some important associated enzymes are transferred to chloroplast
for performing their function [60, 61]. C. reinhardtii has been the
main subject of genetic engineering attempts in microalgae over the
last decades. The main reasons for introducing C. reinhardtii as a
model microalgae to genetic and metabolic engineering studies are
(a) its known genome, (b) easy to work with, and (c) the long
history of use in research [16, 62, 63].
Low expression rates of genes of interest is the main disadvantage of nuclear transformation [1]. In addition to that, one of the
other obstacles associated with nuclear transformation in algae is
the efficient gene silencing microRNA systems present in nuclear
genomes of some algae which can prevent and/or reverse the
artificial manipulating [64, 65]. In contrast, recombinant gene
expression in chloroplast shows higher rates of protein accumulation [66, 67].
It should be noted that metabolic engineering is beholden of
developments in genetic engineering tools such as genome
sequencing, transformation, gene targeting, and selection methods. These tools have substantially facilitated the design of new
pathways or the manipulation of existing ones to achieve improved
biofuel production by microalgae. The omics technologies are also
considered as promising methods available to accomplish this aim
[68–72].
Among these tools, transformation and selection methods are
key steps of microalgae genetic engineering to achieve the modification of a specific target gene. The main methods for microalgae
transformation include (1) electroporation, (2) agitation and shaking with glass beads, (3) Agrobacterium construction, and (4) particle gun bombardment (biolistic) [73, 74].
5 Methods and Protocols of Metabolic Engineering
The following protocol (in three steps) is used for metabolic engineering of various microorganisms including microalgae [75]:
Step 1: Primary Lab Experiments
1. Selection of host strain and determination/formulation of
nutrient requirements: as mentioned earlier, expression
and/or silencing of the same genes in different microalgae
species might not lead to similar results. Therefore, it is very
important to select the right species/strain as well as the culture medium.
2. Collection of information on host features: most of wild strains
have low production yields, low productivities, or low resistance to environmental conditions. Therefore, it is important
to acquire thorough data on the various features of the selected
host algae.
Metabolic Engineering of Microalgae
163
for performing their function [60, 61]. C. reinhardtii has been the
main subject of genetic engineering attempts in microalgae over the
last decades. The main reasons for introducing C. reinhardtii as a
model microalgae to genetic and metabolic engineering studies are
(a) its known genome, (b) easy to work with, and (c) the long
history of use in research [16, 62, 63].
Low expression rates of genes of interest is the main disadvantage of nuclear transformation [1]. In addition to that, one of the
other obstacles associated with nuclear transformation in algae is
the efficient gene silencing microRNA systems present in nuclear
genomes of some algae which can prevent and/or reverse the
artificial manipulating [64, 65]. In contrast, recombinant gene
expression in chloroplast shows higher rates of protein accumulation [66, 67].
It should be noted that metabolic engineering is beholden of
developments in genetic engineering tools such as genome
sequencing, transformation, gene targeting, and selection methods. These tools have substantially facilitated the design of new
pathways or the manipulation of existing ones to achieve improved
biofuel production by microalgae. The omics technologies are also
considered as promising methods available to accomplish this aim
[68–72].
Among these tools, transformation and selection methods are
key steps of microalgae genetic engineering to achieve the modification of a specific target gene. The main methods for microalgae
transformation include (1) electroporation, (2) agitation and shaking with glass beads, (3) Agrobacterium construction, and (4) particle gun bombardment (biolistic) [73, 74].
5 Methods and Protocols of Metabolic Engineering
The following protocol (in three steps) is used for metabolic engineering of various microorganisms including microalgae [75]:
Step 1: Primary Lab Experiments
1. Selection of host strain and determination/formulation of
nutrient requirements: as mentioned earlier, expression
and/or silencing of the same genes in different microalgae
species might not lead to similar results. Therefore, it is very
important to select the right species/strain as well as the culture medium.
2. Collection of information on host features: most of wild strains
have low production yields, low productivities, or low resistance to environmental conditions. Therefore, it is important
to acquire thorough data on the various features of the selected
host algae.
Metabolic Engineering of Microalgae
163