Development of biosensors highly responsive to N-acetylneuraminic acid in Bacillus subtilis / 生物工程学报

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.

Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7 / 生物工程学报

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

Strategies and tools for metabolic engineering in Bacillus subtilis / 生物工程学报

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.

Directed evolution of chitinase Chisb and biosynthesis of chitooligosaccharides / 生物工程学报

Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.

Transcriptome analysis and gene function annotation of Bambusa emeiensis shoots based on high-throughput sequencing technology / 生物工程学报

Bambusa emeiensis is one of the preponderant species of sympodial bamboos in Sichuan province of China, and has excellent fiber length and quality as raw materials for papermaking, textile and other industries. In this study, with the application of Illumina HiSeq™ 2000 platform, we analyzed transcriptome in B. emeiensis with different heights of 10, 50, 100 and 150 cm. A total of 69.28 M reads were obtained, and a sum up of 111 137 bands of Unigenes were acquired following de novo stitching, assembly and clustering, among which there were 63 094 bands that had been integrated in the COG, GO, KEGG, Swiss-Prot and Nr databases using annotated methods. These Unigenes not only had general functions, such as transcription and signal transduction, but were also involved in sucrose transport and metabolism, secondary metabolites and cell wall biosynthesis. There was significant difference regarding the expression of cellulose synthase gene in B. emeiensis at different heights, relevant genes were found that might be responsible for the regulation of the growth and development of B. emeiensis as well as the biosynthesis of cellulose and lignin. Our findings could provide some elementary theories for breed improvement of B. emeiensis.

Study on the relationship between degree of venous injury and the indwelling time by injection of chemotherapy drugs with intravenous catheter system / 中国实用护理杂志

Objective To observe the relationship between degree of venous injury and the indwelling time by injection of chemotherapy drugs with intravenous catheter system. Methods Totally 43 New Zealand rabbits were selected as the study sample. Among them, three rabbits were chosen by lot as a blank control, the others were evenly divided into two groups randomly. The veins at the edges of the two ears were taken out as the tested blood vessels for the infusion with intravenous catheter system. The two groups were injected respectively with physiological saline and chemotherapy drugs once daily, and then blocked with heparin salt water. After intraperitoneal anaesthesia, pathological sections were prepared by taking live samples from the ear vein where intravenous catheter system puncturing on the 2nd, 4th, 6th,8th and 10th day of transfusion. And inflammation and thrombosis in the vein had been observed under the light microscope. Results In the same indwelling time, the inflammatory response of the two groups was compared: no difference on the 2nd day; difference was seen on the 4th、6th and 8th day; but again no difference on the 10th day. Thrombosis compared: no difference on the 2nd, 4th day, but difference was seen on the 6th, 8th and 10th day. Conclusions Chemotherapy drugs has a strong irritant on blood vessels.The indwelling time should not be too long, generally advisable 2 days,even if no abnormal response is observed locally by the naked eye.