Recombineering is an in vivo genetic engineering method that relies on the ability to induce homologous DNA recombination. Short for recombination-mediated genomic engineering, recombineering relies on bacteriophage homologous recombination proteins that induce and catalyze homologous recombination. These proteins are introduced into the cell by plasmids or bacteriophage vectors, are endogenously expressed, or are expressed in transformed strains. Being based on homologous recombination inducing proteins give recombineering the prime advantage of inde­pendence from the use of restriction enzymes to produce changes in DNA. Those changes in DNA can be in the form of insertions, deletions, or alterations through the introduction of a synthesized oligonucleotide substrate that contains the desired change and that replaces the native DNA (Court et al. 2002). To function properly, a recombineering system must have the ability to perform three tasks. First, it must be able to prevent nucleases from degrading the foreign oligonucleotides; second, it must have an exonuclease function to cleave the foreign DNA creating sites for DNA recombination; third, it must promote the annealing of DNA strands to ensure the foreign DNA recombines and is restored into the native DNA (Sharan et al. 2009).

One example of a system that provides all the requirements for recombineering is the X Red system, commonly used for bacterial recombineering. The X Red system consists of three bacteriophage recombination proteins from three respective genes: Gam protein produced by the gam gene; Beta protein produced by the bet gene; and Exo protein produced by the exo gene (Sharan et al. 2009). A simple description of the mechanism of the X Red system is that the Exo binds to ends of the oligonucleotide and cleaves the 5′ DNA ends. This transforms the oligonucleotide to an oligonucleotide with a 3′ overhang. The Beta protein then binds to the overhangs and facilitates annealing with the native DNA, thus completing the recombination process (Fig. 9.2) (Datta et al. 2008).

The design of the produced DNA construct should depend on the ultimate objective of recombineering. Recombineering has been successfully used to insert genetic markers, retrieve DNA fragments, insert non-selectable markers, and pro­duce point mutations (Court et al. 2002).

Homologous recombination has already been shown to be a viable mean of genetically engineering algae, albeit still at a lesser efficiency than bacteria. One such example is the utilization of homologous recombination to knockout nitrate and nitrite reductase genes in Nannochloropsis sp. In this case, the required proteins appeared to be expressed endogenously (Kilian et al. 2011). A study on homolo­gous recombination in C. reinhardtti showed that recombination occurs readily between overlapping plasmids and requires around 230 homologous base pairs (bp) only, but is lacking when the recombination targets endogenous DNA (Gumpel et al. 1994). The required proteins appear to also be expressed endogenously, but the introduction of exogenous homologous recombination proteins increases the rate of recombination. Homologous recombination has also been demonstrated in

the multicellular green algae Volvox carteri (Hallmann et al. 1997) and in the red alga Cyanidioschyzon merolae (Minoda et al. 2004). Although those examples of homologous recombination in algae clearly show the complexity that arises from the significant difference in the mechanism of homologous recombination and its efficiency in different species, they also clearly demonstrate the viability of engi­neering a homologous recombination system (recombineering) in algae.