The expression of the apo-lipoyl domain in strains with various vectors.

<p>Indicated strains were cultured in LB medium to OD₆₀₀ = 0.3, and the expression of the apo-lipoyl domain was induced for 3 hours. A dash (-) indicates that neither IPTG nor arabinose was added, while a plus sign (+) indicates IPTG (TM245, YS17 and YS28) or arabinose (YS19) was added for induction. The lipoyl domain was analyzed as described in the Materials and Methods section. The gel assay was conducted using non-denaturing polyacrylamide gel electrophoresis.</p

The pathway of lipoic acid synthesis in <i>E</i>. <i>coli</i>.

<p>The roundish rectangle represents an <i>E</i>. <i>coli</i> cell. Two complementary systems for protein lipoylation in <i>E</i>. <i>coli</i> are shown. Exogenous lipoic acid or octanoic acid is transferred to unlipoylated apo proteins in an ATP-dependent manner by LplA. The second <i>E</i>. <i>coli</i> pathway requires LipB to transfer endogenously synthesized octanoic acid to apo proteins, which then becomes the substrate for sulfur insertion by LipA. The carboxyl group of octanoic acid and lipoic acid is linked to the ε-amino group of a specific lysine residue of the lipoyl domain of the E2 subunit.</p

Overproduction of α-Lipoic Acid by Gene Manipulated <i>Escherichia coli</i>

<div><p>Alpha-lipoic acid (LA) is an important enzyme cofactor widely used by organisms and is also a natural antioxidant for the treatment of pathologies driven by low levels of endogenous antioxidants. In order to establish a safer and more efficient process for LA production, we developed a new biological method for LA synthesis based on the emerging knowledge of lipoic acid biosynthesis. We first cloned the <i>lipD</i> gene, which encodes the lipoyl domain of the E2 subunit of pyruvate dehydrogenase, allowing high levels of LipD production. Plasmids containing genes for the biosynthesis of LA were subsequently constructed utilizing various vectors and promotors to produce high levels of LA. These plasmids were transformed into the <i>Escherichia coli</i> strain BL21. Octanoic acid (OA) was used as the substrate for LA synthesis. One transformant, YS61, which carried <i>lipD</i>, <i>lplA</i>, and <i>lipA</i>, produced LA at levels over 200-fold greater than the wild-type strain, showing that LA could be produced efficiently in <i>E</i>. <i>coli</i> using genetic engineering methods.</p></div

Overproduction of α-Lipoic Acid by Gene Manipulated <i>Escherichia coli</i> - Fig 5

<p>(A) Growth of TM179 in E minimal medium containing 1 μL of purified LA from YS56 (a), 1 μL of the purified LA from BL21 (b), 1 ng/mL of standard LA (c), or without any addition of LA (d). (B) The determination of LA concentration by HPLC. Ten microliters of the extracts from YS56 (a) and BL21 (b) were applied. Red lines were obtained with 7.5 μg of standard LA.</p

Production of apo-, octanoylated- and holo-lipoyl domains in strains with a plasmid carrying <i>lipD</i> and <i>lplA</i>.

<p>Indicated strains were cultured in LB medium and then cultured with OA or LA for 3 hours to produce the respective domains. The domains were analyzed as described in the Materials and Methods section. The gel shift assay was conducted using non-denaturing polyacrylamide gel electrophoresis. AD, apo-lipoyl domain; OD, octanoylated-lipoyl domain; LD, holo-lipoyl domain.</p

Strains and plasmids.

<p>Strains and plasmids.</p

Octanoylated-lipoyl domain production in YS25 and YS47.

<p>(A) The production of the octanoylated-lipoyl domain under different culture conditions. ‘9h’ indicates 10 μM IPTG plus 1 mM OA were added, and cells were cultured for 9 hours; ‘3h/4h’ indicates cells were cultured for 3 hours and then 4 hours in the presence of 1 mM IPTG plus 1 mM OA; ‘3h/5h’ indicates cells were cultured for 3 hours and then 5 hours in the presence of 1 mM IPTG plus 1 mM OA; ‘6h/3h’ indicates cells were cultured for 6 hours and then 3 hours in the presence of 1 mM IPTG plus 1 mM OA; ‘6h/4h’ indicates cells were cultured for 6 hours and then 4 hours in the presence of 1 mM IPTG plus 1 mM OA. LB medium was used. (B) The production of the octanoylated-lipoyl domain in the presence of various concentrations of OA. YS25 and YS47 were cultured for 6 hours in LB medium and then 3 hours in the presence of 1 mM IPTG plus indicated concentrations of OA. Yield of the octanoylated—lipoyl domain were measured as described in Materials and Methods. Data from three independent experiments are expressed as mean ± S. D.</p

Primers.

<p>Primers.</p