Development of an Inosine Hyperproducer from Bacillus licheniformis by Systems Metabolic Engineering.

Inosine is widely used in food, chemical, and medicine. This study developed Bacillus licheniformis into an inosine hyperproducer through systems metabolic engineering. First, purine metabolism was activated by deleting inhibitors PurR and YabJ and overexpressing the pur operon. Then, the 5-phosphoribosyl-1-pyrophosphate (PRPP) supply was increased by optimizing the glucose transport system and pentose phosphate pathway, increasing the inosine titer by 97% and decreasing the titers of byproducts by 36%. Next, to prevent the degradation of inosine, genes deoD and pupG coding purine nucleoside phosphorylase were deleted, accumulating 0.91 g/L inosine in the culture medium. Additionally, the downregulation of adenosine 5'-monophosphate (AMP) synthesis pathway increased the inosine titer by 409%. Importantly, enhancing the glycine and aspartate supply increased the inosine titer by 298%. Finally, the guanosine synthesis pathway was blocked, leading to strain IR-8-2 producing 27.41 g/L inosine with a 0.46 g inosine/g glucose yield and a 0.38 g/(L·h) productivity in a shake flask.

Enhanced propionic acid production from Jerusalem artichoke hydrolysate by immobilized Propionibacterium acidipropionici in a fibrous-bed bioreactor.

Propionic acid is an important chemical that is widely used in the food and chemical industries. To enhance propionic acid production, a fibrous-bed bioreactor (FBB) was constructed and Jerusalem artichoke hydrolysate was used as a low-cost renewable feedstock for immobilized fermentation. Comparison of the kinetics of immobilized-cell fermentation using the FBB with those of fed-batch free-cell fermentation showed that immobilized-cell fermentation gave a much higher propionic acid concentration (68.5 vs. 40.6 g/L), propionic acid yield (0.434 vs. 0.379 g/g) and propionic acid productivity (1.55 vs. 0.190 g/L/h) at pH 6.5. Furthermore, repeated batch fermentation, carried out to evaluate the stability of the FBB system, showed that long-term operation with a high average propionic acid yield of 0.483 g/g, high productivity of 3.69 g/L/h and propionic acid concentration of 26.2 g/L were achieved in all eight repeated batches during fermentation for more than 200 h. It is thus concluded that the FBB culture system can be utilized to realize the economical production of propionic acid from Jerusalem artichoke hydrolysate during long-term operation.

Genome-Scale Mining of Novel Anchor Proteins of Corynebacterium glutamicum.

The display of recombinant proteins on the surfaces of bacteria is a research topic with many possible biotechnology applications-among which, the choice of host cell and anchoring motif is the key for efficient display. Corynebacterium glutamicum is a promising host for surface display due to its natural advantages, while single screening methods and fewer anchor proteins restrict its application. In this study, the subcellular localization (SCL) predictor LocateP and tied-mixture hidden Markov models were used to analyze all five known endogenous anchor proteins of C. glutamicum and test the accuracy of the predictions. Using these two tools, the SCLs of all proteins encoded by the genome of C. glutamicum 13032 were predicted, and 14 potential anchor proteins were screened. Compared with the positive controls NCgl1221 and NCgl1337, three anchoring proteins-NCgl1307, NCgl2775, and NCgl0717-performed better. This study also discussed the applicability of the anchor protein screening method used in this experiment to other bacteria.

Disruption of lactate dehydrogenase through homologous recombination to improve bioethanol production in Thermoanaerobacterium aotearoense.

To enhance ethanol production in Thermoanaerobacterium aotearoense, the lactate dehydrogenase (ldh) gene, which is responsible for lactic acid production in a key branch pathway, was successfully disrupted via homologous recombination. ldh-up and ldh-down were designed and amplified based on JW/SL-YS485-AY 278026, and they were subsequently used as homologous fragments with an inserted erythromycin resistance gene to construct the targeted vector based on pBLUESCRIPT II SK(+). Southern hybridization and PCR-based assay definitely confirmed that the ldh gene in the Δldh mutant was disrupted by the insertion of the erythromycin resistance gene. Compared with the wild type, the Δldh mutant exhibited increases of 31.0% and 31.4% in cell yield under glucose and xylose cultivation, respectively, probably because knocking out the ldh gene results in increased acetate and ATP levels. Knockout of lactate dehydrogenase produced 2.37- and 2.1-fold increases in the yield of ethanol (mole/mole substrate) under glucose and xylose cultivation, respectively. Moreover, no lactic acid was detected in Δldh mutant fermentation mixtures (detection limit of HPLC: 0.5 mM), but lactic acid was readily detected for growth of the wild-type strain on both glucose and xylose, with final concentrations up to 59.24 mM and 56.06 mM, respectively. The success of this process thoroughly demonstrates the methodological possibility of gene knockout through homologous recombination in Thermoanaerobacterium.

High efficiency hydrogen production from glucose/xylose by the ldh-deleted Thermoanaerobacterium strain.

A strictly anaerobic, thermoacidophilic, H(2)-producing bacterium was isolated and designated as Thermoanaerobacterium aotearoense. The optimized cultivation conditions for H(2) production are 55 degrees C, pH 6.5 and 10gl(-1) of glucose or xylose. A metabolic pathway analysis showed that lactate occupied most of the liquid metabolites and consumed a large amount of NADH. To increase the efficiency of hydrogen production, the gene encoding the l-lactate dehydrogenase was knocked out to redirect the NADH flow. Genetic manipulation resulted in the 2 and 2.5 folds increase of the H(2) yield and production rate, respectively. The maximum H(2) yields using the Deltaldh mutant were 2.71, 1.45 and 2.28molH(2)mol(-1) sugar under glucose, xylose and glucose/xylose mixture tests, respectively. The recombinant Deltaldh strain could ferment the mixture of glucose and xylose to produce H(2) effectively, indicating that the performance of Thermoanaerobacterium in H(2) production can be significantly improved by metabolic engineering technique.