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The selection of ideal algal strain from the huge diverse group is the
vital step in microalga based fuel production. Compared to animal and land
plants, algae have larger genetic pool and potential of which is revealed in
the diversity of algal species being explored for example, cyanobacteria, green
algae and diatoms (Georgianna and Mayfield,
2012). The algal strains should be tolerant to variation in light,
temperature, salinity and pathogen load. Seasonal optimization of algal strains
for cultivation in various geographical regions is needed to ensure their
widespread use. In this context, native strains may be a favorable choice
because they have all the characteristics required for agriculture and
industrial production. A number of wild type strains have been isolated from
different environmental sites and were tested for growth rates and lipid
accumulation. Out of 30 native strains of microalgae, Nannochloropsis sp. F&M-M26 achieved maximum lipid production
of 61 mg L-1 per day through a biomass productivity of 0.21 g L-1
per day and lipid content of 29.6% of the biomass (Larkum et al. 2012). Another researcher
have isolated two different native isolates
of Scenedesmus dimorphus, which showed higher growth rate with lipid
content of ~30% of dry biomass compare to other isolates (Gour et al. 2016). Although the native strains
have potential however, the production efficiencies are much less
from industrial point of view. Therefore, recent techniques of
genetic/metabolic engineering may be successful for cost-effective production.


Metabolic engineering approach

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In this technique, metabolic pathway in a cell is altered in such a way
so that it can activate the target metabolite production. This can be achieved
by various approaches/ strategies as given below (Banerjee et al. 2016):

1. Flux
balance analysis

2. Improving
photosynthetic proficiency (Decreasing cell shading/ Increasing light

3. Enzyme
engineering for lipid biogenesis

4. Identification
of rate limiting enzymes or committed step

5. Carbon

6. Mathematical

7. Over expression
of multiple gene or a single gene

8. Engineering the
transcription factor


1.4.3 In silico metabolic engineering

In this approach large scale models could be designed
through numerous in silico tools to
interpret the role of crucial enzymes, genes, transcripts and different
metabolites accountable for metabolic fluxes (Banerjee
et al. 2016; Schmidt et al. 2010). Computational techniques can be
crucial in understanding the key components of lipid regulation for the
researchers working on biofuel. However,
literature reports showed that flux balance analysis and metabolic network
modeling can play a vital role in designing new pathways for enhanced recovery
of lipids from microalgae (Schuhmann et al.
2012). The accessibility of metabolic model and in silico strategies can provide sufficient information in
identifying key residue of lipid metabolism. Other than this, transcriptomics/
proteomics/ metabolomics data can be used for improvement of the existing
models to attain crucial components toward good quality biofuel however, in silico information need to be
verified through wet lab experiments (Banerjee et
al. 2016).


1.4.4    Genetic engineering

In the recent years, genetic engineering has attracted
researchers towards itself because genome can be edited more accurately through
new and powerful genetic tools (Song et al.
2015). The synthetic biology have opens up new possibilities for
industrialization of microalgae. The basic gene manipulation process (host
selection, gene target, plasmid construction, transformation tools, selection
system and DNA editing tools) remains same in synthetic biology (Bashir et al. 2016). To accomplish target gene
modification in microalgae transformation and selection methods are important
steps. Other than this, gene interfering and genome editing tools are
prerequisite to proficiently target the gene. The recent genome editing tools
include Clustered Regularly Interspaced Short Palindromic Repeats–CRISPR
associated protein 9 (CRISPR-Cas9) and Transcription Activator-Like (TAL)
Effector Nucleases (TALEN) (Kasai et al. 2015;
Shin et al. 2016). The important steps involved in algal genetic
engineering are (Ng et al. 2017):

1.    Transformation

2.    Transgenic
microalgal strains, stability and antibiotic resistant

3.    CRISPR
technology for genome editing in microalgae Transformation techniques

In the molecular biotechnology approach, gene delivery through
transformation in to host is an important technique. The transformation of gene
occurs through four different methods (Ng et al.

with glass beads



Agrobacterium construction

 The oldest method for microalgal
transformation is agitation with glass beads (Kindle,
1990). The method is simple and cost effective but only for cell wall
less strains for example Chlamydomonas reinhardtii (Leon and
Fernández, 2007). The method is not suitable for the strains having
rigid cell wall for example, Chlorella and Phaeodactylum. The
electroporation method uses electric impulses to insert DNA to cells and is
applicable for genetic manipulation in prokaryotic microalgae like
cyanobacteria. This method is well established and most widely and dominantly
used for microalgae nuclear transformation. Agrobacterium
mediated transfection in microalgae is challenging and only have been reported
for C. reinhardtii, Haematococcus pluvialis, and Chlorella vulgaris (Kathiresan et al. 2009; Kumar et al. 2004; San Cha et
al. 2012). At present, the most successful method of transformation of
foreign DNA fragment in to algal chloroplast genome is the microparticle
bombardment using a biolistic device. Other approaches like sonication in the
presence of polyethylene glycol and agitation with silicon carbide whiskers
have also been used for nuclear transformation of algae. All the genetic
manipulations are random insertion or deletion, thus making transformants stability
a cause of concern (Ng et al. 2017). Transgenic microalgal strains, stability and antibiotic

After successful transformation of microalgae, the genetic
stability and effect of continuation were highly uncertain. Among different algae,
Chlamydomonas, Dunaliella, Chlorella and Nannochloropsis are highly stable after
transformation whereas, Ulva lactuca,
Thalassiosira weissflogii, Poryhyra miniata, and Kappaphycus alvarezii were unstable (Ng et al. 2017).

The microalgal nuclear transformants are screened through
two different mechanisms:

Developing auxotrophic defective mutants followed by
transformation of these with the wild-type authentic gene

antibiotic/ herbicide resistance gene

Among the two, antibiotic
screening method is most frequently used. The details about achievement of
antibiotic marker for genetic transformation in microalgae and precise dose of
antibiotics were reviewed by Ng et al. (2017).  CRISPR technology
for genome editing in microalgae

From the last two decades, zinc finger nucleases (ZFNs) and
transcription activator-like effector nucleases (TALENs) were the two major
genetic editing tools (Christian et al. 2010;
Kim et al. 1996; Ng et al. 2017). A number of reports showed that both
the techniques are successful for plants, mammals and insects but rarely in the
diatoms Phaeodactylum tricornutum (Daboussi et al. 2014; Jankele and Svoboda, 2014;
Palpant and Dudzinski, 2013). Discovered in 2013, CRISPER system belongs
to adative immune system of bacteria and majority of Archaea and works through
the corporation of many Cas proteins (Ng et al.
2017; Wang et al. 2016a).

Generally, the CRIPSER technique operates/works in three
different stages to achieve full immune response to invade foreign DNA. The
three stages are: spacer acquisition, CRISPR RNA (crRNA) biogenesis, and
interference stages, which are discussed below (Wang
et al. 2016a):

In the acquisition stage, target DNA fragment (DNA
fragments of invading plasmids/ phages) known as protospacers is inserted into
the host CRISPR array as spacers between crRNA repeats. In the subsequent
stage, the CRISPR array having acquired spacers is transcribed to pre-crRNA
followed by its processing into mature crRNAs via Cas proteins and host
factors. The processed crRNA is a guide, which contain a spacer sequence and all
or part of the crRNA; accountable for targeting it to the invading genome and
permits for recognition of the crRNA by Cas proteins and other RNA components. In
the final interference stage, with the guidance of crRNA the Cas proteins
identify the right target and mediate the cleavage of the invading genome, thus
protecting the host cells from infection (Deltcheva
et al. 2011; Hsu et al. 2014; Wang et al. 2016a).

The beginning of microalgae genome editing has been started
with the demonstrations of CRISPR/Cas9-mediated genome editing in C. reinhardtii cells (Jiang et al. 2014). However, toxicity of Cas9
nuclease is a challenge in microalgae genome editing but can be overcome by
Cas9 protein gRNA ribonucleoproteins (RNPs) (Baek
et al. 2016).  Other demonstration includes genome editing of
marine diatom Phaeodactylum tricornutum through CRISPR/Cas9 vector (Nymark et al. 2016). The technology has also
been implemented for a new model algae (carbon sequestration and oil-producing
variety) Nannochloropsis sp. (Wang et al.
2016b). However, the metabolite production from microalgae using CRIPSER
technology is not yet demonstrated practically. Till 2017, CRISPRi (CRISPER
interference) to increase the lipid production using repression of gene CrPEPC1
is first applied for C. reinhardtii
CC400 (Kao and Ng, 2017).

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