1
ADDITIONAL EXPERIMENTAL PROCEDURES
2
3
Description
4
Astaxanthin analysis
5
Astaxanthin (both free astaxanthin and astaxanthin esters) was quantified by HPLC (Li, et al.
6
2010). Briefly, algal cells were harvested and extracted in a solvent mixture of dichloromethane
7
and methanol (25:75, v/v). The pigment extracts (20 mL aliquots) were separated and analyzed by
8
using a Beckman Ultrasphere C18 column (250 mm long, 4.6 mm i.d.; 5mm; Beckman
9
Instruments, Fullerton, CA, USA) at 25 oC. The mobile phase consisted of solvent A
10
(dichloromethane/ methanol/ acetonitrile/ water, 5.0:85.0:5.5:4.5, v/v) and solvent B
11
(dichloromethane/ methanol/ acetonitrile/ water, 25.0:28.0:42.5:4.5, v/v). The flow rate was 1.0
12
mL/min. The three dimensional chromatogram was monitored from 250 to 750 nm. Peaks were
13
measured at a wavelength of 480 nm to facilitate the detection of astaxanthin species, lutein and
14
β-carotene. Major pigment compounds were identified according to the spectra and retention time
15
of available authentic standards and by comparing of our data with relative literature data.
16
17
Fatty acid quantification
18
Fatty acids were determined by gas chromatography (GC) (Chen et al. 2007). Freeze-dried algal
19
cells were directly transmethylated with 2% sulphuric acid in methanol. The fatty acid methanol
20
esters (FAMEs) were extracted with hexane and analyzed by the HP-6890 GC (Hewlett-Packard)
21
equipped with a HP-INNOWAXTM capillary column (HP 19091N-133, 30 m  0.25 mm  0.25
22
m). The inlet and detector temperatures were kept at 250 oC and 270 oC, respectively, and the
23
oven temperature was programmed from 170 oC to 230 oC increasing at 1 oC min-1. FAMEs were
24
identified by comparison of their retention times with those of the authentic standards (Sigma),
25
and were quantified by comparing their peak areas with that of the internal standard (C17:0).
1
26
27
RNA isolation, cDNA synthesis, and real time RT-PCR
28
H. pluvialis cells cultured under various conditions were harvested by centrifugation, washed
29
with distilled water, flash frozen with liquid nitrogen, and stored at -80 oC until RNA isolation.
30
Total RNA was isolated according to the modified mini-prep RNA extraction procedure (Li, et al.
31
2010). First strand cDNA synthesis was carried out with 250 ng of total RNA with a TaqMan
32
Reverse Transcription system (Applied Biosystems, Foster City, CA, USA). Real time RT-PCR
33
analysis was performed on an ABI Prism 7500 sequence detection system (Applied Biosystems,
34
Foster City, CA, USA) according to the method reported previously (Li, et al. 2010). Primers
35
(Table S1) were designed with Primer Express Software V2.0 (Applied Biosystems). The primer
36
concentrations were determined when specific amplification relative to primer-dimers was
37
maximal in a positive versus negative control experiments. Plasmid containing cDNAs from each
38
gene was used as the templates for standards. To standardize the results, the relative abundance of
39
18S rRNA gene was employed as a normalization standard. The entire experiments including
40
RNA isolation, cDNA synthesis, and real time RT-PCR were repeated three times.
41
42
Isolation of Haematococcus pluvialis biotin carboxylase and stearoyl ACP desaturase
43
As shown in Figure S2, a 658 bp fragment of biotin carboxylase (BC) cDNA (GenBank accession
44
no. EF523480) was isolated from Haematococcus pluvialis with degenerated PCR and 5′- rapid
45
amplification of cDNA ends (RACE), which encoded 201 amino acids (Gene Bank accession no.
46
ABS45106) sharing 71.0% identical with that of Chlamydomonas reinhardtii BC. We tried
47
multiple times but could not amplify out the 3`- end of HpBC. However, this partial cDNA is
48
long enough for designing real time RT-PCR primers to detect its transcriptional expression.
49
A full-length stearoyl ACP desaturase (SAD) cDNA was cloned from H. pluvialis
50
(Genbank accession no. EF523479). As shown in Figure S3, HpSAD encoded a protein of 397
51
amino acids (Genbank accession no. ABP57425) with two (E/D) EXXH motifs which were
2
52
separated by about 100 amino acids. HpSAD showed high homology to the desaturase genes
53
isolated from algae and plants. For instance, it shared 68.5% and 58.9% of the sequence identity
54
with that of C. reinhardtii and A. thaliana, respectively. As a typical diiron-oxo enzyme, HpSAD
55
had two EXXH motifs (Figure S3).
56
57
Generation of real time RT-PCR standard curves of HpBC and HpSAD
58
Primers for HpBC and HpSAD were designed with Primer Express software (Version 2.0,
59
Applied Biosystems), which could produce 50-150 bp PCR products with a melting temperature
60
of about 60 oC and length of 18-25 nucleotides (Table S1). RT-PCR with these primers obtained
61
single band for HpBC and HpSAD, respectively, which were confirmed by sequencing.
62
According to the dissociation curve analysis, when 250 nm of HpBC and HpSAD primers were
63
used in real time RT-PCR, none of them produced nonspecific amplification. HpBC and HpSAD
64
were ligated to pCR21 vectors (Invitrogen, Carlsbad, CA), individually, and the plasmid DNAs
65
were used to prepare the templates for standard curves, which have high correlation coefficients
66
(R2 > 0.99).
67
68
Optimization of enzymatic assay conditions
69
Fraser et al. (1997, 1998) established a method to determine the activity of the recombinant
70
proteins involved in astaxanthin synthesis (Fraser et al. 1997, Fraser et al. 1998). When crude
71
membranes isolated from Haematococcus pluvialis cells were used as the enzymes, no the newly
72
synthesized astaxanthin could be detected, which might be due to the low concentration of target
73
enzymes in algal cell comparing to the heterologous proteins produced in E. coli. To improve the
74
efficiency of enzymatic reaction, ER membrane fraction was used to replace the crude
75
membranes, and a low activity of 0.09 µg mg protein-1 h-1 was detected with astaxanthin esters as
76
the predominant product. Since oxygen molecular is needed for the synthesis of astaxanthin from
77
beta-carotene, the effect of nitrogen gas, air, and oxygen gas was compared. The enzymatic
3
78
activity with the supply of oxygen gas was 2.6 folders higher than that of air, whereas nitrogen
79
gas completely inhibited the reaction (Figure S4a). Subsequently, three cofactors, NADPH, ATP
80
and NADH, were tested with the supply of oxygen. The enzymatic activity was stimulated by
81
NADPH or ATP but inhibited by NADH (Figure S4b). As shown in Figure S4b, the maximum
82
enzymatic activity was achieved with both NADPH and ATP, which was about 10 folders of the
83
original one.
84
85
REFERENCES
86
Fraser, P.D., Miura, Y. and Misawa, N. (1997) In vitro characterization of astaxanthin
87
biosynthetic enzymes. J. Biol. Chem., 272, 6128-6135.
88
Fraser, P.D., Shimada, H. and Misawa, N. (1998) Enzymic confirmation of reactions
89
involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro
90
assay. Eur. J. Biochem., 252, 229-236.
91
92
93
4