While microalgal lipids, specifically triacylglycerides (TAGs), and carbohydrates have been of

interest for biofuel production, lipids and also proteins are of nutritional interest. Some species
can produce high-value long-chain polyunsaturated fatty acids (PUFAs), such as the ω-3 PUFAs
docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Furthermore, many microalgae are
rich in bioactive compounds and high-value pigments including chlorophylls, carotenoids, and
terpenes.

In some cases the accumulation or composition of the targeted metabolite can remain largely
unchanged.

transcriptional engineering (TE)

TFs regulate the expression of specific target genes by binding to specific DNA motifs within cis
elements of the target gene and by interacting with the RNA polymerase to activate or repress
transcription.

Recent studies have begun to use omic approaches to identify TFs in microalgal species, with a
focus on lipid metabolism. For example, TFs have been identified as regulators of TAG biosynthesis
by acting on components of fatty acid and glycerolipid synthesis, and lipid degradation/
remobilization.

Many eukaryotic microalgae modify their metabolism in response to nutrient stresses such as
phosphorus (P) starvation, which substantially induces storage metabolite biosynthesis, but the
genetic mechanisms regulating this response are poorly understood. Here, we show
that P starvation-induced lipid and starch accumulation is inhibited in a Chlamydomonas
reinhardtii mutant lacking the transcription factor Pi Starvation Response1 (PSR1). Transcriptomic
analysis identified specific metabolism transcripts that are induced by P starvation but
misregulated in the psr1 mutant. These include transcripts for starch and triacylglycerol synthesis
but also transcripts for photosynthesis-, redox-, and stress signaling-related proteins. To further
examine the role of PSR1 in regulating lipid and starch metabolism, PSR1complementation lines in
the psr1 strain and PSR1 overexpression lines in a cell wall-deficient strain were
generated. PSR1 expression in the psr1 lines was shown to be functional due to rescue of
the psr1phenotype. PSR1 overexpression lines exhibited increased starch content and number of
starch granules per cell, which correlated

with a higher expression of specific starch metabolism genes but reduced neutral lipid content.
Furthermore, this phenotype was consistent in the presence and absence of acetate.(Bajhaiya,
Dean, Zeef, Webster, & Pittman, 2015).

Overexpression of PSR1 increases TAG accumulation without inhibiting growth [5], and PSR1
overexpression can also increase starch biosynthesis

In this study, GmDof4 from soybean (Glycine max), a transcription factor affecting the lipid content
in Arabidopsis, was transferred into Chlorella ellipsoidea. We then investigated the molecular
mechanism underlying the enhancement of the lipid content of transformed C. ellipsoidea.

We constructed a plant expression vector, pGmDof4, and transformed GmDof4 into C. ellipsoidea
by electroporation. The resulting expression of GmDof4 significantly enhanced the lipid content by
46.4 to 52.9%, but did not affect the growth rate of the host cells under mixotrophic culture
conditions. Transcriptome profiles indicated that 1,076 transcripts were differentially regulated: of
these, 754 genes were significantly upregulated and 322 genes were significantly downregulated
in the transgenic strains under mixotrophic culture conditions. There are 22 significantly regulated
genes (|log2 ratio| >1) involved in lipid and fatty acid metabolism. Quantitative real-time PCR and
an enzyme activity assay revealed that GmDof4 significantly up-regulated the gene expression and
enzyme activity of acetyl-coenzyme A carboxylase, a key enzyme for fatty acid synthesis, in
transgenic C. ellipsoidea cells.

The hetero-expression of a transcription factor GmDof4 gene from soybean can significantly
increase the lipid content but not affect the growth rate of C. ellipsoidea under mixotrophic
culture conditions. Ubiquitin promoter and the selection marker gene nptII.(Bajhaiya et al., 2015)

To identify factors that govern cellular quiescence, we screened for mutants of the model alga
Chlamydomonas reinhardtii that, in contrast to wildtype cells placed under conditions of nitrogen
deprivation, were unable to degrade triacylglycerols following nitrogen resupply. One of the
mutants described here in detail, compromised hydrolysis of triacylglycerols 7 (cht7), was severely
impaired in regrowth following removal of different conditions inducing cellular quiescence.

Toward this end, we searched for mutants of C. reinhardtii unable to rapidly exit quiescence,
readjust their metabolism, and resume growth following N resupply after a period of N
deprivation.

Fig. 1. Phenotypes of cht7. (A) Immunoblot of MLDP in the PL (dw15) and cht7 mutant following N
resupply (NR) at times indicated (hours). (B) TAG degradation in dw15 and cht7 following N
resupply. (C) Confocal microscopy images (Top) and lipid droplet quantification (Bottom) of Nile
red-stained dw15 and cht7 cells following N resupply. Chlorophyll fluorescence (C) and Nile red
fluorescence of lipid droplets (LD); scale bar, 5 μm. Lipid droplets of 5–20 cells, depending on the
sample, were counted and their area was quantified with imaging software and their volume
calculated. No lipid droplets were observed in dw15 cells at NR24. SD is indicated.

endogenous CHT7 promoter in the cht7 mutant background(Tsai et al., 2014)

Of the microalgal CRT genes identified, many are transcriptionally regulated by environmental
stresses. Although carotenoid TFs are yet to be experimentally confirmed, several TFs in
Chlamydomonas correlate positively with CRTs, including zeaxanthin epoxidase and carotenoid
isomerase.

Chlamydomonas reinhardtii accumulates oil (triacylglycerols, TAG) in response to nitrogen (N)

deprivation. Although a few important regulatory genes have been identified that are involved in
controlling this process, a global understanding of the larger regulatory network has not been
developed. In order to uncover this network in this species, a combined omics (transcriptomic,
proteomic and metabolomic) analysis was applied to cells grown in a time course experiment after
a shift from N-replete to N-depleted conditions. Changes in transcript and protein levels of 414
predicted transcription factors (TFs) and transcriptional regulators (TRs) were monitored relative
to other genes. The TF and TR genes were thus classified by two separate measures: up-regulated
versus down-regulated and early response versus late response relative to two phases of polar
lipid synthesis (before and after TAG biosynthesis initiation). Lipidomic and primary metabolite
profiling generated compound accumulation levels that were integrated with the transcript
dataset and TF profiling to produce a transcriptional regulatory network. Evaluation of this
proposed regulatory network led to the identification of several regulatory hubs that control many
aspects of cellular metabolism, from N assimilation and metabolism, to central metabolism,
photosynthesis and lipid metabolism.

In this investigation, we analysed a correlation network generated using a time course of

Chlamydomonas grown under N deprivation, with a focus on the transition in metabolism that
occurs when the cells move from the before TAG synthesis (BTS) phase to the after TAG synthesis
initiation (ATS) phase. More importantly, our objective was not to construct a sparse network but
to identify all (or as close to that as possible) key regulators that collaborate to tune lipid synthesis
under N stress conditions.

When the N stress is prolonged, dramatic metabolic changes and/or the BTS phase TRs induce a
limited number of novel transcription family members, which appear to execute more specific
functions related to the induction of genes directly involved in TAG metabolism and formation
of lipid droplets, this includes AP2-15, FHA10 and MYBL13. (Gargouri et al., 2015)

we show that addition of thiamine to cultures of the model green alga Chlamydomonas reinhardtii
alters splicing of transcripts for the THI4 and THIC genes, encoding the first enzymes of the
thiazole and pyrimidine branches of thiamine biosynthesis, respectively, concomitant with an
increase in intracellular thiamine and TPP levels

Thiamine (vitamin B1) is synthesized via a branched pathway (Fig. 1) from the condensation of a
thiazole and a pyrimidine moiety to make thiamine monophosphate (TMP). This is then
phosphorylated to make thiamine pyrophosphate (TPP), the active cofactor, either directly in
prokaryotes, or by dephosphorylation followed by pyrophosphorylation in eukaryotes. Like many
cofactors, the level of TPP within the cell is low, the majority of it being associated with its cognate
enzymes, which are part of the central respiratory pathways. The biosynthetic pathway is carefully
regulated to ensure that TPP production meets cellular demand, usually by regulation of gene
expression for one or more of the enzymes, and in bacteria and fungi exogenous thiamine severely
represses gene expression (1, 2). In bacteria, an important TPP regulatory mechanism is mediated
via riboswitches in one or more of the thiamine biosynthesis genes. Riboswitches are short
sequences in mRNAs that bind metabolites directly, without the need for intermediary proteins.
Binding of the ligand alters the secondary structure of the RNA, thereby regulating expression of
the gene, typically by premature transcription termination and/or initiation of translation (3, 4).

In the higher plant Arabidopsis thaliana, a TPP riboswitch was identified in the 3 UTR, and this has
been characterized structurally (7, 8). However, the presence of riboswitches in other eukaryotes
has not been established. We have taken advantage of recent genome sequencing projects in two
related green algal species, Chlamydomonas reinhardtii and Volvox carteri (9, 10), to discover TPP
riboswitches in these organisms by sequence comparison.

Pδ (DELTA)εEPSILON