Abstract:The background for developing metabolic engineering was reviewed, followed by a discussion on analyzing the driving force for developing metabolic engineering. Twelve papers published in this special section were briefly introduced with the aim to stimulate further developments in this fast evolving field.

Abstract:Metabolic engineering has been developed for nearly 20 years since its beginning on 1990s, and it has significantly promoted the improvement of microbial fermentation industry. This review summarized the technology development and their applications in fermentation industry in each of the three important phases during the development of metabolic engineering. Finally, the key issues for future development and solving strategies were discussed.

Abstract:In the last few decades, with the development of recombinant DNA technology, metabolic engineering has made tremendous advances. Synthetic biology is a newly and rapidly emerging discipline. It has great potential in assisting and simplifying the study of metabolic engineering. This review focuses on the recent development of synthetic biology and its application in optimizing metabolic pathway and engineering cellular chassis.

Abstract:Intracellular metabolic fluxes are important to understand metabolic characteristics of cells and to direct metabolic engineering strategies. Because intracellular fluxes cannot be directly assessed, isotope experiments are usually conducted to trace metabolic fluxes. The flux-ratio analysis can reflect high biochemical veracity, be employed to identify the topology of the networks, and offer greatly reduced computational expense for flux determination. In order to apply this metabolic analysis method to better elucidate more cell systems, we discussed in this study the principles, experiments and assays, data interpretation, and other issues that should be considered in flux ratio determination, metabolic flux quantification and its metabolic engineering applications.

Abstract:Directed evolution, which is also called molecular evolution, or artificial evolution, combines random mutagenesis and directed selection. In previous studies, it has been extensively applied for the improvement of enzyme catalytic properties and stability, as well as the expanding of substrate specificity. In recent years, directed evolution was also employed in metabolic engineering of promoters for improving their strength and function, and the engineering of global transcription machinery. These techniques contribute to breeding more tolerant strains against environmental stress, as well as strains with improved fermentation efficiency. In this article, we reviewed the applications of directed evolution in the metabolic engineering of promoters and global transcription machinery. These techniques enabled fine-tuning of gene expression and simultaneous alternation of multiple gene transcription inside the cells, and thus are powerful new tools for metabolic engineering.

Abstract:Filamentous fungi are important industrial microorganisms. The focus on its metabolic engineering is to optimize the metabolic pathway with gene expression regulation technology to meet with the industrial production needs. Antisense RNA technology due to its simplicity compared with the gene knock-out technology has great perspectives in filamentous fungal metabolic control. It is an efficient method for regulating gene expression and a key tool for metabolic engineering. In this article, we addressed the mechanism of antisense RNA technology and its applications in filamentous fungal metabolic engineering. Additionally, future perspectives were discussed.

Abstract:The pretreatment of raw materials is necessary for ethanol production from lignocellulose, however, a variety of compounds which inhibit the fermenting microorganism such as Saccharomyces cerevisiae are inevitably formed in this bioprocess. Based on their chemical properties, the inhibitors are usually divided into three major groups: weak acids, furaldehydes and phenolic compounds. These compounds negatively affect the growth of S. cerevisiae, ethanol yield and productivity, which is one of the significant hurdles for the development of large-scale ethanol production from lignocellulose. We address here the origins of the three kinds of inhibitors and their mechanisms to S. cerevisiae. We also discuss the strategies of improving the fermentation performance of yeast, including detoxification of the pretreated substrates, enhancement of yeast tolerance and also fermentation control to reduce the effects of the inhibitors. The methods used in enhancing the yeast tolerance are traditional mutagenic breeding integrated with strains evolution under the suitable selective pressure, and metabolic engineering by introducing and/or overexpressing genes encoding enzymes such as furfural reductase, laccase and phenylacrylic acid decarboxylase, that confer the S. cerevisiae strains resistance towards specific inhibitors.

Abstract:In order to save energy consumption for the downstream processes, consecutive very-high-gravity batch fermentation was developed for ethanol production with the self-flocculating yeast Saccharomyces cerevisiae flo. The fermentation system exhibited a high ethanol productivity of 8.2 g/(L·h) with average ethanol concentration around 120 g/L. However, deterioration of the sedimentation performance of yeast flocs was observed as the consecutive fermentation process was prolonged, which significantly extended the time required for yeast flocs to separate from fermentation broth, and exaggerated the inhibition of high ethanol concentration on the yeast flocs, making them quickly lost viability and the fermentation system interrupted after 11 consecutive batches. Experimental results illustrated that decrease of the size of yeast flocs was the main reason, which could be prevented by stimulating the propagation of the yeast flocs. Thus, yeast was purged from the fermentation system at the end of each batch, and the concentration of yeast flocs within the fermentor was maintained at a relatively low level to stimulate their propagation. Although the ethanol productivity was decreased to 4.0 g/(L·h), the size of yeast flocs was stabilized after 10 consecutive batches and maintained for another 14 batches without further decrease, indicating the fermentation system could be operated reliably.

Abstract:Escherichia coli strain DC1515, deficient in glucose phosphotransferase (ptsG), lactate dehydrogenase (ldhA) and pyruvate:formate lyase (pflA), is a promising candidate for the fermentative production of succinate. To further improve the succinate producing capability of DC1515, we constructed plasmid pTrchisA-pyc with heterogenous pyruvate carboxylase (pyc) from Bacillus subtilis 168 under the Trc promoter and introduced it into DC1515. We used lactose as a substitute of IPTG to induce pyc. We optimized the culture conditions such as the lactose addition time, the lactose concentration and the culture temperature after induction for succinate production. We also explored the effect of lactose supplement during the fermentation. The results showed that pyc can be expressed under lactose induction in the fermentative medium with 15 g/L glucose due to the deficient of ptsG in DC1515. Under optimized conditions, the final succinate concentration reached to 15.17 g/L, which was 1.78-fold higher than that of control strain. If complementing lactose twice to the concentration of 1 g/L during the fermentation, the final succinate concentration could further reach to 17.54 g/L. This work might provide valuable information for gene expression in E. coli strains using lactose as inducer for succinate production in a glucose-medium. Due to the reduced cost, E. coli is becoming a more promising strain for succinate production through fermentation.

Abstract:We studied the ability of lpdA gene knockout Escherichia coli to ferment different sugars in mineral salts medium for the production of pyruvate. The sugars studied were glucose, fructose, xylose and mannose at a concentration of 10 g/L. At the same time, effect?of?inoculum?size?on lpdA fermentation with glucose was studied. The strain was able to use all sugars for biomass generation and pyruvate production. The lpdA knockout mutant converted glucose, fructose, xylose and mannose to pyruvate with yields of 0.884 g/g, 0.802 g/g, 0.817 g/g and 0.808 g/L, respectively. The pyruvate accumulation curve coupled with cell growth except for mannose as carbon source. When the inoculation size increased, the rate of glucose consumption, pyruvate accumulation and cell growth increased but lower pyruvate concentration. This study demonstrates that E. coli lpdA mutant has the potential to produce pyruvic acid from xylose and mannose.

Abstract:Metabolic flux analysis is a very powerful tool to understand CO2 fixation and light energy utilization of microalgae during photoautotrophic cultivation. A comprehensive network structure for the autotrophic growth of Synechococcus sp. PCC7942 was proposed, and the carbon and energetic metabolism under different incident light intensity was investigated based on metabolic flux analysis in this paper. These results showed that CO2 fixation was the main energy and reducing potential trap which accounted for 85% and 70% of the total energy and reducing potential consumption respectively. We also found that the cell yield and the maximum cell yield based on ATP synthesis were maintained 2.80 g/mol and 2.97 g/mol respectively under the appointed incident intensity. But the cell yield on absorbed light energy their corresponding energy conversion efficiency were descended with the increasing of incident intensity.

Abstract:The genus Rhodococcus is of considerable interest in recent years, stemming from their diverse applications in biodegradation, bioremediation, biotransformation and biosurfactant. Using Nocardia/Rhodococcus-Escherichia coli shuttle plasmid pNV18.1 as the backbone vector, we tested the driven efficiency of promoters Ptac and PlacZ of E. coli and Pami-1/Pami-2 of R. ruber in host R. rhodochrous ATCC 33278 by overexpression of nitrile hydratase. Results showed that the specific activity of nitrile hydratase per dry cell weight in engineered Rhodococcus strains driven by Ptac, Pami-1, Pami-2 and PlacZ was 7.5, 6.3, 5.3 and 1.8 times of that in the wild, respectively. It indicated that these promoters could be well recognized by RNA polymerase of Rhodococcus. We further expressed the β-galactosidase reporter gene (lacZ) in R. ruber driven by promoter PlacZ. Results indicated that lacZ was an appropriate reporter gene for genetic or metabolic engineering research of Rhodococcus.

Abstract:In this study, we found that when Pseudomonas mendocina NK-01 accumulated intracellular carbon reserve, medium chain length poly (3-hydroxyalkanoates), it also synthesized extracellular saccharides, alginate oligosaccharides. The high carbon nitrogen ratio of culture medium facilitated alginate oligosaccharides production. We analyzed the structure of alginate oligosaccharide by Ultraviolet-Visible Spectrophotometry, Fourier Transform Infrared Spectroscopy, 1H and 13C of Nuclear Magnetic Resonance, and found that it was compounded in line from β-D-mannuronic acids and α-L-gluronic acids via β-(1→4)/ α-(1→4) bonds, which acetylated partly on the 2- and/or 3-hydroxy. In addition, we determined the weight-average molecular weight of alginate oligosaccharides by gel permeation chromatography to be 2054.

Abstract:The potential effects of recombinant shark hepatical stimulator analogue (r-sHSA) in liver disease have been revealed in our previous studies. In order to further evaluate its clinic application, we carried out high cell-density fermentation in 5 L fermentor to get enough products. Based on the trials in shaking flask, we optimized the parameters for 5 L fermentor, including medium composition, medium supplement, inducer concentration and induction time, etc. In detail, the improved LB medium (0.97% glycerol, 0.91% yeast extract, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4·3H2O, 0.062% MgSO4·7H2O, 0.5% NaCl, pH 7.0) is chosen to cultivate the engineering bacteria with the constant fermentation condition (pH 7.0, and the dissolved oxygen concentration is about 25%-30%). When bacterial culture reaches exponential phase, the modified feeding medium (620 g/L glycerol, 94.8 g/L tryptone, 3.3 mL/L trace elements, and 7.5 g/L MgSO4·7H2O) is then supplied through the method of exponential fed-batch mode. After the optical density (OD600) of engineering bacterial culture reaches to 23, the ultimately concentration of 0.5 mmol/L IPTG is added to induce the expression of r-sHSA for 6 h. Results show that the amount of r-sHSA production is (2.662±0.041) g/L, which is about 13.7 folds of the one optimized before.

Abstract:Lipase-mediated biodiesel production becomes increasingly important because of mild reaction conditions, pollution free during the process and easy product separation. Compared with traditional immobilized lipase, whole cell biocatalyst is promising for biodiesel production because it is easy to prepare and has higher enzyme activity recovery. Rhizopus oryzae IFO4697 can be used as the catalyst for biodiesel production. To further study the stability of the whole cell biocatalyst is extremely important for its further application on large scale. This paper focuses on the stability study of Rhizopus oryzae IFO4697 when used for the methanolysis of renewable oils for biodiesel production. The results showed that water content was important for achieving high catalytic activity and good stability of the biocatalyst. The optimum water content was found to be 5%-15%. Both particle size and desiccation methods showed no obvious effect on the stability of the biocatalyst. With GA cross-linking pretreatment, the stability of the biocatalyst could be improved significantly. When Rhizopus oryzae IFO4697 repeatedly used for next batch reaction, direct vacuum filtration was found to be a good way for the maintenance of good stability of the biocatalyst. Under the optimum reaction conditions, the methyl ester yield could keep over 80% during 20 repeated reaction batches.

Abstract:A xylosidase gene, labeled as BH1068 in genome of Bacillus halodurans C-125, was successfully cloned and overexpressed in Escherichia coli JM109. The purified enzyme was thoroughly characterized and its xylosidase function was unambiguously confirmed. It has maximum activities in neutral condition and is stable over a wide range of pH (4.5–9.0). The enzyme has a broad temperature optimal (35°C–45°C) and is quite stable at temperature up to 45°C. The unique pH and temperature profiles of the enzyme should allow a wide range of xylanolytic operational conditions. With high specific activity of 174 mU/mg protein for its artificial substrate (p-nitrophenyl-b-xylose) and low xylose inhibition (inhibitor constant Ki = 300 mmol/L), this enzyme is among the most active and high tolerant bacterial xylosidase to xylose inhibition. Its high synergy with commercial xylanase has been demonstrated with beechwood xylan hydrolysis, achieving a hydrolysis yield of 40%. Its neutral pH optimal and high tolerance to product inhibition complements well with its fungal counterparts that are only optimal at acidic pH and susceptible to xylose inhibition. In conclusion, this enzyme has high potential in the saccharification of xylan and xylan-containing polysaccharides.

Abstract:The preliminary role of calcineurin B-like protein-interacting protein kinases (CIPKs) in stress response is defined but the exact function of OsCIPK10 gene in rice stress response and its expression pattern yet unclear. In this study we explored the possible functions of OsCIPK10 gene by reverse genetics approaches and also revealed its expression pattern by GUS staining. From the preliminary study of this gene we presumed its function to assist plant to resist stress but over-expressed OsCIPK10 rice transgenic lines showed no significant phenotypic differences from the wild type either under high salt or low potassium conditions however the gene knockdown plants using inverted repeat strategy presented meaningful healthy plants compared to wild type under the stress of salt. Further we checked the expression profile under high salt and low potassium conditions in wild type and found that OsCIPK10 decreases under high salt and increases on low potassium conditions. So we speculate that OsCIPK10 is actually going to function in response to high salt and low potassium stress. We also explored the expression pattern of this gene using Gus staining and found that gene expresses in all plant tissues, the only exception observed was its higher expression in the vascular tissues.

Abstract:Filamentation temperature-sensitive H (FtsH) is an ATP-dependent metalloprotease in prokaryotes and eukaryotes. Homology-based analysis was applied to determine 12 ftsH genes in Arabidopsis genome and 9 members in rice genome. Distribution of these ftsH genes on each chromosome displayed a clear preference for some chromosomes such as chromosome 1, 2, 5 of Arabidopsis and chromosome 1, 5 of rice. All 21 FtsH proteins were subcellularly targeted to chloroplast or mitochondria. These members could be phylogenetically assorted as eight groups, of which no ortholog of AtFtsH12 in rice was detected. Paralogs in each group shared similarity higher than 80% and orthologs higher than 70%. This strongly indicated that the members from single group were descended from a common ancestral gene. Four pairs of paralogs, AtftsH1/5, AtftsH2/8, AtftsH7/9 and AtftsH3/10 were found in Arabidopsis genome. However, only two pairs of ftsH paralogs, OsftsH3/8 and OsftsH4/5, resided in rice genome. The highly homologous members in each group performed striking conservation of exon-intron boundaries and preference for the variable residues in function domains. By contrast, there was significant difference in base composition and sequence length of introns. The comparative analysis of the ftsH gene families of Arabidopsis and rice provided the basis for characteristic and function research of ftsH genes in other plants.

Abstract:We constructed a His-tagged prokaryotic expression vector of WUSCHEL gene of Arabidopsis thaliana, pET-31b(+)-WUS-His(6). The induction condition of the fusion protein expression in Escherichia coli was optimized. After purified by affinity chromatography, the recombinant WUS protein was resolved by renaturation of gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The rabbit anti-WUS antibody titer and specificity were analyzed and confirmed by agarose immunodiffusion testing; the antiserum sensitivity was assayed by dot blot and Western blotting. The results showed that the A. thaliana WUS prokaryotic expression vector was successfully constructed, and the optimized protein expression induction condition in E. coli was 0.5 mmol/L IPTG (isopropy-β-D-thiogalactoside) at 28°C for 10 hours. The purity of the affinity purified protein was higher than 96%, and the prepared polyclonal antibody was with high specificity and sensitivity, it was able to detect protein antigen at ng level.

Abstract:We studied the effect of photodynamic therapy with phycobiliproteins on human liver cancer cells in vitro. With 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay), we used two phycobiliproteins, R-phycoerythrin (R-PE) and C-phycocyanin (C-PC) prepared from Porphyra yezoensis, to determine the killing rates and apoptosis rates of human liver cancer cells (SMMC-7721) mediated by laser. When the concentration of R-PE was 120 mg/L, the survival rate of human liver cancer cells was 27% after treated by Argon laser with 100 J/cm2 doses, while the survival rate in the control group (without adding R-PE) was 65%. When the C-PC concentration was 120 mg/L, the survival cell rate was 47% after treated by He-Ne laser with 35 J/cm2 dose, while the survival rate in the control group (without adding C-PC) was 70%. After handled only with these two kinds of phycobiliproteins for 72 h, the growth of cancer cells presented significant inhibition. The maximal inhibition rates reached up to 31% with R-PE (120 mg/L concentration) and 27% with C-PC (250 mg/L concentration) respectively. After irradiated by laser for 8 h, the maximal cell apoptosis rates were 31.54% with R-PE and 32.54% with C-PC, respectively. It indicated that R-PE and C-PC extracted from Porphyra yezoensis could develop to new photosensitizers for cancer photodynamic therapy.

Abstract:To optimize program of bovine somatic nuclear transfer, we used two different enucleation procedures (by Spindle-view system & Hoechst 33342 staining), two different procedures to introduce donor nuclei (by ooplasm microinjection & electrofusion), and three different group electrofusion parameters (group 1: 1.9 kV/cm, 10 μs, two; group 2: 1.5 kV/cm, 25 ms, two; group 3: 0.6 kV/cm, 100 ms, one) to reconstruct bovine cloned embryos. The cleavation rates and blastocyst development rates of cloned embryos were used to assess the efficiency of different operational procedure. Finally, the best combination of operational procedure, that the spindle-viewer system was used for oocytes enucleating, and donor cell was electrofused into ooplasm by electrical pulse (1.9 kV/cm, 10 ms, two) to reconstruct bovine cloned embryos. Then the excellent blastocysts were transferred to fosters for producing cloned cattle 80 high-quality cloned blastocysts were transferred into 33 fosters, two cloned calves were produced. According to the results, the optimized program could be used to produce cloned cattle.