**3.3 Product storage and metabolism**

Following the metabolic engineering paradigm for increasing product yield, i.e., push, pull, sequester and block storage product turnover, less attention has been directed towards the metabolic engineering of storage and product turnover in microalgae. As stated previously, energy reserves in algae fall into two classes, carbohydrates, and lipids. The genetic manipulation of starch accumulation in algae has received much attention. The chloroplast is the site of starch synthesis and storage in plants and algae. In contrast to plant cells, however, microalgae typically have only a single chloroplast per cell since chloroplast division must be synchronized with cell division to ensure that each progeny has a chloroplast [59]. Thus, there is no differentiation of plastids in single-celled microalgae into specialized starch storing amyloplasts as occurs in plants. As a result, increasing starch storage sites is not a viable strategy for increasing starch accumulation. Starch accumulation in a plastid can be genetically manipulated, however. Structurally, starch is composed of two types of glucose polymers, amylose and amylopectin, that differ in their degree of branching. The glucose density of starch granules and their size is controlled by the levels of starch branching and debranching enzyme activities. Genetic manipulations of enzymes controlling starch branching has been shown to substantially impact biomass production [58].

Enhanced lipid storage in microalgae has been achieved by over-expression of enzymes implicated in fatty acid and TAG biosynthesis [60–63], or by repression of lipid catabolism [62, 63]. Additionally, genetic manipulations to decrease starch accumulation also leads to substantial increases in storage lipid accumulation per cell. A *C. reinhardtii* mutant blocked in starch accumulation nearly doubled the amount of lipids accumulated under nitrogen deprivation relative to the control strain, indicating that TAG can act as an alternate sink for excess carbon and photosynthetic reducing equivalents [62]. High energy dense hydrocarbons are primarily stored as TAGs in microalgae and contained in membrane bound lipid droplets. Lipid droplet size and numbers are regulated in part by the production of lipid droplet proteins which are present in the membranes surrounding lipid droplets. Reductions in the expression of major lipid droplet proteins using RNA silencing techniques has been shown to significantly decrease the size of lipid droplets [63]. However, genetic manipulations to increase TAG accumulation by enhancing lipid droplet protein production to our knowledge has not been reported to date. Overall, genetic manipulation of genes controlling select aspects of source, sink, storage, metabolism, and cell growth rates have all proven to enhance biomass yields. Integration of multiple aspects of carbon metabolism, storage and growth leading to enhanced biomass yields have been achieved by alterations in mastery regulatory genes. But much remains to be characterized to achieve maximum thermodynamic efficiency for conversion of photons to the chemical energy of biomass.
