Transcriptomics analysis reveals shared precursors in the biosynthesis of hydrocarbons
in photosynthetic algae and plants
Adarsh Jose1,2, Wenmin Qin1, Marna Yandeau-Nelson1 and Basil Nikolau1
1Department
of Biophysics, Biochemistry and Molecular Biology, Iowa State University, Ames, IA, United States
2Bioinformatics and Computational Biology, Iowa State University, Ames, IA, United States
1. Introduction
3. Bioinformatics Analysis Pipeline
fastx toolkit
It is generally well accepted that fatty acids are the metabolic precursors of simple,
linear hydrocarbons (HCs) such as n-alkanes and n-alkenes.
However, the
mechanisms and genetic elements of this metabolic conversion are still unknown.
(Figure 1).
Illumina PE
Transcriptome raw data
Quality filter
Protein sequences previously identified to be involved in synthesis of precursors of
hydrocarbons and those hypothesized to be involved in synthesis of hydrocarbons were
curated from different sources. (Figure 4)
Trinity Assembler
Pool reads and
Assemble Transcriptome
No
Reference Transcriptome
Genome
sequence
available ?
Yes
Figure 1. Four possible mechanisms of hydrocarbon biosynthesis. The fatty acid
head-to-head condensation mechanism (1), the elongation-decarboxylation (2), and the
fatty acid elongation-decarbonylation (3) pathways produce odd-numbered alkanes
and alkenes. The final pathway (4) involves a primary alcohol intermediate and would
result in even-numbered hydrocarbons.
Map PE Reads to the
Reference Sequences
Statistical Analysis –
List Enrichment
Statistical Analysis –
Diff Exp
BLASTX
The two algae, Botryococcus braunii (Figure 2.A) and Emiliania huxleyi (Figure 2.B)
were found to accumulate hydrocarbons differentially across different levels of Nitrogen,
Phosphate and Carbon in the growth media. The Pisum sativum accumulates ~ 10 x levels
of carbon on its abaxial surface when compared to adaxial surface (Figure 2.C) while the
corn silks emerged from the husk of Zea Mays was shown to accumulate ~3 x more
hydrocarbon when compared to those encased in the husk (Figure 2.D).
A.
Tophat PE Aligner /
Bowtie PE Aligner
CuffLinks as FPKM /
RSEM as FPKM
Handpicked protein models
from uniprot protein
database
Pathway Mapping
Figure 4: Querying for curated genes and pathway: Genes involved in key breakpoints
in carbon flux, synthesis of precursors of hydrocarbons and those hypothesized to be
involved in synthesis of hydrocarbons identified by sequence similarity. BLAST scores are
used to summarize gene expression levels.
5. Pathway diagram overlaid with fold changes across the hydrocarbon
accumulation conditions
Order of systems represented in the fold change boxes
20
15
10
5
0
NP+/C-
FC < 1/5
5
NP-/C+
3
2
1
NP-/C+
NP+/C-
D
12%
upper
lower
10%
8%
6%
4%
2%
0%
B17_2B17B15_2B15B13_2B13B11_2 B11 B9_2 B9 B7 B5
Hydrocarbon content of leaf epidermis
(older branches to the right)
Pea leaves E. huxleyi B. braunii.
FC > 5
Continuous color coding of Fold Change
From Green -> High Expression in the Low HC condition
through Yellow -> No change
To Red -> High Expression in the high HC condition
4
0
C.
Hydrocarbon content
(g/g dry weight)
E. huxleyi-1516
6
1
Hydrocarbon (umol/g)
Hydrocarbon (umol/g)
25
Hydrocarbon(umol/g)
Corn Silk
B. Braunii – UTEX 572
Heuristics using
BLAST scores
Figure 3: Bioinformatics pipeline: The reads were quality filtered and mapped to
reference transcript/genome sequences. Reference transcriptomes were assembled de-novo
when genomes were not available. The mapped short reads were counted and normalized
to estimate expression levels.
B.
30
BLASTX
Summarize Expression data for each
Enzyme/Transporter and estimate fold
change across conditions
Diff Exp usind Cuffdiff /
DESeq R package
Curated Protein Sequences
Denovo assembled
contigs/ Gene Models
from the four organisms
TAIR and aralip databases
Chlamydomonas reinhardtii
Protein models
Estimate Normalized
Read Counts
Global Results
The Arabidopsis Information Resource (TAIR)
The Arabidopsis Acyl-Lipid Metabolism (ARALIP) website
The Chlamydomonas reinhardtii genome v.4 portal from joint genome institute.
Handpicked set of proteins from the uniprot database based on literature.
Functional Annotations from
the Genome Annotation
Uniprot-KB,
Conserved Domain Database,
Sequenced Phylogenetic Neighbors
2. Hydrocarbon Accumulation Conditions
Reference genome
•
•
•
•
Reference Sequences
BLASTX
HCs are known to occur in algae and the epidermis of plants. In this project, four
experimental systems are being investigated: two microalgae, Emiliania huxleyi and
Botryococcus braunii, as well as vascular plants (leaf epidermis of Pisum sativum, and
Zea mays)(Figure 2).
4. Curating candidate protein sequences
6
5
4
3
2
1
0
-4
-3
-2
-1
1
2
3
4
Segmentation of Corn Silk
6 Days post emergence
Figure 2: Hydrocarbon accumulation conditions of the four organisms:
A. Braunii – UTEX 572 was grown in Waris medium (low nutrients(NO3-PO4-), high
C(HCO3-)) and modified B3N medium(High nutrients(NO3-PO4-), low C(HCO3-)). B.
E. huxleyi-1516 grown in C+/NP- and C+/NP- media after reaching early stationary phase.
C. Abaxial and adaxial surface of pisum sativum leaves. BX indicates the branch from
which the leaves were obtained. D. Silk obtained from Zea mays encased within and
emergence from the corn husk.
Pathway drawn using pathvisio (http://pathvisio.org/)