Abstract:Microbe is extremely abundant in nature, and its size has a very wide coverage from nano- to micro-scale making it suitable to be processed at multi-scale level as natural "building blocks" and "chassis cells". Biofabrication based on microbes is an artificial manipulation on microbes to assemble functional materials and devices by using the specific structures and various biological functions of microbes. In the meantime, the novel strategies of biofarication enables us to study the behavioral details of microbes, which will provide new platforms for uncovering the unsolved basic scientific problems of microbes. In this paper, we reviewed the frontier and progress in biofabrication from nano- and micro-scale in microbes that were manipulated as structured "building blocks" or functional "micro/nano robots".

Abstract:a-ketogluratate is one of the key intermediates in the TCA cycle, playing an important role in the connection of carbon and nitrogen metabolism. This article aims at stating recent research progress in the production of a-ketoglutarate by microbial fermentation. First, a large group of microbes have been screened to accumulate a-ketoglutarate including prokaryotes and eukaryotes. Second, physiological characterization of over-accumulation of a-ketoglutarate is caused by thiamine defect and nitrogen starvation. Third, the process of fermentation was controlled and optimized by the manipulation of pH, dissolved oxygen and cofactors. Fourth, many metabolic engineering strategies were also presented for a-ketoglutarate production focusing on regeneration of cofactor and manipulation of the pathway. Last, we discussed the limitation of current progress and proposed the future research needs for microbial production of a-ketoglutarate.

Abstract:Taxol has clinical efficacy on many malignant tumors, thus having a good market prospects. The anti-cancer mechanism of taxol is to arrest the cell cycle of tumor cells, leading to apoptosis. Based on our research over the years, we reviewed the latest developments in signaling pathways, the effects of related genes and proteins on apoptosis during paclitaxel-induced apoptosis process, including the paclitaxel-producing gene in microbial fermentation process, cyclin, telomerase of apoptosis.

Abstract:In this study, we investigated reductive degradation of nitroaromatic antibiotic chloramphenicol to non-effective antibacterial amine product in fed-batch biocatalyzed electrolysis systems (BES) (applied voltage was 0.5 V) under low temperature (12±2 °C). The ohm resistance of the whole BES reactor increased when the phosphate buffer solution concentrations decreased. Efficiencies (ErCAP) of chloramphenicol reduction with biocathode (PBS, 25 mmol/L) in presence of glucose was (86.3±1.69)% within 24 h and sludge fermentation liquor was (74.1±1.44)% within 24 h. While the ErCAP of abiotic cathode under the same condition was only (57.9±1.94)% within 24 h. It suggested that biocathode could be a promising technology for reductive biodegradation of nitroaromatic antibiotics-containing wastewater in areas with relatively low annual mean temperature.

Abstract:Carbonyl reductases catalyze carbonyl compounds to chiral alcohols that are important building blocks in fine chemical industry. To study carbonyl reductase from Pichia pastoris GS115 (ppcr), we discovered a new gene (ppcr) encoding an NADPH-dependent carbonyl reductase by genomic data mining. It was amplified by PCR from the genomic DNA, and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity. The optimum temperature was 37 ○C and the optimum pH of PPCR was 6.0. PPCR was stable below 45 ○C. The Km and kcat value of the enzyme for ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s-1, respectively. The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, α-ketoesters, β-ketoesters and aryl ketones to give the corresponding alcohols with >97% ee with only a few exceptions, showing its application potential in the synthesis of chiral alcohols.

Abstract:Direct secretory expression of active microbial transglutaminase (MTG) using heterologous hosts is a promising strategy, although its production level still needs to be improved for industrial production. Pichia pastoris is one of the most efficient expression systems developed in recent years. In this study, secretory expression of active MTG was successfully achieved by co-expressing the pro sequence and mature MTG genes in P. pastoris. Furthermore, we optimized the copy number of pro/MTG expression cassettes and the fermentation conditions. MTG production level reached 7.3 U/mL in 1-liter fermentor through high density fermentation, providing the feasiblity for industrial scale preparation of MTG.

Abstract:Most current research in the field of adventitious shoot formation is focused on the regulatory function of a single gene. However, a systematic transcriptomic analysis of the early adventitious shoot formation is still lacking. Here, we analyzed the transcriptome profiling of the early adventitious shoot formation in Arabidopsis by RNA-seq high throughput sequencing technology, and identified 2 457 differentially expressed genes. Detailed categorization revealed that these genes were mainly involved in hormone homeostasis or signal transduction, callus and lateral root formation, shoot apical meristem development and photosynthesis. Further pathway enrichment analysis showed that genes involved in phenylalanine metabolism and phenylpropanoid biosynthesis were significantly enriched. Moreover, exogenous phenylalanine could repress adventitious shoot formation, indicating that phenylalanine metabolism and phenylpropanoid biosynthesis might be important for adventitious shoot formation.

Abstract:In order to identify genes involved in floral transition and development of the orchid species, a full-length APETALA1/FRUITFULL-like (AP1/FUL-like) MADS box cDNA was cloned from Cymbidium faberi (C. faberi) sepals and designated as C. faberi APETALA1-like (CfAP11, JQ031272.1). The deduced amino acid sequence of CfAP11 shared 84% homology with a member of the AP1/FUL-like group of MADS box genes (AY927238.1, Dendrobium thyrsiflorum FUL-like MADS box protein 3 mRNA). Phylogenetic analysis shows that CfAP11 belonged to the AP1/FUL transcription factor subfamily. Bioinformatics analysis shows that the deduced protein had a MADS domain and a relatively conservative K region. The secondary structure of CfAP11 mainly consisted of alpha helices (58.97%), and the three-dimensional structure of the protein was similar to that of homologues in Roza hybrida, Oryza sativa and Narcissus tazetta. Real-time quantitative PCR (qRT-PCR) results reveal low levels of its mRNA in roots, lower levels in leaves during reproductive period than vegetative period, and higher levels in pedicels at full-blossom stage than at bud stage. These results suggest that CfAP11 is involved in floral induction and floral development. Additionally, we observed higher levels of CfAP11 expression in pedicels and ovaries than in other tissues during full-blossom stage, which suggests that CfAP11 may also be involved in fruit formation in certain mechanism.

Abstract:In order to investigate the effects of fungal elicitor and macroporous adsorption resin on shikonin accumulation in hairy roots of arnebia euchroma (Royle) Johnst, we used spectrophotometry to determine the total naphthoquinone content of the hairy roots, by adding different volume ratio of Aspergillus niger elicitor, Aspergillus oryzae elicitor, and the macroporous resin into the M-9 liquid medium at different culture time. The results show that the total naphthoquinone content was 2.28 times higher than the control when we added mixed elicitors of Aspergillus niger and Aspergillus oryzae at the ratio of 2.5:50 in the 10th day of hairy roots cultivating. The total naphthoquinone content was 3.71 times higher than that of the control, when we added macroporous adsorption resin NKA-9. Aspergillus niger elicitor exhibited synergistic effect with Aspergillus oryzae elicitor to enhance the naphthoquinone. Also, the total naphthoquinone level was 4.17 times higher than that of the control by adding mixed fungal elicitor and macroporous adsorption resin NKA-9 in the bioreactor. Aspergillus oryzae and mixed elicitor could promote the hairy roots proliferation, and macroporous adsorption resin NKA-9 and mixed elicitor increased the total naphthoquinone content. In summary, the measure developed for Arnebia euchroma (Royle) Johnst hairy roots cultivating in bioreactors may potential for large-scale production of naphthoquinone.

Abstract:Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100×g 2 min and the protoplasts yield was 7×106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, 100×g 2?min and the protoplasts yield was 6×106 cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000×g 2 min and the protoplasts yield was 6×106 cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%?80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.

Abstract:Adeno-associated virus (AAV) has many advantages for gene therapy over other vector systems. However, after the production of recombinant AAV (Raav) vectors, the biological titration of rAAV stocks is still cumbersome. Different investigators used laboratory-specific methods or internal reference standards that may limit preclinical and clinical applications. The inverted terminal repeats (ITR) sequences are the only cis-regulated viral elements required for rAAV packaging and remain within viral vector genomes. ITR is the excellent target sequences for qPCR quantification of rAAV titer. In this study, we developed a novel qPCR strategy to quantify rAAVs’ vector genome titer via targeting the ITR2 or ITR2-CMV element. In conclusion, the method is fast and accurate for the titration of rAAV2-derived vector genomes. It will promote the standardization of rAAV titration in the future.

Abstract:In order to improve ferment efficiency of biocontrol agent Burkholderia pyrrocinia JK-SH007, the fermentation conditions of this strain were optimized. The optimal fermentation conditions were corn steep liquor (13.88?g/L) and glucose (3.37 g/L) by screening test, steepest ascent experiments and response surface analysis. The results showed that the cell density of JK-SH007 (1.18×109 CFU/mL) increased 1.35 times than before, and there was a 28.84% increase in antifungal activity.

Abstract: