The document discusses genetic engineering approaches for microalgae. It describes forward genetics methods like chemical and physical mutagenesis that generate random mutants for screening. It also describes reverse genetics methods like insertional mutagenesis that allow targeted modification of specific genes. The goal is to generate strains with desired traits like increased lipid or starch production for industrial applications.

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2.  Emerging economies and the growing world
population rely heavily on the natural resources.
Outlook for future energy source (IEA, 2010)
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3.  Sustainable production methods for food and energy are
necessary, if we do not want to convert all available nature
into agricultural land. So, there is a need for alternatives.
 The society focuses increasingly on sustainability and
therefore on the recycling of waste streams, sustainable
production and efficient use of energy.
 The cultivation of microalgae can make an important
contribution to the transition to a more sustainable society
or bio based economy.
Barbosa (2003)
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4. • Microalgae, also called phytoplankton by biologists, they are
very small plant-like organisms between 1-50 micrometres in
diameter without roots or leaves.
• Together with the sea weeds (large aquatic plants),
microalgae are so called aquatic biomass.
• Only a few tens of thousands out of a total have been
described in literature and more to be known.
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5.  The genetic analysis and ranking of all types of microalgae is
still in progress and there is not yet a complete and consistent
classification. At the moment taxonomists have distinguished
the following main groups:
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8.  Microalgae have traditionally been used in many biotechnological
applications, where each new application required a different species
or strain expressing the required properties;
 The challenge therefore is to isolate or develop, characterize and
optimize species or strains that can express more than one specific
property.
 However, many biotechnologically relevant algae do not possess the
sexual characteristics required for traditional breeding/crossing,
although they can be modified by chemical and physical mutagens.
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9.  The resulting mutants are not considered as genetically modified
organisms (GMOs) and their cultivation is therefore not limited by
legislation.
 On the other hand, mutants prepared by random or specific
insertion of foreign DNA are considered to be GMOs.
 This review will compare the effects of two genetic approaches
on model algal species and will summarize their advantages in
basic research.
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10.  Genetic material of all living organisms is transferred from parents
to progeny, thus forming the basis of hereditary traits.
 Genes, the simplest units of heredity, are transferred to the next
generation.
 Naturally occurring mutants arise by interactions between
environmental effectors such as UV irradiation or metabolically
produced ROS and the genetic material.
 Such mutations, and the resulting mutants, are a major source of
genetic variability with potential for evolution (Barton, 2010; Eyre-Walker
and Keightley, 2007).
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11.  Mutation frequency can be increased by several orders of magnitude
using different mutagens (Table 1), leading to the production of
mutant populations.
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Mutagen Mode of Action Most Common Mutation
Caused
EMS, MNNG Alkylation of DNA base,
particularly Guanine
Point Mutations
UV Radiation
Photochemical reaction
leading to Cyclobutane
ring
Point Mutations,
Deletions
Gamma Radiation Ionization leading to
double stranded break
Deletions
Heavy Ion Beams Ionization leading to
double stranded break
Chromosome breaks and
Exchange
T-DNA Antibiotics
Resistant Gene
DNA Fragment Insertion Insertions, Deletions

12.  To maximize these, thousands to ten thousands of independent mutants
must be generated in order to cover the entire genome.
 The required phenotypes can be complex, including increased cell size,
improved growth, resistance to different compounds or improved
productivity of a specific compound, all of which will require specific
screening protocols.
 Both breeders and researchers can search for similar phenotypes, but
with different motivations
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13.  The first step in characterizing a mutational mechanism is to
identify the mutated gene(s).
 This is traditionally done by crossing mutants to other strains, and
requires the existence of sexual reproduction in the specific
species.
 While generally in higher plants, this prerequisite is sometimes
complicated to fulfill in algae, where the conditions needed for
sexual reproduction are sometimes not known and the strains are
usually maintained asexually.
 In basic research, this obstacle can be overcome by sequencing the
mutant genome using next-generation sequencing and comparing
this to the parental strain (Dutcher et al., 2011).
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14.  Mutant strains can be selected based on phenotype and reproduced
asexually, giving rise to a culture expressing that phenotype.
 Traditionally, only strains with desired phenotype/s are selected from the
mutant population.
 However, it is also possible to save and characterize mutants of all genes
in the collection, irrespective of phenotype.
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15.  These are most widely used, both in basic and applied science.
 They are easy to apply at different doses and their mutagenic
potentials are well characterized (Table 1).
 Alkylating agents such as Ethyl methane sulfonate (EMS) and
Methylnitronitrosoguanidine (MNNG).
 They were also the first ones used in mutagenic screenings to
increase EPA production in Nannochloropsis oculata (Chaturvedi and
Fujita, 2006) and to enhance growth properties of Chlorella (Ong et al.,
2010).
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MNNG EMS

17.  Physical mutagens include different types of irradiation such as UV,
gamma or heavy ion beams.
 The mode of action and mutagenic potential of each type of radiation on
cells depends on the energy while the frequency of their use depends on
ease of application.
 Mutagenesis by UV is very simple, since it requires neither specialized
equipment nor chemicals and can be very easily performed, essentially by
exposing cells to germicidal UV lamps in a sterile hood.
 Given its simplicity and potential, this method has been used both in basic
research to prepare algal strains with specific features (Neupert et al., 2009),
and in applied science to produce strains with increased production of oil
(de Jaeger et al., 2014; Vigeolas et al., 2012).
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18.  Essential genes are less amenable to a classical genetic approach
since an inactivating mutation cannot be recovered due to its
lethality.
 Conditional mutants show the phenotype only under specific,
restrictive conditions, while they behave as wild type under other,
permissive conditions.
 The most widely used types of conditional mutants are temperature
sensitive ones.
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19.  Typical examples of temperature-sensitive mutants have mutations
in cell cycle regulators, e.g. genes essential for processes related to
nuclear and/or cell division.
 Such mutants will grow and divide normally at a permissive
(usually lower) temperature, whereas they will stop growth and cell
division at restrictive (higher) temperatures (Harper et al., 1995; Hartwell et
al., 1974; Nurse et al., 1976; Thuriaux et al., 1978).
 Such mutants in two staple algal species, Chlamydomonas
reinhardtii and Chlorella vulgaris, were tested for lipid production
at a restrictive temperature (Yao et al., 2012).
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20.  Firstly, in order to be able to perform insertional mutagenesis in a certain
organism, an established method of DNA transformation is required.
 Secondly, the insertion of a large piece of DNA usually has detrimental
effects at the insertional locus/gene, so only mutations in non-essential
genes will be recovered.
 Thirdly, the identification of the mutation site is simple since the inserted
piece of DNA represents a well defined tag.
 Inserted pieces of DNA range from naturally occurring transposons or
viruses, to laboratory derived DNA vectors like modified transposons,
viruses, T-DNA, artificial DNA plasmids or antibiotic resistance cassettes.
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21.  It is a common approach to study gene function in microalgae,
although to achieve this, the inserted piece of DNA must contain
either a
1. selection marker (antibiotic resistance cassette) or
2. an amino acid synthesis gene (to complement an auxotrophic
mutation).
 Recently, a tagged mutant library of C. reinhardtii was prepared
with T-DNA insertion into A. thaliana (Zhang et al., 2014).
 This library was successfully used to isolate mutants with increased
lipid production (Terashima et al., 2014) and with appropriate screening
procedure/s it should serve as a basis for isolating mutants with
diverse phenotypes.
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22.  In a basic research project, an insertional mutagenesis screen was
performed to reveal new aspects of algal starch synthesis in C.
reinhardtii.
 In the screen, a novel mutant was isolated and denoted sta6. The
mutant lacked the small subunit of adenosine diphosphate (ADP)-
glucose pyrophosphorylase, leading to the accumulation of less
than 0.01% of the wild type amount of starch (Zabawinski et al., 2001) and
was denoted as starchless.
 Increased interest in using microalgae as a feedstock for biodiesel
production (Chisti, 2007) brought about new efforts to increase oil
production by microalgae.
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23.  Since the metabolic pathways for starch and oil metabolism share a
common precursor, inactivation of starch synthesis was the prime
research target (Li et al., 2010b).
 Indeed, blockage of starch synthesis in the sta6 (Li et al., 2010a,
2010b;Wang et al., 2009; Work et al., 2010) and sta7-10 (Work et al., 2010)
starchless mutants increased the accumulation of lipids compared
to wild type; lipid accumulation could be further improved by
nitrogen deprivation of the cells.
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24.  Numerous benefits arise from this research:
1) Establishing a direct interdependency link between starch and oil
metabolic pathways,
2) Identification of conditions leading to hyper-accumulation of oil
in the mutants unable to produce starch,
3) Unraveling the molecular mechanisms responsible for TAG
hyper-accumulation.
Importantly, it is evident that studies on a model organism such as
C. reinhardtii can unravel growth and genetic conditions required for
increased lipid production and thus can be used to optimize
conditions further.
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25.  Starchless mutants were recently generated by UV mutagenesis in the
starch producing alga Chlorella pyrenoidosa (Ramazanov and Ramazanov, 2006)
and in the oleaginous alga Scenedesmus obliquus (de Jaeger et al., 2014).
 Both mutants had increased TAG accumulation. Biomass productivity
was lower in slm1 (the starchless mutant of S. obliquus) compared to wild
type
 In the case of C. pyrenoidosa STL-PI mutant, growth rates of wild type
and mutant were similar, with the mutant saturating at slightly higher cell
concentrations (Ramazanov and Ramazanov, 2006)
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26. Forward Genetics Reverse Genetics
Targeted on desired feature,
phenotype is selected for
causative gene primarily unknown
Targeted on modification of a
known gene, phenotype primarily
unknown, desired feature
predicted not guaranteed
Results in mutants, not GMO Results in GMO
No prior information required Requires prior information
Available in any organism Available in selected organisms
only
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Reverse Genetics

27.  Natural products, such as oil or starch, isolated from an alga are not
considered GM. e.g. starch isolated from a mutant raised by insertional
mutagenesis is not considered GM.
 Limited number of algal species available for a this approach
 Used as basic research models,
the green alga C. reinhardtii,
the primitive red alga Cyanidioschyzon merolae,
the primitive green alga Ostreococcus tauri, and
algae with more biotechnological potential such as diatoms
(Phaeodactylum tricornutum, Thalassiosira pseudonana) or
other heterokonts (Nannochloropsis sp.) (Table 3).
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28. Species Taxonomic group Available tools
Chlamydomonas reinhardtii Green algae, Chlorophyceae
Genome sequence, genetic
transformation, the most
developed molecular toolkit,
chloroplast gene targeting
available, lacks nuclear gene
targeting.
Cyanidioschyzon merolae Red algae, Cyanidiophyceae
Genome sequence, genetic
transformation, developed
molecular toolkit, gene
targeting available.
Nannochlorolsis sp. Heterokonts,
Eustigmatophyceae
Ostreococcus tauri Green algae, Prasinophyceae
Phaeodactylum tricornutum Heterokonts,
Bacillariophyceae
Genome sequence, genetic
transformation, developed
molecular toolkit, gene
targeting by TALE available
Thalassiosira pseudonana Heterokonts,
Coscinodiscophyceae
Genome sequence, genetic
transformation, developed
molecular toolkit.
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29.  The goal of reverse genetics is to alter gene expression in order to
either study gene function or to obtain a defined line with a
predicted phenotype.
 In principle, there are two possible outcomes of altered gene
expression:
1) the target gene expression is lower or absent,
2) the target gene expression is higher than in the wild type or
parental strains.
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30.  The traditional way to decrease gene expression involves RNAi or a
miRNA constructs or the creation of deletion mutants, either by
homologous recombination or insertional mutagenesis.
 Gene over expression can be achieved by driving expression of the
target gene with a strong promoter, derived either from a virus or
from a naturally strongly expressed gene.
 With the sole exception of homologous recombination, all the
methods involve integration of a transgene into a genomic locus
different from its natural one.
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31.  The methods of gene editing have been developed based on the
combination of a
DNA binding protein and zinc-finger nuclease (ZFN) (Bibikova et al.,
2001; Kim et al., 1996),
Transcription activator-like effectors (TALEs) (Boch et al., 2009;Moscou
and Bogdanove, 2009) or
Clustered regularly interspaced short palindromic repeats
(CRISPR) (Horvath and Barrangou, 2010; Jinek et al., 2012)
that allow almost seamless activation of a gene, gene deletion,
gene replacement by another variant, either natural or recombinant,
introduction of a specific mutation in a gene, and creation of specific
gene variants.
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33.  Lies at the crossroads of engineering, chemistry and biology.
 Its aim is to build a biological system using artificial genetic,
regulatory and metabolic systems (Benner, 2003; Lee et al., 2008).
 While just the building and testing of such a system will largely
improve understanding of the functions of biological systems, the
ultimate goal in biotechnology is the production of a desired
metabolite.
 Uses a bottom up approach and thus requires preliminary
information on the organism.
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34.  Proof of concept experiments with amenable model organisms,
bacteria and yeasts, have demonstrated the potential of synthetic
biology
 Synthetic biology can only be developed in organisms with reverse
genetic tools and methodologies such as genetic transformation,
selection markers, specific plasmid vectors, different promoters, and
optimally, the existence of advanced molecular biology techniques
such as ZFN, TALEs and CRISPR.
 Therefore, in algae it is limited to a handful of established model
organisms (Table 3).
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35.  The chloroplast has been easier to tackle by reverse genetics since it
was amenable to gene editing, even before the new technologies
such as ZFN, TALEs and CRISPR.
 This is because, upon genetic transformation, the DNA is inserted
by homologous recombination into a specific, homologous locus.
 Information on metabolic pathways and the metabolome can be
built into metabolic networks.
 Such networks are a prerequisite for engineering an artificial
metabolic pathway/s in the organism of choice using tools of
reverse genetics.
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Commercially Important Metabolic Pathways in Microalgae
http://ipec.utulsa.edu/Conf2010/Powerpoint%20presentations%20and%20papers%20received/Noor_Sam_90_received9-8-10.ppt

37.  The most critical parameter for any mutagenesis is the method of
screening.
 For any of the mutant screens it is important to maintain the same
conditions for all mutants in order to compare them directly.
It is not always easy since a large number of mutants are involved.
 A more general procedure to avoid mutant reversion is to limit
multiple sub culturing, e.g. by keeping mutants in storage under
liquid nitrogen and taking out fresh aliquots periodically.
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38.  The major issue for any strain, newly constructed by mutagenesis or
any new production protocol is its suitability for large scale
production.
 Upon transfer to large scale cultivation, the strains experience
conditions that are more diverse than those controlled precisely in
the laboratory.
 The design of the photo bioreactor can also affect cell behavior and
fitness.
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39.  A specific issue related to amenability of reverse genetics and
synthetic biology approaches is the cost of strain development.
 Reverse genetics (and synthetic biology) require specialized know-
how, equipment and the process is time consuming.
 All these factors will affect the final cost.
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40.  We believe that ultimately the most cost effective way lies in further
improvements of current strains through at least two systems:
1) breeding and classical genetic approaches,
2) reverse genetics and synthetic biology systems.
 Breeding & mutagenesis with chemical and physical mutagens is
phenotype-based, can be used for any algal species, and yields
strains that are not considered to be GMOs.
 Reverse genetics, and its most sophisticated application, synthetic
biology, start with a gene, the manipulation or introduction of which
should cause a desired phenotype.
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41.  The application of the two methodologies is distinct.
 Breeding and mutagenesis being relatively simple & cheap
 Can be used to improve productivity of compounds natural to algae, such
as carotenoids, TAGs, starch and to increase biomass productivity.
 Reverse genetics & synthetic biology require specific knowledge and
optimization is more expensive in time and money.
 The production of such compounds can take advantage of fast growth,
low-cost production & simple scalable cultivation.
 In this way, the potential of algal based products will broaden and algae
could become available to both industry and pharma.
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42.  Anarat-Cappillino G, Sattely ES. The chemical logic of plant natural
product biosynthesis. Curr Opin Plant Biol 2014;19:51–8
 Blatti JL, Michaud J, Burkart MD. Engineering fatty acid biosynthesis in
microalgae for sustainable biodiesel. Curr Opin Chem Biol 2013;17:496–
505.
 Gong Y, Hu H, Gao Y, Xu X, Gao H. Microalgae as platforms for
production of recombinant proteins and valuable compounds: progress and
prospects. J IndMicrobiol Biotechnol 2011;38:1879–90.
 Kroth PG. Genetic transformation: a tool to study protein targeting in
diatoms. Methods Mol Biol 2007;390:257–67.
 Ramazanov A, Ramazanov Z. Isolation and characterization of a starchless
mutant of Chlorella pyrenoidosa STL-PI with a high growth rate, and high
protein and polyunsaturated fatty acid content. Phycol Res 2006;54:255–9.
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