초록
<P><B>Abstract</B></P> <P>An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6–10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (<I>bktB</I>) and thioesterase (<I>ydiI</I>) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C<SUB>6</SUB>–C<SUB>10</SUB> carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the <I>Pseudomonas putida</I> alkane monooxygenase system, encoded by <I>alkBGT</I>, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, <I>chnD</I> and <I>chnE</I> from <I>Acinetobacter</I> sp. strain SE19 were expressed in conjunction with <I>alkBGT</I> to demonstrate the synthesis of the C<SUB>6</SUB>–C<SUB>10</SUB> dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8g/L C<SUB>6</SUB>–C<SUB>10</SUB> ω-hydroxyacids or about 0.5g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.</P> <P><B>Highlights</B></P> <P> <UL> <LI> ω-functionalized products through β-oxidation reversal and ω-oxidation pathways. </LI> <LI> <I>P. putida</I> AlkBGT expression facilitated synthesis of C<SUB>6</SUB>, C<SUB>8</SUB>, and C<SUB>10</SUB> ω-hydroxyacids. </LI> <LI> ~0.8 g/L of C<SUB>6</SUB>, C<SUB>8</SUB>, and C<SUB>10</SUB> ω-hydroxyacids produced in glycerol minimal media. </LI> <LI> Expression of dehydrogenases enabled oxidation of ω-hydroxyacids to diacids. </LI> <LI> ~0.5 g/L of C<SUB>6</SUB>, C<SUB>8</SUB>, and C<SUB>10</SUB> dicarboxylic acids produced in glycerol minimal media. </LI> </UL> </P>