No abstract

The sucrose isomerase (SIase) gene from an efficient strain of Erwinia rhapontici NX-5 for isomaltulose hyperproduction was cloned and overexpressed in Escherichia coli. Protein sequence alignment revealed that SIase was a member of the glycoside hydrolase 13 family. The molecular mass of the purified recombinant protein was estimated at 66 kDa by SDS-PAGE. The SIase had an optimal pH and temperature of 5.0 and 30 °C, respectively, with a K (m) of 257 mmol/l and V (max) of 48.09 μmol/l/s for sucrose. To the best of our knowledge, the recombinant SIase has the most acidic optimum pH for isomaltulose synthesis. When the recombinant E. coli (pET22b- palI) cells were used for isomaltulose synthesis, almost complete conversion of sucrose (550 g/l solution) to isomaltulose was achieved in 1.5 h with high isomaltulose yields (87%). The immobilized E. coli cells remained stable for more than 30 days in a "batch"-type enzyme reactor. This indicated that the recombinant SIase could continuously and efficiently produce isomaltulose.

Succinic acid synthesized from glucose shows potential as a bio-based platform chemical. However, the need for a high glucose concentration, and the accompanying low yields, limit its industrial applications. Despite efficient glucose uptake by the phosphotransferase system (PTS), 1 mol of phosphoenolpyruvate is required for each mole of internalized glucose. Therefore, a PTS-defective Corynebacterium glutamicum mutant was constructed to increase phosphoenolpyruvate availability for succinic acid synthesis, resulting in a lower glucose utilization rate and slower growth. The transcriptional regulator iolR was also deleted to enable the PTS-defective mutant to utilize glucose via iolT-mediated glucose transport. Deletion of iolR and overexpression of iolT1 and ppgk (polyphosphate glucokinase) in the PTS-deficient C. glutamicum strain completely restored glucose utilization, increasing production by 11.6% and yield by 32.4% compared with the control. This study revealed for the first time that iolR represses the expression of the two glucokinase genes (glk and ppgk).

Corynebacterium glutamicum wild type lacks the ability to utilize the xylose fractions of lignocellulosic hydrolysates. In the present work, we constructed a xylose metabolic pathway in C. glutamicum by heterologous expression of the xylA and xylB genes coming from Escherichia coli. Dilute-acid hydrolysates of corn cobs containing xylose and glucose were used as a substrate for succinic acid production by recombinant C. glutamicum NC-2. The results indicated that the available activated charcoal pretreatment in dilute-acid hydrolysates of corn cobs could be able to overcome the inhibitory effect in succinic acid production. Succinic acid was shown to be efficiently produced from corn cob hydrolysates (55 g l(-1) xylose and 4 g l(-1) glucose) under oxygen deprivation with addition of sodium carbonate. Succinic acid concentration reached 40.8 g l(-1) with a yield of 0.69 g g(-1) total sugars within 48 h. It was the first report of succinic acid production from corn cob hydrolysates by metabolically engineered C. glutamicum. This study suggested that dilute-acid hydrolysates of corn cobs may be an alternative substrate for the efficient production of succinic acid by C. glutamicum.

In the previous study, we unraveled the unique “erasure strategy” during the mouse spermiogenesis. Chromatin associated proteins sequentially disassociated from the spermatid chromosome, which led to the termination of transcription in elongating spermatids. By this process, a relatively naïve paternal chromatin was generated, which might be essential for the zygotic development. We supposed the regulation of histone acetylation played an important role throughout this “erasure” process. In order to verify this hypothesis, we treated mouse spermatids in vitro by histone acetylase (HAT) inhibitor Curcumin. Our results showed an inhibiting effect of Curcumin on the growth of germ cell line in a dose-dependent manner. Accordingly, the apoptosis of primary haploid spermtids was increased by Curcumin treatment. As expected, the acetylated histone level was downregulated. Furthermore, we found the transcription in spermatids ceased in advance, the dynamics of chromatin associated factors was disturbed by Curcumin treatment. The regulation of histone acetylation should be one of the core reprogramming mechanisms during the spermiogenesis. The reproductive toxicity of Curcumin needs to be thoroughly investigated, which is crucial for its further clinical application.