Figure 1.
Overview of lipid metabolism in yeast and the enzymes deleted within this work.
Formation of lyso-PA from dihydroxyacetone phosphate (DHAP) is mediated by synthesis and reduction of 1-acyl-DHAP. Compounds in alphabetic order: Acyl-CoA, acyl-coenzymeA; CDP-DAG, cytidinediphosphate-diacylglycerol; DAG, diacylglycerol; DHAP, dihydroxyacetone phosphate; FFA, free fatty acid; Glycerol-3-P, glycerol-3-phosphate; Lyso-PA, lyso-phosphatidic acid; Lyso-PL, lyso-phospholipid; PA, phosphatidic acid; PL, phospholipid; SE, steryl ester; TAG, triacylglycerol. Enzymatic activities in alphabetic order: Are1,2, acyl-CoA:sterol acyltransferase 1 and 2; Dga1, diacylglycerol acyltransferase 1; Faa1,2,3,4 and Fat1, fatty acid activation 1 to 4 and fatty acid transporter 1 (acyl-CoA synthetases); Lro1, phospholipid:diacylglycerol acyltransferase (PDAT); Nte1, phosphatidylcholine phospholipase B; Plb1,2,3, phospholipase B 1 to 3; Tgl1,Yeh1,Yeh2, steryl ester hydrolases; Tgl2,3,4,5, triacylglycerol lipase 2 to 5.
Figure 2.
FFA content in phospholipases B deficient mutant strains.
Presented is a comparison of the total (sum of 14∶0 to 18∶1) FFA in YB526 (ACS deficient) cells and YB526 cells additionally deficient for phospholipases B. The genotypes of the individual mutant strains are indicated in this and the following figures in table format below the bars. Δ is indicating the deletion of the respective gene and several Δ in one column indicate the combined deletion of the respective genes in one strain. The last column, for example, is referring to the eight-fold mutant deficient of all FAA genes and all phospholipases B. The values presented for FFA in this and the following figures include both FFA retained in the cells and secreted to the media. The FA species composition corresponding to these results and those in Figures 3, 5, 6 and 7 are presented in Table S2. Cells were grown to late stationary phase (136 h) in YPR media. Mean values of at least three independent experiments. Error bars correspond to standard deviation.
Figure 3.
Impact of TAG metabolism on FFA homeostasis.
Given are the content of total FFA in YB526 cells and YB526 cells additionally deficient for combinations of the DAG acyltransferases LRO1 and DGA1, TAG lipases and phospholipases B. Cells were grown to late stationary phase (136 h) in YPR media. Mean values of at least three independent experiments. Error bars correspond to standard deviation. Asterisks indicate significantly different values between the reference strain YB526 and the individual mutant strain (P≤0.05).
Figure 4.
Changes in lipid class composition of mutant strains.
Given are the contents of PC (A), PE (B), TAG (C), DAG (D) and SE (E) in YB526 cells and YB526 cells additionally deficient in TAG or SE metabolism. Cells were grown to late stationary phase (136 h) in YPR, lipid classes were separated by TLC and subjected to transmethylation. The resulting FA methyl esters were quantified by GC. Error bars represent the standard deviation in three independent experiments. Asterisks indicate significantly different values between the reference strain YB526 and the individual mutant strain (P≤0.05).
Table 1.
Relative FA composition (in percentage) of specific lipid classes.
Figure 5.
Impact of SE and TAG synthesis and degradation on FFA homeostasis.
Displayed are the total FFA in YB526 cells and YB526 cells additionally deficient for enzymes of SE and TAG synthesis and degradation. Cells were grown to late stationary phase (136 h) in YPR media. Mean values of at least three independent experiments. Error bars correspond to standard deviation. Asterisks indicate significantly different values between the reference strain YB526 and the individual mutant strain (P≤0.05).
Figure 6.
Analysis of specific mutants at the end of exponential phase.
Given are the content of total FFA in YB526 cells and YB526 cells additionally deficient for phospholipases B or enzymes of TAG synthesis and degradation. Cells were grown to the end of exponential phase (35 h) in YPR media. Mean values of three independent experiments. Error bars correspond to standard deviation. Asterisks indicate significantly different values between the reference strain YB526 and the individual mutant strain (P≤0.05).
Figure 7.
Impact of Fat1p on the FFA pool.
Given are the content of total FFA in YB526fat1Δ cells and YB526fat1Δ cells additionally deficient for enzymes of TAG synthesis and degradation. Cells were grown to late stationary phase (136 h) in YPR media. Mean values of three independent experiments. Error bars correspond to standard deviation. Asterisks indicate significantly different values between the strain YB526fat1Δ and the individual mutant strain (P≤0.05).
Table 2.
Yeast strains.