Plant Science 296 (2020) 110475
Contents lists available at ScienceDirect
Plant Science
journal homepage: www.elsevier.com/locate/plantsci
The possibility of using marine diatom-infecting viral promoters for the
engineering of marine diatoms
T
Takashi Kadonoa, Yuji Tomarub, Kengo Suzukic, Koji Yamadac, Masao Adachia,*
a
Laboratory of Aquatic Environmental Science, Faculty of Agriculture and Marine Science, Kochi University, Otsu-200, Monobe, Nankoku, Kochi, 783-8502, Japan
National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima, 7390452, Japan
c
euglena Co., Ltd., G-BASE Tamachi 2nd and 3rd Floor 5-29-11 Shiba Minato-ku, Tokyo, 108-0014, Japan
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Marine diatom
Diatom-infecting virus
Promoter
Marine diatoms constitute a major group of unicellular photosynthetic eukaryotes. Diatoms are widely applicable for both basic studies and applied studies. Molecular tools and techniques have been developed for diatom
research. Among these tools, several endogenous gene promoters (e.g., the fucoxanthin chlorophyll a/c-binding
protein gene promoter) have become available for expressing transgenes in diatoms. Gene promoters that drive
transgene expression at a high level are very important for the metabolic engineering of diatoms. Various marine
diatom-infecting viruses (DIVs), including both DNA viruses and RNA viruses, have recently been isolated, and
their genome sequences have been characterized. Promoters from viruses that infect plants and mammals are
widely used as constitutive promoters to achieve high expression of transgenes. Thus, we recently investigated
the activity of promoters derived from marine DIVs in the marine diatom, Phaeodactylum tricornutum. We discuss
novel viral promoters that will be useful for the future metabolic engineering of diatoms.
1. Marine diatoms
Marine diatoms constitute a major group of unicellular photosynthetic eukaryotes. These diatoms comprise 200,000 extant species
found in aquatic environments [1]. Diatoms are of broad interest in
both basic studies and applied studies. They account for 20 % of global
photosynthetic CO2 fixation and 40 % of primary production in the
oceans [2]. In addition to their ecological role, the complex evolutionary background of diatoms as secondary endosymbionts [3] and
their unique ability to produce silica-based cell walls [4] are also of
interest to diatom biologists. Moreover, diatoms present great potential
as a source of beneficial chemicals for use in human activities [5].
Diatoms produce biofuel precursors such as fatty acids and hydrocarbons in some cases that may prove useful for solving ecological
problems such as the energy crisis [6]. In addition to producing endogenous molecules, they present great potential as novel protein factories for medical applications via genetic engineering [7].
To understand diatom biology and advance the metabolic engineering of diatoms, various molecular tools and techniques have been
developed in recent years. Databases of genome sequences and expressed sequence tags (ESTs) of the model diatoms such as pennate
diatom Phaeodactylum tricornutum and centric diatom Thalassiosira
⁎
pseudonana have been released for public use, allowing the identification of distinct metabolic characteristics of diatoms [8–10]. The
genomic sequences of other diatom species such as Fragilariopsis cylindrus [11] and Pseudo-nitzschia multiseries are available in public databases such as the Ensemble Protists (https://protists.ensembl.org/) and
U.S. Department of Energy Joint Genome Institute (https://jgi.doe.gov/
) databases. The NCBI genome resources database (https://www.ncbi.
nlm.nih.gov/genome) contains the genomic sequences of Thalassiosira
oceanica [12,13] and Fistulifera solaris [14]. Methods for the transformation of diatoms via biolistic transformation [15–19], electroporation
[20–22], and bacterial conjugation [23–25] have been published. A
number of expression vectors containing promoters, terminators, and
selectable marker/reporter genes have been developed for effective
DNA delivery to the genome of diatoms [26,28]. More recently,
genome-editing techniques have been developed for diatoms via the
application of TALEN (transcription activator-like effector nuclease)
[27,29,30] and CRISPR/Cas9 (clustered regulatory interspaced short
palindromic repeats/CRISPR-associated protein 9) [31,32] technologies. Among molecular tools, several endogenous gene promoters,
bacterial promoter, and viral promoters have become available for
expressing transgenes in diatoms (Tables 1 and 2). Among viral promoters, we recently investigated the activity of promoters derived from
Corresponding author.
E-mail address: madachi@kochi-u.ac.jp (M. Adachi).
https://doi.org/10.1016/j.plantsci.2020.110475
Received 2 November 2019; Received in revised form 26 February 2020; Accepted 18 March 2020
Available online 20 March 2020
0168-9452/ © 2020 Elsevier B.V. All rights reserved.
2
I (iron depletion-inducible)
I (phosphate depletion-inducible)
U
U
U
U
PFld
pPhAP1
Clf(P1)
Clf(P2)
Pt202
Pt667
iron starvation induced protein 1 (-1021 to +214 from the
transcription start site)
ferrichrome binding protein 1 (-1097 to +9 from the
transcription start site)
flavodoxin (-688 to +407 from the transcription start site)
alkaline phosphatase gene
clumping factor A gene (-1538 to -1038 from the translation
initiation site)
clumping factor A gene (-616 to -116 from the translation
initiation site)
hypothetical protein gene (PHATRDRAFT_49202*) gene
hypothetical protein (PHATRDRAFT_47667*) gene
ammonium transporter gene
acyl-CoA diacylglycerol acyltransferase gene
hypothetical protein (PHATRDRAFT_49211*) gene
vacuolar ATP synthase 16-kDa proteolipid subunit gene
beta-carbonic anhydrase 1 gene (-1292 to +61 from the
transcription start site)
nitrate reductase gene
purine permease-like transporter gene
tubulin gamma chain gene
histone H4 gene
actin-like gene
elongation factor-1 alpha gene
highly abundant secreted protein 1 gene
40S ribosomal protein S8 gene
ribulose-1,5-bisphosphate carboxylase/oxygenase small
subunit N-methyltransferase I gene
elongation factor 2 gene
glutamine synthetase gene
fucoxanthin chlorophyll a/c-binding protein D gene
fucoxanthin chlorophyll a/c-binding protein E gene
fucoxanthin chlorophyll a/c-binding protein F gene
fucoxanthin chlorophyll a/c-binding protein B gene
fucoxanthin chlorophyll a/c-binding protein C gene
Associated gene (description regarding promoter region)
stationary
unknown
unknown
Ω leader
Ω leader
log
unknown
stationary
log
log
no
no
no
no
no
no
log
unknown
unknown
no
no
no
log
unknown
stationary
unknown
log
log
log
log
log
unknown
log
stationary
stationary
log
unknown
unknown
unknown
stationary
unknown
unknown
unknown
unknown
unknown
Growth phase
no
Ω leader
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
Additional regulatory
elements
conjugation
conjugation
conjugation
conjugation
electroporation
electroporation
biolistic
biolistic
biolistic
biolistic
biolistic
biolistic
biolistic
biolistic
biolistic
biolistic
electroporation
biolistic
biolistic
biolistic
bacterial conjugation
bacterial conjugation
biolistic
bacterial conjugation
biolistic
biolistic
biolistic
biolistic
bacterial conjugation
biolistic
biolistic
bacterial
biolistic
bacterial
biolistic
biolistic
bacterial
bacterial
Transformation methods
[80]
[69]
[69]
[92]
[25]
6.18B (m)
0.44A (f)
5.64A (f)
44A (m)
1.87B (c), 8.65C (c), N.A.C
(m)
N.A.A (w)
1.16B (c), 5.35C (c), N.A.C,F
(m)
2.12B (c), 9.83C (c), N.A.C,F
(m)
N.A.A (w)
0.08B (c), 0.39C (c), N.A.C,F
(m)
N.D.C (lipid content)
2.73A (m)
4.22A (g)
[93]
[93]
[67]
0.53A (m)
70.0C (m)
22.9C (m)
[99]
[70]
[67]
[99]
[99]
[72]
[58]
[70]
[128]
[127]
[67]
[94]
[25]
[25]
[25]
N.D. (g)
9.3A (f)
0.80A (m)
N.D. (g)
24.61A (f) under nitrate
containing medium
0.89A (f) under nitrogen
depletion medium
N.A.A (w)
0.22A (m) not under
nitrogen starvation
N.D. (g)
[15]
[15]
[25]
[67]
[25]
[15]
[16]
[25]
[25]
1.22A (c)
2.21A (c)
0.22A (c)
0.73A (m)
2.20B (c), 10.19C (c)
0.74A (c)
0.2−2.0A (c)
1.20B (c), 5.54C (c)
0.89B (c), 0.97C (c)
[72]
[25]
References
Relative promoter activityb
(methods)
b
Cited from articles or estimated from associated genes. I, inducible type; C, constitutive type; U, unknown type.
Relative promoter activity of each promoter compared with those of the P. tricornutum fcp promoters, which is cited from articles or calculated from reported data. A,B,C,FCompared with the activities of the fcpA
promoter, fcpB promoter, fcpC promoter, and fcpF promoter, respectively. Method for the comparison of promoter activity: c, colony formation; g, GUS activity; f, GFP fluorescence intensity; m, mRNA expression level; w,
western blot. Italicization of figures show that the evaluation of promoter activity was carried out using only one transformant. N.A.: not calculated. N.D.: cannot compare.
* Gene ID in the U.S. Department of Energy Joint Genome Institute database.
a
I (iron depletion-inducible)
PFBP1
C
C
pPUP
pγ Tubulin
I (iron depletion-inducible)
C
pH4–1B
PIsi1
C
C
pAct2
pEF-1α
I (nitrate depletion-inducible)
I (nitrate depletion-inducible)
C
C
HASP1 promoter
p40SRPS8
pAMT
pDGAT1
C
C
EF2 promoter
GLNA promoter
I (nitrogen depletion/nitrate-inducible
and ammonium-suppressible)
C (light-dependent)
pRBCMT
nr promoter (pNR)
C (light-dependent)
C (light-dependent)
C (light-dependent)
fcpD promoter
fcpE promoter
fcpF promoter
C
C
I (low [CO2]-inducible)
C (light-dependent)
C (light-dependent)
fcpB promoter
fcpC promoter
Pt211
V-ATPaseC promoter
ptca1 promoter
Typea (dependency/inducer/suppressor)
Promoter
Table 1
Relative activity of endogenous promoters used in the marine diatom species Phaeodactylum tricornutum compared with that of the P. tricornutum fcp promoters.
T. Kadono, et al.
Plant Science 296 (2020) 110475
Plant Science 296 (2020) 110475
[33]
0.12 (m), 0.48 (f)
biolistic
stationary
no
capsid protein gene
Relative promoter activity of each promoter compared with that of the P. tricornutum fcpA promoter, which is cited from articles or calculated from reported data. Method for the comparison of promoter activity: g,
GUS activity; f, GFP fluorescence intensity; m, mRNA expression level. N.A.: not calculated. Related accession numbers of DIV promoters: CdP1; JA784022, ClorDNAV complete genome: AB553581, and TnitDNAV
replication-associated protein (VP3); AB781284.
Thalassionema
nitzschioides
TnitDNAV
marine diatom-infecting viruses (DIVs) in the marine diatom P. tricornutum [33] (Table 2). In this review, we discuss DIV promoters that
are useful for the future metabolic engineering of diatoms.
2. Diatom endogenous promoters
Gene promoters that drive transgene expression at a high level are
very important for the metabolic engineering of diatoms. Among the
available endogenous promoters, the fucoxanthin chlorophyll a/cbinding protein (FCP) gene (fcp, now referred to as light-harvesting
complex containing fucoxanthin, Lhcf) promoter has been frequently
used in biotechnological applications involving the transformation of
diatoms [28]. LHCF is a member of a family of proteins known as lightharvesting complexes (LHC), which are essential components of photosynthesis in photosynthetic organisms [34]. In addition to the LHCFs,
other LHC families such as the LHCR and LHCX gene families, are
present in both pennate diatom and centric diatom [35]. In the genome
of pennate diatom P. tricornutum, 17 LHCF genes, 14 LHCR genes, and 4
LHCX genes are found [36,37]. In the genome of centric diatom T.
pseudonana, 11 LHCF genes, 14 LHCR genes, and 7 LHCX genes have
been identified [9]. The promoters of the following LHCF genes are
available for the transformation of diatoms: lhcf5 [20,38] and lhcf14
[20] derived from Chaetoceros gracilis; fcpA-1A [39,40] derived from
Cylindrotheca fusiformis; fcpB [18,41–43] derived from Fistulifera solaris;
fcpA [15,27,33,44–72], fcpB [15,16,23,25,32,64,68,73–81], fcpC
[15,21,67,82,83], fcpD [25], fcpE [15] and fcpF [16,23,24,29,80] derived from P. tricornutum; and fcp8 [84] and lhcf9 [17,31,85–91] derived from T. pseudonana. The fcp promoters have been categorized as
constitutive-type promoters [28], although their transgene expression
activity depends on light and dark conditions [74,80].
Recently, endogenous promoters that drive the high expression levels of introduced genes in P. tricornutum have been reported (Table 1),
including an elongation factor 2 (EF2) gene promoter [80], a vacuolar
ATP synthase 16-kDa proteolipid subunit (V-ATPase C) gene promoter
[67], a glutamine synthetase (GLNA) gene promoter [69] and a highly
abundant secreted protein 1 (HASP1) gene promoter [92], all of which
provide constitutive expression of introduced genes (constitutive promoters). In addition, some 5′ upstream regions (Pt202 and Pt667)
whose regulation is unknown can achieve high expression levels of
introduced genes [93]. Among the endogenous promoters listed in
Table 1, promoters such as the EF2 promoter, V-ATPase C promoter,
Pt202, Pt211 and Pt667 were isolated from highly expressed genes
characterized from transcriptome data of P. tricornutum. In the case of
the GLNA gene promoter, the corresponding gene encodes one of most
abundantly expressed proteins during and the stationary phase of P.
tricornutum. The HASP1 promoter was isolated from the gene that encodes the most abundant secreted protein from the proteome profile of
the culture medium of P. tricornutum. Endogenous promoters isolated
from highly expressed genes may be a useful molecular tool for the
overexpression of introduced genes. Inducible endogenous promoters
that can be activated only under specific conditions have also been
isolated to drive transgene expression in P. tricornutum (Table 1). For
example, the nitrogen-responsive promoters, such as the nitrate reductase gene (nr) promoter [45] and acyl-CoA diacylglycerol acyltransferase gene promoter [58], phosphate depletion-inducible promoters, the alkaline phosphatase gene promoter (pPhAP1) [46], and
CO2 concentration-responsive promoter, the beta-carbonic anhydrase 1
gene promoter [94], can drive transgene expression under specific
conditions.
In some studies [69,80,92,93], the evaluation of promoter activity
has been carried out using only a single transformant that shows high
activity (Table 1). In general, the expression levels of transgenes can
vary among transformants due to differences in the copy numbers of
transgenes integrated within a host genome [95] and the position of the
introduced gene cassette in the genome (i.e., the position effect) [96],
which is likely to cause misinterpretation of promoter activity. To avoid
a
plants
Chaetoceros debilis
C. lorenzianus
Agrobacterium tumefaciens
CdebDNAV
ClorDNAV
nos promoter
CdP1
ClP1
ClP2
TnP1
TnP2
0.48
0.86
2.17
1.22
0.43
(f)
(f)
(f)
(f)
(f)
[33]
[81]
[33]
[33]
[33]
[33]
[33]
0.42 (f)
(m),
(g)
(m),
(m),
(m),
(m),
(m),
0.10
N.A.
0.24
0.96
4.97
1.49
0.08
biolistic
biolistic
biolistic
biolistic
biolistic
biolistic
biolistic
no
no
no
no
no
no
no
35S
35S (core region)
nopaline synthase gene
replication-associated protein gene
replication-associated protein gene
capsid protein gene
replication-associated protein gene
stationary
unknown
stationary
stationary
stationary
stationary
stationary
[33]
[104]
[104]
0.17 (m), 0.53 (f)
2.73 (g)
2.03 (g)
biolistic
biolistic
biolistic
Rous sarcoma virus
cauliflower mosaic virus
CMV promoter
PRSV-LTR
CaMV35S promoter
(PCaMV35s)
chicken
Brassicaceae family
plants
no
no
no
stationary
log
log
[104]
0.33 (g)
biolistic
log
no
immediate early gene (minimal
region)
immediate early gene
long terminal repeat
35S
cytomegalovirus
mPCMV
human
Transformation methods
Growth phase
Additional regulatory
elements
Associated gene (description
regarding promoter region)
Host
Isolated virus
Promoter
Table 2
Relative activities of viral and bacterial promoters used in the model marine diatom species Phaeodactylum tricornutum compared with that of the P. tricornutum fcpA promoter.
Relative promoter activity
(methods)a
References
T. Kadono, et al.
3
Plant Science 296 (2020) 110475
T. Kadono, et al.
we identified at least two open reading frames (ORFs) in each DIV
(Fig. 1). One encodes a replication-associated protein (VP3) gene, and
the other encodes a capsid protein (VP2) gene. Other ORFs are present
within the genome of DIVs. However, the functions of these ORFs remain unknown. Our group considered the approximately 500 base
upstream regions of the VP2 and VP3 genes as potential promoter regions (Fig. 1). To test viral promoter activity levels in the model diatom
species P. tricornutum, cells were transformed with specific vectors that
contained the enhanced green fluorescence protein (eGFP) gene (egfp)
driven by each tested promoter [33]. The promoter regions of the
Chaetoceros debilis-infecting DNA virus (CdebDNAV) VP3 gene (CdP1)
and that of the Chaetoceros lorenzianus-infecting DNA virus (ClorDNAV)
VP2 gene (ClP2) showed the same activity level as the fcpA promoter,
while the activities of the Thalassionema nitzschioides-infecting DNA
virus (TnitDNAV) VP3 gene promoter (TnP1) and VP2 gene promoter
(TnP2) were extremely low when they were applied to P. tricornutum
(Table 2). Among the DIV promoters, the activity of the promoter region of the ClorDNAV VP3 gene (ClP1) was significantly higher than the
activity of the endogenous fcpA promoter (Table 2). The activity of ClP1
was almost identical in P. tricornutum under low-nutrient culture conditions and standard nutrient culture conditions [33]. The ClP1 could
therefore be a useful metabolic engineering tool for maximizing the
productivity of bioproducts to minimize nutrient utilization.
misinterpretation of promoter activity, the analysis of numerous
transformants is preferred for the evaluation of promoter activity.
In addition to the development of promoters with high transcriptional activity, conserved motifs that may be involved in the initiation
of gene expression in diatoms have been investigated. Bhaya and
Grossman’s group [97] and our group [33] identified a potential initiator (Inr)-like sequence. Our group proposed TCAH+1W (the degenerate bases described according to the International Union of Pure and
Applied Chemistry nucleotide code) as a novel potential Inr-like sequence located upstream of the translation site in some P. tricornutum
fcp genes [33]. This potential Inr-like sequence is present in approximately 68 % of the 5′-flanking sequences (80 bases) of the P. tricornutum genes (12,237 sequences), whose sequences are available in
Ensembl Protists BioMart (Dataset: ASM15095v2) [33].
Twelve types of cis-regulatory elements have been reported in P.
tricornutum: CRE1 (nucleic acid sequence: ATACGTCA), the p300binding element (GGGAGTG), CRE2 (TGACGGCA) [94], CCRE1
(TGACGT), CCRE2 (ACGTCA), and CCRE3 (TGACGC) [98], which are
responsive to changes in cAMP or CO2 concentrations; motif A (AMGSCGCRTG or AMGSCCRTG) and motif B (CACGTGYC), which are responsive to iron deficiency [99]; Motif 17 (BBNKDHHVNHDHBVVWMDWR) and Motif 6 (HGVAAWWCKRG) elements, which are
responsive to nitrogen deficiency [100]; the HMG-box binding site (
CCCCAGCTGGG), which is a potential light-responsive cis-regulatory
element [81]; and GAATCT, which is the binding site of the P. tricornutum phosphorus starvation response transcription factor (PtPSR)
within the promoter region of genes induced by phosphorus limitation
[101]. These cis-regulatory elements might aid in the design of synthetic inducible promoters for controlling the transcription of transgenes in diatoms.
4. Regulation of diatom-infecting viral promoters
The activity of DIV promoter such as ClP1 has been observed in
transformants grown under both light and dark conditions [33], which
suggests that ClP1 constitutively induces the expression of transgenes
without an effect of light and dark periods. The DIV promoters include
plant-type light-responsive cis-regulatory elements [33]. Endogenous
promoter, the V-ATPase C promoter, also possesses plant-type light-responsive cis-regulatory elements [67]. However, the expression of reporter genes controlled by these promoters can be detected in both light
and dark periods [33,67]. These findings suggest that plant-type lightresponsive cis-regulatory elements cannot respond to light in P. tricornutum.
The activity levels of ClP1 are influenced by the growth phase of
diatoms. The expression level of the transgene controlled by ClP1 in the
stationary phase seem to be higher than that in the log phase of
P.tricornutum [33]. In the relationship between the Chaetoceros tenuissimus-infecting DNA virus (CtenDNAV) and the growth phase of C.
tenuissimus, the replication of the viral genome of CtenDNAV is activated when the host cells reach the stationary phase [117]. In the case
of the relationship between the PpV01 virus and the haptophyte
Phaeocystis pouchetii, the production of the virus in infected host cells is
higher in exponentially growing cultures than in stationary phase cultures [118]. Transgene expression mediated by the CaMV 35S promoter
in Schizosaccharomyces pombe is induced during the log phase rather
than the stationary phase [119]. In P. tricornutum, the expression level
of a transgene controlled by the CaMV 35S promoter seemed to be
higher in the log phase than in the stationary phase (Table 2). Although
there is a lack of knowledge regarding the mechanisms of DIV replication in a host diatom, the elucidation of the mechanism of DIV
promoter activation might help to understand DIV replication in a host
diatom.
The mechanisms of DIV infection are not fully understood; however,
DIVs such as DNA viruses are thought to have a specific host range
[106–109,112,113]. In contrast, DIV promoters such as the CdP1 and
ClP1 can be applied to both the centric diatom Chaetoceros sp. strain
CCK09 and the pennate diatom P. tricornutum [33]. Transcription factor
(TF) contents in the pennate diatom P. tricornutum and the centric
diatom T. pseudonana are similar [120]. Endogenous promoters such as
fcp promoters derived from P. tricornutum can drive the expression of
introduced genes in centric diatom such as Thalassiosira weissflogii
[16,23]. These findings suggest that the DIV promoter region contains
3. Viral and bacterial promoters used for diatom transformation
Promoters from viruses that infect plants or mammals have generally been used to transform a wide range of higher plants and
mammals, allowing the high constitutive expression of transgenes. For
example, the cauliflower mosaic virus 35S (CaMV 35S) promoter and
cytomegalovirus (CMV) immediate early (IE) gene promoter efficiently
facilitate transformation in plants and mammals, respectively
[102,103]. Some viral promoters, such as the CaMV 35S promoter
[81,104], the minimal region of the CMV IE gene promoter [104], and
the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter
[104], can drive transgene expression in P. tricornutum (Table 2). In
addition to viral promoters, bacterial promoter, the nopaline synthase
gene (nos) promoter of Agrobacterium tumefaciens, has been used extensively for transformation in plants [105] and exhibit the promoter
activity in P. tricornutum [33] (Table 2). Among these promoters, the
activities of the CMV IE gene promoter and the nos promoter are lower
than that of the fcpA promoter (Table 2). In the case of the CaMV 35S
promoter, the expression level of a transgene controlled by the CaMV
35S promoter was found to be lower than that produced by the fcpA
promoter in the stationary phase of P. tricornutum (Table 2). In contrast,
at the log phase of P. tricornutum, the activity of the CaMV 35S promoter was higher than that of the fcpA promoter (Table 2). The RSVLTR promoter also shows higher activity than the fcpA promoter in the
log phase of P. tricornutum (Table 2).
Recently, our group isolated various DIVs, including both DNA
viruses and RNA viruses, and characterized their genome sequences
[106–113]. Another group identified a similar RNA virus [114]. Because DIVs can infect host diatoms and cause their lysis, it has been
suggested that DIVs may influence the composition of marine communities and may act as a major force driving biogeochemical cycles
[115,116]. In most of the DNA viruses among DIVs, the genome
structure consists of a covalently closed circular single-stranded DNA
molecule that includes a partially double-stranded DNA region (Fig. 1),
while RNA viruses consist of single-stranded RNA. Via in silico analysis,
4
Plant Science 296 (2020) 110475
T. Kadono, et al.
Fig. 1. Typical genome structure of marine diatom-infecting DNA viruses. Modified from the ClorDNAV genome [107].
expression in diatoms (Table 2). The activities of promoters from
viruses that infect plants or mammals have generally been found to be
higher than those of endogenous promoters. The ongoing discovery of
DIVs is expected to lead to the isolation of various types of promoters,
including strong constitutive promoters.
DIV promoters are expected to be useful for the metabolic engineering of diatoms because their activity is detectable in both light
and dark periods and under low-nutrient culture conditions [33]. In
addition, understanding DIV promoters may help to elucidate the mechanism of native DIV gene expression in host diatoms, which may shed
light on the formation/decay of diatom blooms related to ocean ecosystems.
conserved cis-regulatory elements that are recognized by conserved TFs
in both pennate diatoms and centric diatoms.
Within DIV promoters, in silico analyses have revealed the presence
of Myb [121], bZIP [122], CCAAT-binding [123], homeobox [124], and
E2F-DP [125] cis-regulatory elements recognized by TFs, which have
also been found in genomic sequences of the pennate diatom P. tricornutum and the centric diatom T. pseudonana [33,120]. In endogenous
promoters that drive high expression levels of introduced genes, such as
the nr promoter, pPhAP1, HASP1 promoter, Pt202, and Pt667, one or
more kinds of these cis- regulatory elements have been found
[70,92,93,128]. The CMV IE gene promoter and the nos promoter,
which show low-level promoter activity, also contain one or more kinds
of these cis- regulatory elements [33]. Among these cis-regulatory elements, in the minimal region of the CMV IE gene promoter and core
region of the CaMV promoter, only the Myb cis-regulatory element is
found [33,81,104]. These findings suggest that unknown novel motifs
that contribute to high promoter activity may exist among endogenous
promoters, bacterial promoter, and viral promoters, including DIV
promoters. Among DIV promoters such as CdP1, ClP1, and ClP2, conserved motifs that may be involved in promoter activity have been
found [33]. The number, direction, and proximity of the conserved
motifs might be related to DIV promoter activity [33]. However, the
mechanisms of transcriptional control by DIV promoters are not fully
understood in diatoms.
Acknowledgments
Our study of DIV promoter activity was supported by the past/
present laboratory members Dr. Arisa Miyagawa-Yamaguchi, Takuma
Okami, Takamichi Yoshimatsu, Kohei Ohno, Yumi Watanabe, Dr.
Kazunari Fukunaga, Dr. Nozomu Kira, and Assoc. Prof. Haruo
Yamaguchi and the researchers/research groups of Prof. Keizo Nagasaki
(Faculty of Agriculture and Marine Science, Kochi University), Dr.
Masanori Okauchi (National Research Institute of Aquaculture, Japan
Fisheries Research and Education Agency), Dr. Liyuan Hou and Prof.
Takeshi Ohama (Kochi University of Technology), Prof. Kohei Ohnishi
(Research Institute of Molecular Genetics, Kochi University), Prof.
Angela Falciatore (Institut de Biologie Physico-Chimique), and Dr.
Nicole Poulsen and Prof. Nils Kröger (Technische Universität Dresden).
This study was supported by JSPS KAKENHI Grant Number
JP15K14804 to M.A.
5. Conclusions
Endogenous promoters, especially fcp promoters, have been frequently used in the transformation of diatoms [28]. Recently, various
endogenous promoters of diatom, viral and bacterial origin have been
characterized and used for transgene expression in diatoms (Tables 1
and 2). Strong endogenous promoters that are expected to be useful for
the metabolic engineering of diatoms were subsequently isolated
(Table 1).
In plants, the induction of transcriptional gene silencing (TGS) by
multiple use of the same promoter can result in the suppression of both
the introduced gene and the endogenous gene controlled by the same
promoter [126]. To avoid TGS, the arbitrary selection of promoters
among numerous promoters may have present advantages for the genetic engineering of diatoms. Recently, we developed several DIV
promoters, among which the ClP1 drove the highest level of transgene
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