Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage A with S amber mutation (S-RRz) and poly(B-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coli cell, simultaneousl...Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage A with S amber mutation (S-RRz) and poly(B-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coli cell, simultaneously or respectively. Six novel strains containing phbCAB and vgb with or without lytic genes were constructed. Strain VG1 (pTU14), in which vgb, phbCAB and S-RRz could all be successfully expressed, has superior characteristics in cell growth and PHB accumulation, while the results of strains containing vgb and phbCAB without S- RRz were not better than that of strains harbored ph&CAB only. The simultaneous expression of vgb and S- RRz in the recombinant VG1 (pTU14) showed a great potential for low-cost production of PHB.展开更多
The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability harrier against the diffusion of substrates and products. Although common chemical or physical permeahiliza...The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability harrier against the diffusion of substrates and products. Although common chemical or physical permeahilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, as well as impede large scale processing. To increase membrane permeability and cell- bound glutamate decarboxylase （GAD） activity of recombinant Escherichia coil （BL21 （DE3）-pET28a-gadB） cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or suffactants to the culture media. Ampidllin was the most effective at improving cell-bound GAD activity of the BL21 （DE3）-pET28a-gadB, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 pg. ml-1. Using this concentration, the cell hiomass did decrease by 40.58%, but the cell-bound GAD activity of BL21 （DE3）-pET28a-gadB and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment ofBL21 （DE3）-pET28a-gadB cells with 5 tag.ml 1 ampicillin resulted in structural changes to the cell envelope, but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol （PVA）, alginate, and boric acid, the transfor- mation rate of γ-aminobutyric acid （GABA） at the 10th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after 30 days of storage at 4 ℃.展开更多
（R）-2-hydroxy-3-phenylpropionic acid （PIP,） is an ideal antimicrobial compound with broad-spectrum activity against a wide range of Gram-positive bacteria, some Gram-negative bacteria, and fungi. We studied the bi...（R）-2-hydroxy-3-phenylpropionic acid （PIP,） is an ideal antimicrobial compound with broad-spectrum activity against a wide range of Gram-positive bacteria, some Gram-negative bacteria, and fungi. We studied the bioconversion of phenylpyruvate （PPA） to PLA using whole recombinant Escherichia coli cells in a series of buffer/organic solvent systems. Octane was found to be the best organic solvent. The optimum volume ratio of the water phase to the n-octane phase, conversion temperature, substrate concentration, and cell concentration were 6：4, 40 ℃, 12.5 g/L, and 30 g/L wet cells, respectively. Under the optimized conditions, the average PLA productivity in the aqueous/ n-octane system was 30.69% higher than that in the aqueous system, and 32.31 g/L PLA was obtained with the use of a stirred reactor （2-L scale）. Taken together, our findings indicated that PLA biosynthesis was more efficient in an aqueous/n-octane biphasic system than in a monophasic aqueous system. The proposed biphasic system is an effective strategy for enhancing PLA yield and the biosynthesis of its analogues.展开更多
基金Supported by the National Natural Science Foundation of China (No. 29834103, 29876021).
文摘Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage A with S amber mutation (S-RRz) and poly(B-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coli cell, simultaneously or respectively. Six novel strains containing phbCAB and vgb with or without lytic genes were constructed. Strain VG1 (pTU14), in which vgb, phbCAB and S-RRz could all be successfully expressed, has superior characteristics in cell growth and PHB accumulation, while the results of strains containing vgb and phbCAB without S- RRz were not better than that of strains harbored ph&CAB only. The simultaneous expression of vgb and S- RRz in the recombinant VG1 (pTU14) showed a great potential for low-cost production of PHB.
基金Supported by the grants from the National Natural Science Foundation of China(21176220,20876143,31470793)the Natural Science Foundation of Zhejiang Province(Z13B060008)the Key Technology Research and Development Project of Ningbo(2011C11023)
文摘The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability harrier against the diffusion of substrates and products. Although common chemical or physical permeahilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, as well as impede large scale processing. To increase membrane permeability and cell- bound glutamate decarboxylase （GAD） activity of recombinant Escherichia coil （BL21 （DE3）-pET28a-gadB） cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or suffactants to the culture media. Ampidllin was the most effective at improving cell-bound GAD activity of the BL21 （DE3）-pET28a-gadB, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 pg. ml-1. Using this concentration, the cell hiomass did decrease by 40.58%, but the cell-bound GAD activity of BL21 （DE3）-pET28a-gadB and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment ofBL21 （DE3）-pET28a-gadB cells with 5 tag.ml 1 ampicillin resulted in structural changes to the cell envelope, but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol （PVA）, alginate, and boric acid, the transfor- mation rate of γ-aminobutyric acid （GABA） at the 10th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after 30 days of storage at 4 ℃.
基金Project supported by the National Natural Science Foundation of China(Nos.31470092 and 31501459)the Natural Science Youth Foundation of Jiangsu Province(No.BK20130380)China
文摘（R）-2-hydroxy-3-phenylpropionic acid （PIP,） is an ideal antimicrobial compound with broad-spectrum activity against a wide range of Gram-positive bacteria, some Gram-negative bacteria, and fungi. We studied the bioconversion of phenylpyruvate （PPA） to PLA using whole recombinant Escherichia coli cells in a series of buffer/organic solvent systems. Octane was found to be the best organic solvent. The optimum volume ratio of the water phase to the n-octane phase, conversion temperature, substrate concentration, and cell concentration were 6：4, 40 ℃, 12.5 g/L, and 30 g/L wet cells, respectively. Under the optimized conditions, the average PLA productivity in the aqueous/ n-octane system was 30.69% higher than that in the aqueous system, and 32.31 g/L PLA was obtained with the use of a stirred reactor （2-L scale）. Taken together, our findings indicated that PLA biosynthesis was more efficient in an aqueous/n-octane biphasic system than in a monophasic aqueous system. The proposed biphasic system is an effective strategy for enhancing PLA yield and the biosynthesis of its analogues.
