Control analysis of lipid biosynthesis in tissue cultures from oil crops shows that flux control is shared between fatty acid synthesis and lipid assembly

Top-Down (Metabolic) Control Analysis (TDCA) was used to examine, quantitatively, lipid biosynthesis in tissue cultures from two commercially important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). A conceptually simplified system was defined comprising two blocks of reactions: fatty acid synthesis (Block A) and lipid assembly (Block B), which produced and consumed, respectively, a common and unique system intermediate, cytosolic acyl-CoA. We manipulated the steady-state levels of the system intermediate by adding exogenous oleic acid and, using two independent assays, measured the effect of the addition on the system fluxes (JA and JB). These were the rate of incorporation of radioactivity: (i) through Block A from [1-14C]acetate into fatty acids and (ii) via Block B from [U-14C]glycerol into complex lipids respectively. The data showed that fatty acid formation (Block A) exerted higher control than lipid assembly (Block B) in both tissues with the following group flux control coefficients (C):(i) Oil palm: ∗CJTLBlkB = 0.64±0.05 and ∗CJTLBlkB = 0.36±0.05(ii) Olive: ∗CJTLBlkB =0.57±0.10 and ∗CJTLBlkB = 0.43±0.10where ∗C indicates the group flux control coefficient over the lipid biosynthesis flux (JTL) and the subscripts BlkA and BlkB refer to defined blocks of the system, Block A and Block B. Nevertheless, because both parts of the lipid biosynthetic pathway exert significant flux control, we suggest strongly that manipulation of single enzyme steps will not affect product yield appreciably. The present study represents the first use of TDCA to examine the overall lipid biosynthetic pathway in any tissue, and its findings are of immediate academic and economic relevance to the yield and nutritional quality of oil crops.

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Use of metabolic control analysis in lactation biology

The Journal of Agricultural Science ◽

10.1017/s002185960800782x ◽

2008 ◽

Vol 146 (3) ◽

pp. 267-273 ◽

Cited By ~ 2

Author(s):

T. C. WRIGHT ◽

J. P. CANT ◽

B. W. MCBRIDE

Keyword(s):

Sensitivity Analysis ◽

Fatty Acid ◽

Metabolic Control ◽

Fatty Acid Synthesis ◽

Acid Synthesis ◽

Metabolic Control Analysis ◽

Control Analysis ◽

Acetyl Coa Carboxylase ◽

Rate Limiting ◽

Control Coefficients

SUMMARYSensitivity analysis is routinely carried out in the evaluation of simulation models to identify the degree to which parameters influence model outputs. This type of sensitivity analysis is much less frequently applied to real systems, but a technique called metabolic control analysis (MCA) was developed in the 1970s for the purpose of experimentally identifying the degree to which individual enzymes in a metabolic pathway influence flux through the pathway. MCA is applied to the results of inhibition, activation or genetic manipulation of enzymatic steps in a biochemical pathway. Flux control coefficients for each enzyme are defined as the fractional change in steady-state flux through the entire pathway for an infinitesimal change in the activity of that one enzyme. The sum of control coefficients in a linear, non-branching pathway is equal to one. It is a common finding in MCA that the control, or sensitivity, is distributed over multiple enzymes and not in a single rate-limiting enzyme. The fundamental principles of MCA are reviewed and an overview of experimental methods to measure control coefficients is provided, with the objective of introducing this approach to the fields of agricultural biochemistry and modelling, where it is little known. The application of MCA to the study of glucose metabolism and fatty acid synthesis in bovine mammary tissue are reviewed. The analyses indicated that mammary hexokinase activity exerts more control than transmembrane transport of glucose over lactose synthesis, and that control of cytosolic fatty acid synthesis is shared between acetyl-CoA carboxylase and fatty acid synthase, contrary to the widely held view that acetyl CoA carboxylase is the rate-limiting enzyme. It is suggested that MCA could be a valuable aid in the integration of proteomic and metabolomic data with metabolic flux measurements to engineer desired changes in the composition of milk from dairy animals.

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Control mechanisms operating for lipid biosynthesis differ in oil-palm (Elaeis guineensis Jacq.) and olive (Olea europaea L.) callus cultures

Biochemical Journal ◽

10.1042/bj20010202 ◽

2002 ◽

Vol 364 (2) ◽

pp. 385-391 ◽

Cited By ~ 23

Author(s):

Umi S. RAMLI ◽

Darren S. BAKER ◽

Patti A. QUANT ◽

John L. HARWOOD

Keyword(s):

Fatty Acid ◽

Oil Palm ◽

Olea Europaea ◽

Elaeis Guineensis ◽

Lipid Synthesis ◽

Control Analysis ◽

Flux Control ◽

Olea Europaea L ◽

Elaeis Guineensis Jacq ◽

Olea Europaéa

As a prelude to detailed flux control analysis of lipid synthesis in plants, we have examined the latter in tissue cultures from two important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). Temperature was used to manipulate the overall rate of lipid formation in order to characterize and validate the system to be used for analysis. With [1-14C]acetate as a precursor, an increase in temperature from 20 to 30°C produced nearly a doubling of total lipid labelling. This increase in total lipids did not change the radioactivity in the intermediate acyl-(acyl carrier protein) or acyl-CoA pools, indicating that metabolism of these pools did not exert any significant constraint for overall synthesis. In contrast, there were some differences in the proportional labelling of fatty acids and of lipid classes at the two temperatures. The higher temperature caused a decrease in polyunsaturated fatty acid labelling and an increase in the proportion of triacylglycerol labelling in both calli. The intermediate diacylglycerol was increased in olive, but not in oil palm. Overall the data indicate the suitability of olive and oil-palm cultures for the study of lipid synthesis and indicate that de novo fatty acid synthesis may exert more flux control than complex lipid assembly. In olive, diacylglycerol acyltransferase may exert significant flux control when lipid synthesis is rapid.

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Use of control analysis to study the regulation of plant lipid biosynthesis

Biochemical Society Transactions ◽

10.1042/bst0301043 ◽

2002 ◽

Vol 30 (6) ◽

pp. 1043-1046 ◽

Cited By ~ 3

Author(s):

U. S. Ramli ◽

D. S. Baker ◽

P. A. Quant ◽

J. L. Harwood

Keyword(s):

Biosynthetic Pathway ◽

Fatty Acid Biosynthesis ◽

Elaeis Guineensis ◽

Lipid Biosynthesis ◽

Control Analysis ◽

Experimental Conditions ◽

Plant Lipid ◽

Oil Crops ◽

First Time ◽

Single Enzyme

Control analysis is a powerful method to quantify the regulation of metabolic pathways. We have applied it to lipid biosynthesis for the first time by using model tissue culture systems from the important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). By the use of top-down control analysis, fatty acid biosynthesis has been shown to exert more control than lipid assembly under different experimental conditions. However, both parts of the lipid biosynthetic pathway are important, so that attempts to alter oil yield by manipulating the activity of a single enzyme step are very unlikely to produce significant increases.

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Fatty acid synthesis in the oil palm (Elaeis guineensis): Incorporation of acetate by tissue slices of the developing fruit

Lipids ◽

10.1007/bf02534189 ◽

1985 ◽

Vol 20 (4) ◽

pp. 205-210 ◽

Cited By ~ 26

Author(s):

Khaik-Cheang Oo ◽

Sau-Keen Teh ◽

Hun-Teik Khor ◽

Augustine S. H. Ong

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Oil Palm ◽

Elaeis Guineensis ◽

Acid Synthesis ◽

Tissue Slices

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Inhibition of rotavirus replication by downregulation of fatty acid synthesis

Journal of General Virology ◽

10.1099/vir.0.050146-0 ◽

2013 ◽

Vol 94 (6) ◽

pp. 1310-1317 ◽

Cited By ~ 26

Author(s):

Eleanor R. Gaunt ◽

Winsome Cheung ◽

James E. Richards ◽

Andrew Lever ◽

Ulrich Desselberger

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Biosynthetic Pathway ◽

Fatty Acid Synthase ◽

Virus Assembly ◽

Acid Synthesis ◽

Viral Rna ◽

Viral Infectivity ◽

Fatty Acid Biosynthetic Pathway ◽

Infected Cells

Recently the recruitment of lipid droplets (LDs) to sites of rotavirus (RV) replication was reported. LDs are polymorphic organelles that store triacylglycerols, cholesterol and cholesterol esters. The neutral fats are derived from palmitoyl-CoA, synthesized via the fatty acid biosynthetic pathway. RV-infected cells were treated with chemical inhibitors of the fatty acid biosynthetic pathway, and the effects on viral replication kinetics were assessed. Treatment with compound C75, an inhibitor of the fatty acid synthase enzyme complex (FASN), reduced RV infectivity 3.2-fold (P = 0.07) and modestly reduced viral RNA synthesis (1.2-fold). Acting earlier in the fatty acid synthesis pathway, TOFA [5-(Tetradecyloxy)-2-furoic acid] inhibits the enzyme acetyl-CoA carboxylase 1 (ACC1). TOFA reduced the infectivity of progeny RV 31-fold and viral RNA production 6-fold. The effect of TOFA on RV infectivity and RNA replication was dose-dependent, and infectivity was reduced by administering TOFA up to 4 h post-infection. Co-treatment of RV-infected cells with C75 and TOFA synergistically reduced viral infectivity. Knockdown by siRNA of FASN and ACC1 produced findings similar to those observed by inhibiting these proteins with the chemical compounds. Inhibition of fatty acid synthesis using a range of approaches uniformly had a more marked impact on viral infectivity than on viral RNA yield, inferring a role for LDs in virus assembly and/or egress. Specific inhibitors of fatty acid metabolism may help pinpoint the critical structural and biochemical features of LDs that are essential for RV replication, and facilitate the development of antiviral therapies.

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