Construction of "lock-key" biological living material based on double engineered bacteria and its application on intestinal retention in vivo / 生物工程学报

At present, the research of biological living materials mainly focuses on applications in vitro, such as using a single bacterial strain to produce biofilm and water plastics. However, due to the small volume of a single strain, it is easy to escape when used in vivo, resulting in poor retention. In order to solve this problem, this study used the surface display system (Neae) of Escherichia coli to display SpyTag and SpyCatcher on the surface of two strains, respectively, and constructed a double bacteria "lock-key" type biological living material production system. Through this force, the two strains are cross-linked in situ to form a grid-like aggregate, which can stay in the intestinal tract for a longer time. The in vitro experiment results showed that the two strains would deposit after mixing for several minutes. In addition, confocal imaging and microfluidic platform results further proved the adhesion effect of the dual bacteria system in the flow state. Finally, in order to verify the feasibility of the dual bacteria system in vivo, mice were orally administrated by bacteria A (p15A-Neae-SpyTag/sfGFP) and bacteria B (p15A-Neae-SpyCatcher/mCherry) for three consecutive days, and then intestinal tissues were collected for frozen section staining. The in vivo results showed that the two bacteria system could be more detained in the intestinal tract of mice compared with the non-combined strains, which laid a foundation for further application of biological living materials in vivo.

Construction and optimization of microbial cell factories for producing cis, cis-muconic acid / 生物工程学报

cis, cis-muconic acid (MA) is an important platform chemical. Now, majority of reported engineered strains are genetically instable, the exogenous genes are expressed under the control of expensive inducer and the components of their fermentation medium are complex, thus large-scale microbial production of MA is limited due to the lack of suitable strains. Hence, it is still necessary to construct novel high-performance strain that is genetically stable, no induction and grows in simple inorganic fermentation medium. In this study, after 3 exogenous genes (aroZ, aroY, catA) for biosynthesis of MA were integrated into previously constructed 3-hydroshikimate producing Escherichia coli WJ060 strain and combinatorially regulated with 3 constitutive promoters with different strengths, 27 engineered strains were constructed. The best engineered strain, E. coli MA30 could produce 1.7 g/L MA in the simple inorganic fermentation medium without induction. To further enhance the production capacity of MA, the mutant library of E. coli MA30 was constructed by genome replication engineering and screened via high-throughput assay. After two-round screening, the new strain, E. coli MA30-G2 with improved production of MA was obtained, and the titer of MA increased more than 8%. Under the condition of 5 L fed-batch fermentation, E. coli MA30-G2 could produce about 11.5 g/L MA. Combinatorial regulation and high-throughput screening provide important reference to microbial production of other bio-based chemicals.

Fitness analysis between the L-sorbosone dehydrogenase modules and Ketogulonigenium vulgare chassis / 生物工程学报

Ketogulonigenium vulgare is an acid-producing strain in the process of two-step vitamin C fermentation. L-sorbosone dehydrogenase (SNDH) is one of the key enzymes during the biosynthesis of 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. However, the catalytic mechanism of SNDH is unclear. According to the whole genome sequencing of K. vulgare, two genes encoding sorbosone dehydrogenases, one derived from the chromosome (named as sndhg) and one from plasmid (named as sndhp), were introduced into an industrial strain K. vulgare. The overexpression of gene sndhg had hardly effect on 2-KGA production, and the overexpression of gene sndhp produced an obvious byproduct in the fermentation broth. Combinational expression of sndhg/sndhp with pqqA (obtaining sndhg-pqqA and sndhp-pqqA modules) in K. vulgare resulted in the similar fermentation phenotype to two previous strains. After serial sub-cultivation of co-cultured Bacillus endophyticus with each engineered K. vulgare for 50 d, the conversion rate of 2-KGA increased by 15.4%, 179%, 0.65% and 125% compared with that of the parental K. vulgare with B. endophyticus. This study shows that adaptive evolution of microbial consortium is an effective strategy to increase the fitness between functional modules and chassis, thus quickly getting better strains for production of 2-KGA.

Advances and prospects of taxol biosynthesis by endophytic fungi / 生物工程学报

Taxol is one of the most important chemotherapeutic drugs against cancer. Taxol has been mainly extracted from the bark of yews for a long time. However, methods for the extraction of taxol from the bark of Taxus species were inefficient and environmentally costly. As a result of the high ecological toll exacted on trees with the potential for Pacific yew extinction, investigators began to look for other methods of taxol production. Recently, increasing efforts have been made to develop alternative means of taxol production, such as using complete chemical synthesis, semi-synthesis, Taxus spp. plant cell culture and microbe fermentation. Using microbe fermentation in the production of taxol would be a very prospective method for obtaining a large amount of taxol. Therefore, it is necessary to understand the molecular basis and genetic regulation mechanisms of taxol biosynthesis by endophytic fungi, which may be helpful to construct the genetic engineering strain with high taxol output. In this paper, the taxol biosynthesis pathway from Taxus cells and the advantages of taxol biosynthesis by endophytic fungi were discussed. The study on the isolation and biodiversity of taxol-producing endophytic fungi and the taxol biosynthesis related genes are also discussed.

Construction of an Escherichia coli strain for sensitive detection of arsenite ion in water / 生物工程学报

In order to construct an Escherichia coli strain with high sensitivity and specificity to detect arsenic ion using fluorescence as reporter, a sensitive strain to arsenic ion was obtained by knocking out the gene arsB that acts as an arsenic efflux pump. The pET28b vector containing arsenite detecting cassette Pars-arsR-egfp was constructed and then transformed into arsB deleted mutant. Measuring conditions of this constructed whole-cell biosensor were optimized and its linear concentration range, limit of detection and specificity were determined. This modified biosensor was much more sensitive than that using wild-type strain as host. The optimal detection range of As³⁺ concentration was 0.013 to 42.71 μmol/L, and the limit concentration of detection was as low as 5.13 nmol/L. Thus we successfully improved the sensitivity of arsenite detecting biosensor by modification of E. coli genome, which may provide useful strategies for development and optimization of microbial sensors to detect heavy metals.

Advances in tumor-therapy using genetically modified Salmonella / 生物工程学报

Tumor is a neoplasm formed by the abnormal proliferation of local tissue cells under the effects of different tumorigenic factors. Tumor-therapy has always been a difficult clinical issue, while regular cancer treatments, such as radiotherapy, chemotherapy and surgery, have obvious limitations. Earlier studies have shown that some obligate anaerobes or facultative anaerobes have anti-tumor effects, for example, Salmonella typhymurium as facultative anaerobic bacteria can selectively colonize tumors and inhibit their growth. Besides, Salmonella has many advantages in tumor-therapy. In the past decade or two, many researchers have carried out genetic manipulation to attenuate the virulence of Salmonella, to improve their specificity of tumor colonization and specially to use attenuated Salmonella as carriers to deliver a variety of anti-tumor therapeutic molecules, and these genetically modified Salmonella have shown good anti-tumor effects in many animal experiments. Along with further research of Salmonella-mediated antitumor treatment, applications of genetically modified Salmonella for more effective tumor-therapy are promising. We reviewed the anti-tumor mechanisms of Salmonella, the research progress in tumor-therapy using genetically modified Salmonella, and current problems and possible solutions.

Fetoscópica y fotocoagulación láser en el síndrome de transfusión intergemelar: ¿en qué estadio realizarla? una revisión sistemática de la literatura / Fetoscopic and laser photocoagulation in intergemelar transfusion syndrome: in what stage do it? systematic review of the literature

Objetivo: Realizar una revisión sistemática de la literatura que compare la eficacia del tratamiento con fotocoa-gulación láser entre los estadios tempranos vs tardíos en el Síndrome de transfusión intergemelar, según la cla-sificación de Quintero y col. Estrategia de búsqueda: Se realizó una revisión sistemática de la literatura que se llevó a cabo en las bases de datos MEDLINE, PubMed, EMBASE y Cochrane Library, sin limitar la búsqueda de artículos publicados exclusivamente en idioma inglés; las publicaciones se registraron entre enero de 1990 a mar-zo de 2015, con base en las palabras clave: síndrome de transfusión de gemelo a gemelo, la fotocoagulación con láser endoscópica, embarazo de gemelos monocoriales, placenta vasos comunicantes, vasos comunicantes. Resultados: De 67 estudios, con un total de 32 cumplieron los criterios de inclusión, y una revisión sistemática de la literatura técnica se utilizó para estudiar los 8 ensayos controlados aleatorios con 951 mujeres con embarazo gemelar monocorial y síndrome de transfusión de gemelo a gemelo. La supervivencia observada en el síndrome de transfusión de gemelo a gemelo tratados con cirugía láser fetoscópica fue en general el 67 % para ambos fetos y 85 % para un feto; resultados todavía lejos de ser satisfactorios, porque las tasas de mortalidad varían de 20 % a 48 %, sin definir el escenario en el que se debe realizar el procedimiento. Conclusiones: La fotocoagulación con láser bajo guía fetoscópica parece ser el tratamiento óptimo para estadios II a IV, no dependiendo de la técnica quirúrgica; sin delimitar adecuadamente el mejor estadio para realizar este procedimiento.

Objetive: To conduct a systematic review of the literature to define, as rated by Quintero et al., the best stage for treatment with endoscopic laser photocoagulation syndrome twin-twin transfusion. Results: From 67 studies, a total of 32 studies met our inclusion criteria, and a systematic review of the literature technique was used to study the 8 randomized controlled trials involving 951 women with monochorionic twin pregnancy and twin-twin transfusion syndrome. The survival observed in the syndrome of twin-twin transfusion treated with laser surgery fetoscopic was overall 67 % for both fetuses and 85 % for one fetus; results still far from satisfactory, because mor-tality rates range from 20 % to 48 %, without defining the stage on which to perform the procedure. Conclusions: Laser photocoagulation under fetoscopic guide seems to be the optimal treatment for stages II to IV, does not depending on the surgical technique; without adequately delimiting the best stadium to perform this procedure.

Cultivo de la línea celular HEp-2: doblaje poblacional y coloración con Giemsa Perspectivas para el estudio de la infección con Chlamydia trachomatis / Cultivation of the cell line hep-2: dubbing of population and coloring with Giemsa

Los cultivos celulares se han convertido en herramientas esenciales para la investigación básica. Se aplican en inmunología, virología, biología molecular, ingeniería genética y farmacología, entre otras áreas. Se usan también en procesos industriales farmacéuticos, en técnicas de diagnóstico clínico y para estudio de trasplante de tejidos. En bacteriología, estos cultivos permiten confirmar una infección, evaluar la eficiencia de antimicrobianos, realizar estudios de infectividad, investigar sobre nuevas especies, obtener gran cantidad de microorganismos no cultivables para optimizar técnicas y examinar las relaciones entre la célula huésped y los microorganismos intracelulares (virus, bacterias y parásitos). La línea celular HEp-2 (Human Epidermoid Cancer Cells) es utilizada en estudios de infección con diferentes bacterias, entre ellas Chlamydia trachomatis (CT), con el fin de determinar los mecanismos por los cuales este patógeno sobrevive en la célula huésped. También se emplea para observar la acción de péptidos antimicrobianos y de extractos para combatir la infección causada por dicha bacteria. Para este estudio se realizaron curvas de crecimiento en la línea celular HEp-2 con medios DMEM-F12 y MEM. Se estandarizó, además, la coloración con Giemsa y se calculó el doblaje poblacional con diferentes inóculos para evaluar el desarrollo de la línea celular en cultivo y seleccionar las condiciones óptimas para realizar futuros ensayos de infección con parásitos intracelulares, en particular con CT serovar L2.

Cell cultures have become essential tools for basic research. They are applied in immunology, virology, molecular biology, genetic engineering and pharmacology, among other areas. They are also used in pharmaceutical industrial processes, in techniques of clinical diagnostic, and to study tissue transplantation. In bacteriology, these crops allow us to confirm an infection, assess the efficiency of antimicrobials, carry out studies of infectivity, investigate on new species, obtaining a large number of microorganisms non-arable to optimize techniques, and to examine the relationship between the host cell and intracellular microorganisms (bacteria, viruses, and parasites). The HEp-2 cell line (Human epidermoid cancer cells) is used in studies of infection with different bacteria, including Chlamydia trachomatis (CT), in order to determine the mechanisms by which the pathogen survives in the host cell. It is also used to observe the action of antimicrobial peptides and extracts to combat the infection caused by the bacterium. For this study, growth curves of the HEp-2 cell line were carried out with DMEM-F12 and MEM media. In addition, the staining with Giemsa was standardized, and the population dubbing was calculated with different inocula for assessing the development of the cultured cell line and select the optimal conditions for future tests of infection with intracellular parasites, in particular with CT serovar L2.