Sustainable biosynthesis of 1,3-butanediol using crude glycerol derived from biodiesel production by metabolically engineering of Klebsiella pneumoniae GEM167

Most methods used for microbial production of 1,3-butanediol (1,3-BDO) use starch-derived glucose as food resource. Klebsiella pneumoniae can effectively metabolize crude glycerol derived from biodiesel. An artificial PhaA-dependent 1,3-BDO biosynthetic pathway was first constructed and introduced it into K. pneumoniae GEM167. This engineered strain produced 0.35 ± 0.11 g/L 1,3-BDO using pure glycerol after 24 h. Knock-out of genes that functioned in the biosynthesis of major byproducts (lactic acid [ldhA], ethanol [adhE], and 2,3-butanediol [budA]) led to the production of 4.91-fold more 1,3-BDO (1.72 ± 0.14 g/L after 24 h). Our investigation of the effects of agitation speed and pH on 1,3-BDO biosynthesis indicated that maximal production of 2.59 ± 0.20 g/L at 24 h occurred with agitation at 500 rpm and a pH 6.5. Finally, the K. pneumoniae GEM167ΔldhAΔadhEΔbudA/pACYC171-bld-phaA-phaB-yqhD strain was used to produce 1,3-BDO using crude glycerol (3.92 ± 0.23 g/L 1,3-BDO after 30 h). This was slightly more than the amount produced from pure glycerol (3.75 ± 0.16 g/L after 36 h). This is the first study to produce 1,3-BDO using crude glycerol. Our findings contribute to the advancement of glycerol-derived production of 1,3-BDO and provide a better understanding of glycerol metabolism in K. pneumoniae.