Klebsiella pneumoniae HR526, a new isolated 1,3-propanediol (1,3-PD) producer, exhibited great productivity. However, the accumulation of lactate in the late-exponential phase remained an obstacle of 1,3-PD industrial scale production. Hereby, mutants lacking D-lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D-lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed-batch fermentation. In experiments using pure glycerol as feedstock, the 1,3-PD concentrations, conversion, and productivity increased from 95.39 g L(-1), 0.48 and 1.98 g L(-1) h(-1) to 102. 06 g L(-1), 0.52 mol mol(-1) and 2.13 g L(-1) h(-1), respectively. The diol (1,3-PD and 2,3-butanediol) conversion increased from 0.55 mol mol(-1) to a maximum of 0.65 mol mol(-1). Lactate would not accumulate until 1,3-PD exceeded 84 g L(-1), and the final lactate concentration decreased dramatically from more than 40 g L(-1) to <3 g L(-1). Enzymic measurements showed LDH activity decreased by 89-98% during fed-batch fermentation, and other related enzyme activities were not affected. NADH/NAD(+) enhanced more than 50% in the late-exponential phase as the D-lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3-PD industrial production.

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Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.

The glycerol fed-batch fermentation by Klebsiella pneumoniae CGMCC 1.6366 exhibited the sequential synthesis of products, including acetate, 1,3-propanediol (1,3-PD), 2,3-butanediol, ethanol, succinate, and lactate. The dominant flux distribution was shifted from acetate formation to 1,3-PD formation in early- exponential growth phase and then to lactate synthesis in late-exponential growth phase. The underlying physiological mechanism of the above observations has been investigated via the related enzymes, nucleotide, and intermediary metabolites analysis. The carbon flow shift is dictated by the intrinsic physiological state and enzymatic activity regulation. Especially, the internal redox state could serve as a rate-controlling factor for 1,3-PD production. The q(1,3-PD) formation was the combined outcomes of regulations of glycerol dehydratase activity and internal redox balancing. The q(ethanol)/q(acetate) ratios demonstrated the flexible adaptation mechanism of K. pneumoniae preferring ATP generation in early-exponential growth phase. A low PEP to pyruvate ratio corresponded LDH activity increase, leading to lactate accumulation in stationary phase.