초록
<I>Klebsiella pneumoniae</I>is a promising industrial species, however the lack of genetic engineering tools restricts its applications. Here we developed a lambda (λ) Red recombinase system and antisense RNA technology in<I>K. pneumoniae</I>to reshape glycerol metabolism pathways. We deleted the lactate dehydrogenase gene<I>ldh</I>through RecA-dependent recombination to block lactic acid synthesis. Next, the 1,3-propanediol dehydrogenase gene<I>dhaT</I>was replaced by an aldehyde dehydrogenase gene (<I>aldH</I>from<I>E. coli</I>) to repress 1,3-propanediol (1,3-PDO) synthesis and simultaneously convert 3-hydroxypropionaldehyde (3-HPA) to 3-hydroxypropionic acid (3-HP). Specially, we developed a Red recombinase system in<I>K. pneumoniae</I>, by which the enzymes related to glycerol metabolism were mutated by transformed oligos. One positive strain produced 6.39 g L<SUP>−1</SUP>3-HP and 32.6 g L<SUP>−1</SUP>1,3-PDO at 36 h without using any antibiotics and inducers. Sequencing results showed that the mutation occurred mainly in byproduct pathways. Finally, antisense RNA technique was applied to block the synthesis of lactic acid and acetic acid. We found that the increase of 3-HP was approximately proportional to the decrease of lactic acid and acetic acid, indicating their competition for glycerol carbon flux. Overall these results and approaches developed in this study provide basis for basic research and microbial production of 3-HP, 1,3-PDO and 2,3-butanediol in<I>K. pneumoniae</I>..