engineering the expression
of enzymes such as arabinosidase, glucuronidase, acetylxylan esterase and feruloyl
esterase (38).
58
DISCUSSION
The reported efficiency for substrate conversion of these cellulolytic and
xylanolytic systems is very low (77, 79).
degradation
Geobacillus
could
be
To achieve optimal cellulose and xylan
engineered
to
express
and
secrete
a
minicellulosome. Cellulosomes are large, extracellular multi-enzyme complexes, which
are designed for the efficient deconstruction of cellulose and hemicellulose (113).
Minicellulosomes are smaller and less complex artificial versions tailored for optimal
enzymal synergy to act against complex polysaccharide substrates and therefore
increasing enzyme efficiency (114). Minicellulosomes have been engineered in Bacillus
subtilis where they successfully digested the constituents of plant cell walls (115, 116);
this can be used as a starting point for future research.
59
CONCLUSION
5. CONCLUSION
The genomic survey identified a number of cellulose and xylan degrading genes
predicted in the genome of each representative Geobacillus species investigated. The
re-formulated MASM mitigated pH change and delayed aggregation, which allowed the
carbohydrate catabolism of the Geobacillus to be investigated. The four representative
Geobacillus species catabolised a wide range of C5 and C6 saccharides that compose
cellulose and xylan. However, cellulose and xylan were not catabolised to support
growth, except in the case of G. thermodenitrificans that grew on xylan. Engineering
Gtn xynB into G. thermoglucosidans did not allow bacterial growth on xylan, however, it
revealed a signal peptide sequence and suggested the xylanase was induced by
glucose rather than xylan or xylose.
60
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