To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHA(MCL)) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways. Pseudomonas mendocina NK-01 produces PHA(MCL) from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHA(MCL) biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(a dagger alg), NK-U-2 (a dagger alga dagger galU), NK-U-3(alga dagger galUa dagger rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHA(MCL) production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 +/- A 0.16 to 3.5 +/- A 0.11 g/l) and equal PHA(MCL) (from 0.78 +/- A 0.06 to 0.79 +/- A 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 +/- A 0.16 to 3.55 +/- A 0.23 g/l) and PHA(MCL) (from 0.78 +/- A 0.06 to 1.05 +/- A 0.07 g/l); 3) CDW and PHA(MCL) dramatically decreased in NK-U-3 (1.53 +/- A 0.21 and 0.41 +/- A 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 +/- A 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %. The PHA(MCL) biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.