Kenan Jijakli, Rasha Abdrabu, Basel Khraiwesh, David R. Nelson, Joseph Koussa and Kourosh Salehi-Ashtiani

Abstract The uniquely diverse metabolism of algae can make this group of organisms a prime target for biotechnological purposes and applications. To fully reap their biotechnological potential, molecular genetic techniques for manipulating algae must gain track and become more reliable. To this end, this chapter describes the currently available molecular genetic techniques and resources, as well as a number of relevant computational tools that can facilitate genetic manipulation of algae. Genetic transformation is perhaps the most elemental of such techniques and has become a well-established approach in algal-based genetic experiments. The utility of genetic transformations and other molecular genetic techniques is guided by phenotypic insights resulting from forward and reverse genetic analysis. As such, genetic transformations can form the building blocks for more complex genic manipulations. Herein, we describe currently available engineered homologous recombination or recombineering approaches, which allow for substitutions, inser­tions, and deletions of larger DNA segments, as well as manipulation of endogenous DNA. In addition, as reagent resources in the form of cloned open reading frames (ORFs) of transcription factors (TFs) and metabolic enzymes become more readily available, algal genetic manipulations can greatly increase the range of obtainable phenotypes for biotechnological applications. Such resources and a few case studies are highlighted in the context of candidate genes for algal bioengineering. On a final note, tools for computer-aided design (CAD) to prototype molecular genetic tech­niques and protocols are described. Such tools could greatly increase the reliability and efficiency of genetic molecular techniques for algal bioengineering.

Kenan Jijakli and Rasha Abdrabu contributed equally to this work.

K. Jijakli

Division of Engineering, New York University Abu Dhabi,

P. O. Box 129188, Abu Dhabi, United Arab Emirates

R. Abdrabu • B. Khraiwesh • D. R. Nelson • J. Koussa • K. Salehi-Ashtiani (H) Division of Science and Math, Center for Genomics and Systems Biology (CGSB), New York University Abu Dhabi, P. O. Box 129188, Abu Dhabi, United Arab Emirates e-mail: ksa3@nyu. edu

© Springer International Publishing Switzerland 2015

N. R. Moheimani et al. (eds.), Biomass and Biofuels from Microalgae,

Biofuel and Biorefinery Technologies 2, DOI 10.1007/978-3-319-16640-7_9

9.1 Introduction

Microalgae have been described as nature’s very own power cells and can provide alternatives to petroleum-based fuels without competing with food crops (Dismukes et al. 2008; Singh et al. 2011). The heterogeneity and diversity that algae evolved make the molecular mechanisms that different algae have adopted, along with manipulating those mechanisms of tremendous interest. Currently, research is being conducted to develop methods for genetic modification to introduce desirable traits into algae and to develop synthetic biology approaches to re-engineer algal cells (Ferry et al. 2011; Gimpel et al. 2013; Rabinovitch-Deere et al. 2013). The crux of this research is to advance the molecular biology techniques utilized for algae and to ease the modification of the molecular systems of the species of interest.

One powerful example is the alga Chlamydomonas reinhardtii. As a single — celled alga containing a single large chloroplast, C. reinhardtii represents typical soil green algae. Moreover, Chlamydomonas combines powerful genetics with the availability of unique genetic and genomic resources. All three genomes (nuclear, plastid, and mitochondrial) have been fully sequenced (Merchant et al. 2007); large mutant collections have been established; and all three genomes are amenable to genetic manipulation by transformation (Hippler et al. 1998; Neupert et al. 2009). Most tools required for systematic functional genomics studies are available in Chlamydomonas, including high-frequency transformation protocols (Kindle 1990), efficient methods for chemical and insertional mutagenesis (Dent et al. 2005), and workable protocols for RNA interference (RNAi) (Arif et al. 2013; Molnar et al. 2007; Zhao et al. 2007). Overall, this represents a great advance in the molecular techniques and methods, especially with their applications to algae.

In the absence of cell differentiation, some algae such as Chlamydomonas can provide a much simpler system for genetic manipulations compared with higher plants. Manipulation of microalgae by metabolic and genetic methods would both permit (1) selection of beneficial pathways redirecting cellular functions toward the synthesis of preferred products and (2) introduction of non-algal genes for the generation of algal recombinant proteins. The selection of favorable pathways may include increased resistance to environmental or stress changes on the culturing and life cycle of the algae, expedited biomass production, and excretion of valuable products. The potential of such system remains to be optimized as an alternative protein expression system.

In light of all these potentials, and particularly during the past two or three decades, algal biotechnology grew steadily into an important global industry with new entrepreneurs realizing the potential of algae. However, creating profitable industries out of microalgae still remains challenging, and perhaps the development of new molecular techniques might expedite microalgae’s full industrial develop­ment, especially that some microalgal classes have highly complex genetic compositions rendering their modification arduous: Microalgal genome sizes range from 12.6 Mbp in Ostreococcus tauri, a Chlorophyta member, to 168 Mbp in the

Haptophyta Emiliania huxleyi and up to an impressive 10,000 Mbp for the Dinophyta Karenia brevis (Cadoret et al. 2012). Currently, a genome size of 10 Gbp precludes full genome sequencing, and as such, a lesser extent of knowl­edge would be available rendering the modification of such organisms a highly demanding task.