In order to increase productivity of biomass or a desired product, target genes have been selected and manipulated with success using transgenic approaches. These genes may comprise the metabolic pathways under consideration or be part of unrelated pathways that indirectly contribute to higher productivity. Highlighted below are some notable examples of target genes used for genetic manipulation along with examples of potential targets yet to be explored.

Much work has been done to genetically manipulate plants to produce more biomass or more of a specific tissue or compound. The Viridiplantae (green plants) include land plants and two lineages of algae, the chlorophytes and the charophytes (Finet et al. 2010). We therefore can look at gene targets in plants for possible research avenues when considering algae. A number of gene targets have already been picked and utilized with great success from previous knowledge of metabolic pathways. Fruit yield was increased in tomato (Solanum lycopersicum L.) when RNAi was used to decrease ascorbate oxidase activity (Garchery et al. 2013). Basically, the stress response to water or environment was down-regulated thus allowing more energy to be allocated for fruit production even in unfavorable conditions. Likewise, stress response genes in algae, such as those involved in oxidative stress (Perez-Martin et al. 2014) and light-related stress (Kukuczka et al.

2014) , might be down-regulated to allow for more energy to be allocated to biomass production. One strategy to increase biomass is to manipulate photosynthetic light capture. Internal shading is a limiting factor in biomass production in algae, so reducing the size of the light-harvesting antennae may increase biomass production. However, pigment mutants of Cyclotella sp., a diatom, failed to outperform their wild-type counterparts in biomass productivity (Huesemann et al. 2009). The authors note that the mutagenesis procedures (chemical and UV) may have affected other metabolic processes that contributed to the Cyclotella sp.’s unremarkable growth and found no difference in growth of wild and mutated strains of this alga when grown parallel in raceway ponds. Green algae (e. g., C. reinhardtii) have evolved genetic strategies to assemble large light-harvesting antenna complexes (LHC) to maximize light capture under low-light conditions, with the downside that under high solar irradiance, most of the absorbed photons are wasted as fluores­cence and heat generated by photoprotective mechanisms.

An insertional mutant was obtained with a disrupted gene for the antennae protein TLA1 (Polle et al. 2003). Biochemical analyses showed the TLA1 strain to be chlorophyll deficient, with a functional chlorophyll antenna size of photosystem I and II being about 50 and 65 % of that of the wild type, respectively. The TLA1 strain showed greater solar conversion efficiencies and a higher photosynthetic productivity than the wild type under mass culture conditions (Polle et al. 2003). Other researchers have used RNAi to knock down the entire family of LHCs (Mussgnug et al. 2007). The resultant mutant, Stm3LR3, exhibited reduced levels of fluorescence, a higher photosynthetic quantum yield and a reduced sensitivity to photoinhibition. Cultures with these mutants have higher light penetrance, which may lead to more efficient biomass production. Another strategy to increase bio­mass production is to focus on increasing the rate at which the alga assimilates CO2 from the atmosphere or through supplied gas, or to increase the efficiency of the carbon capture mechanism (CCM) (Stephenson et al. 2011). Genes that are involved in the CCM and are possible targets for genetic manipulation include an ABC transporter (Hla3/Mrp1 Cre02.g097800), two low-CO2-inducible chloroplast envelope proteins (Ccp1 Cre04.g223300), an anion transporter (LciA/Nar1.2 Cre06.g309000), and ten different carbonic anhydrases (Winck et al. 2013). When the desired outcome is increased production of a specific product, increased bio­mass yield may or may not be desirable. For example, in engineering algal cells to increase lipid yield, biomass productivity is only important so far as it increases total lipid yield. A variety of genes have been manipulated with this aim with varying degrees of success (Stephenson et al. 2011). One of the most remarkable achievements in lipid production from Chlamydomonas occurred when researchers showed that Chlamydomonas deprived of a nitrogen source accumulates a high degree of lipids (Wang et al. 2009b). This effect is much more pronounced in mutants lacking the small subunit of a heterotetrameric ADP-glucose pyrophos — phorylase (Zabawinski et al. 2001). The normal response of Chlamydomonas under nitrogen deprivation is to accumulate starch; however, the mutants, unable to accumulate starch, instead store energy as lipids (Wang et al. 2009b).

A recent study showed a 12 % increase in the total lipid content of the micro­algae D. salina by transforming it with a bioengineered plasmid comprising specific parts, genes, and inducible promoters, driving the cellular carbon flux into the fatty acid biosynthesis pathway (Talebi et al. 2014).

In addition to lipid production, hydrogen production is also seen as a possible route to biofuel production in Chlamydomonas (Lehr et al. 2012). Increasing hydrogen production does not necessarily follow an increase in biomass; instead, researchers usually aim to increase photosynthetic efficiency or force the cells to more readily assume an anaerobic state.

RNAi knockdown of light-harvesting proteins was found to increase H2 pro­duction in the high-H2-producing C. reinhardtii mutant Stm6Glc4 (Oey et al. 2013). Oey et al. (2013) also stated that the overall improved photon-to-H2 conversion efficiency is due to (1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface, (2) improved light distribution in the reactor, (3) reduced photoinhibition, (4) early onset of HYDA expression, and (5) reduction of O2-induced inhibition of HYDA (Oey et al. 2013). Rubisco has also been used as a target for genetic manipulation to increase hydrogen production. The Rubisco mutant Y67A accounted for 10- to 15­fold higher hydrogen production than the wild type under the same conditions (Pinto et al. 2013). In conclusion, a variety of gene targets are available in algae that when manipulated may increase biomass and biofuel productivity.