|Content of Metabolic Engineering of the Content and Fatty Acid Composition of Vegetable Oils
The gateway to fatty acid synthesis is generally considered the plastidial acetyl
coenzyme A carboxylase (ACCase), which converts acetyl-CoA to malonyl-CoA.
In all plants studied other than grasses, the plastidial form of the enzyme involved
in fatty acid synthesis has four dissociable subunits. A biotin carboxylase subunit
first affixes a carboxyl group to the biotin of a second subunit, biotin carboxyl
carrier protein (BCCP), using bicarbonate and ATP as substrates. The resulting
conformational change brings the biotin arm to a carboxyltransferase domain
formed by the remaining two subunits, where the biotin donates the carboxyl
group to acetyl-CoA (Cronan and Waldrop, 2002; Nikolau et al., 2003). Grass
ACCases possess the same activities as the multisubunit form, but combine
them into a multifunctional homodimer that is the primary target of herbicides
targeting weedy grasses (Zagnitko et al., 2001).
ACCase is considered to be the rate-limiting step in fatty acid synthesis. The
multisubunit ACCase is light-activated by reduction of the carboxyltransferase subunits via the thioredoxin pathway, and is subject to feedback inhibition
by oleic acid (Kozaki et al., 2001; Shintani and Ohlrogge, 1995). Although the
β-carboxyltransferase is plastid-encoded while the remaining subunits are
imported to the plastids, all four subunits are normally coordinately expressed
(Ke et al., 2000). Attempts to upregulate fatty acid synthesis by manipulating
individual subunits of the heteromeric ACCase have had mixed results.
Increased biotin carboxylase has little effect, and overexpression of BCCP actually
decreased fatty acid synthesis, perhaps due to incorporation of unbiotinylated
enzyme into ACCase (Shintani et al., 1997; Thelen and Ohlrogge, 2002).
However, Madoka et al. reported that transformation of tobacco with the plastidial
carboxyltransferase subunit raised overall yield of seed oil by increasing
seed production, although oil per seed remains constant (Madoka et al., 2002).
Alternatively, introduction of homomeric ACCase to rapeseed plastids increased
ACCase activity and, to a lesser extent, seed oil (Roesler et al., 1997).
The availability of bicarbonate and particularly of acetyl-CoA for ACCase can
also impact overall fatty acid synthesis. Reduced carbonic anhydrase activity
inhibited fatty acid synthesis in cotton embryos, presumably by decreasing local
bicarbonate supplies (Hoang and Chapman, 2002). The sources of acetyl-CoA for
ACCase probably vary between tissues and stages of development. In castor seed
endosperm, malate generated by a specific phosphoenolpyruvate carboxylase
isoform appears to be the major source of carbon for fatty acids (Blonde and
Plaxton, 2003). In rapeseed embryos, on the other hand, malate does not contribute
significantly; instead, carbon flows primarily from glycolysis, entering the
plastid via transporters for glucose-6-phosphate, dihydroxyacetone phosphate,
and especially phosphoenolpyruvate (Kubis and Rawsthorne, 2000; Schwender
and Ohlrogge, 2002). There is also potential for increasing flow of carbon into seed
oil via alternative sources of acetyl-CoA. For example, introduction of ATP:citrate
lyase from rat into tobacco plastids increased total leaf fatty acids 16%
(Rangasamy and Ratledge, 2000).