Alternative fates of the acyl aldehyde intermediate produced by Aar.

<p>Acyl-ACP reductase (Aar) converts the end-product of fatty acid biosynthesis, acyl-ACP, into an acyl aldehyde. This intermediate can be converted into alkanes or free fatty acids by the endogenously encoded aldehdye decarbonylase (AD) and AldE enzymes, respectively; or into wax esters by co-expression of alcohol dehydrogenase and wax ester synthase (WS) transgenes. Previously described pathways in <i>S. elongatus</i> are in black; pathways described in this paper are in gray.</p

Overexpression of Aar (encoded by orf1594) leads to increased free fatty acid production in <i>S. elongatus</i> PCC 7942.

<p><b>A.</b> qPCR analysis of <i>aar</i> expression levels. WT and IPTG-inducible Aar strains were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058307#s4" target="_blank">materials and methods</a>, and induced with 1 mM IPTG at t = 0. “I” indicates induction; “U” indicates uninduced. Total cellular RNA was obtained from all samples at 24 hour post-induction. RNA was converted to cDNA and relative <i>aar</i> transcript levels were assessed by quantitative PCR. RNA input for each sample was normalized to <i>rnpB</i>, and <i>aar</i> expression is presented as the fold change in expression compared to endogenous <i>aar</i> from a WT strain. The data is the average of 6 independent experiments; error bars are the standard deviation. <b>B.</b> Growth curves of the indicated strains (log OD₇₅₀ vs. time). The WT strain is the average of 6 independent experiments; Aar_U and Aar_I are the average of 10 independent experiments. Error bars are the standard deviations. <b>C.</b> 0.5 OD₇₅₀-equivalents from samples collected at 24, 48, 72 and 120 hours post-induction were separated by thin layer chromatography (TLC) using polar solvent for the mobile phase. 10 µg of hexadecanol (HD) and 5 µg of palmitic acid (PA; C16∶0 free fatty acid) were included as standards. MGDG: monogalactosyldiacylglycerol; DGDG: digalactosyldiacylglycerol; PG: phosphatidylglycerol. <b>D.</b> Total fatty acid methyl esters (FAMES) assessed by GC. Each timepoint is the average of three measurements; error bars are standard deviations. The numbers above the Aar_I bars indicate the fold-increase of total FAMES compared to WT samples. <b>E.</b> Individual constituent FAMES were quantified. The observed increase in total FAMES is due almost entirely to accumulation of C16∶0 free fatty acids (solid black).</p

orf0489 is required for Aar-induced FFA production and to mitigate Aar toxicity.

<p><b>A.</b> Growth curve of the indicated strains (log OD₇₅₀ vs time). For clarity, only cultures induced with IPTG are shown. There was no significant difference in growth rate in any of the uninduced strains compared to WT. Values are the average +/− SD of three independent experiments. <b>B.</b> Colony forming units per OD₇₅₀ (CFU/OD₇₅₀) of the indicated strains were determined 48 h post-induction. WT is the average of five independent experiments; the remaining strains are the average of three independent experiments. Error bars are the standard deviation. <b>C.</b> 0.5 OD₇₅₀-equivalents from samples collected 24 and 48 hours post-induction were resolved by TLC using polar solvents (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058307#s4" target="_blank">Materials and Methods</a>). PA: palmitic acid. <b>D.</b> Total FAMES 24 and 48 h post-induction of the indicated strains. <b>E.</b> In a separate experiment from A-D, samples from biological triplicates were collected 24 h post-induction, and 1 OD₇₅₀-equivalents were resolved on TLC using non-polar solvents (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058307#s4" target="_blank">Materials and Methods</a>). A C16∶0 fatty aldehyde standard (i.e. hexadecanal) was included between lanes and on the flanking lanes.</p

Kinetic parameters of h₆-AldE from <i>S. elongatus</i> PCC7942¹.

1<p>Results are from three independent experiments and are expressed as average ± standard deviation.</p

Aar-dependent production of wax esters in PCC7942.

<p>Strains containing the WS/DGAT enzyme from <i>A. baylyii</i> (“DGAT”); DGAT and Aar; DGAT, Aar and slr1192 (“triple 1); or DGAT, Aar and ACIAD3612 (“triple 2″) were analyzed 24 hours post-induction by TLC using nonpolar solvents to resolve WE and TAG (<b>A</b>) or polar solvents to resolve fatty acids (<b>B</b>). slr1192 and ACIAD3612 are long-chain aldehyde dehydrogenases from <i>Synechocystis</i> and <i>A. baylyii</i>, respectively. 0.5- OD₇₅₀ equivalents were loaded per well. WE: wax ester; TAG: triacylglycerol; PA: palmitic acid. <b>C.</b> Electron micrographs of the indicated strains 24 hours post-induction. Lipid bodies are present in DGAT-containing samples and appear as white globules.</p

Overexpression of two copies of Aar leads to increased FFA production, but at a growth cost that is alleviated by AldE overexpression.

<p>Strains were constructed containing a single copy of Aar in neutral site 1 (NS1) or neutral site 2 (NS2); two copies of Aar (2X); or two copies of Aar and an additional copy of AldE (“Aar(2X)/AldE”). <b>A.</b> Growth curve of the indicated strains. For clarity only the Aar (2X) and Aar(2X)/AldE strains are presented; Aar(NS1) and Aar(NS2) strains were processed in parallel for FAMES analysis. <b>B.</b> Transcript levels of <i>aar</i> and <i>aldE</i> were assessed by qPCR from samples collected 24 hours post-induction and are presented as the fold increase relative to endogenous expression of each gene from a WT strain. The data are the average of three experiments +/− SD. <b>C.</b> Total FAMES of the indicated strains.</p