Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene

Lycopene is a terpenoid pigment that has diverse applications in the fields of food and medicine. Metabolic engineering in microbial hosts has shown that mevalonate kinase (MK, EC2.7.1.366) is one of the rate-limiting enzymes in the lycopene synthetic pathway. In this study, a directed evolution strategy in Escherichia coli was used to optimize the activity of Saccharomyces cerevisiae MK. Using three rounds of error-prone PCR; screening the development of a lycopene-dependent color reaction; and combinatorial site-specific saturation mutagenesis, three activity-enhancing mutations were identified: V13D, S148I, and V301E. V13D was near the MK catalytic center, in the β-sheet that forms a salt-bridge with nearby Arg-248. S148I was located in the α-helix lid and improved the stability of the α-helix. V301E may increase MK folding by influencing its secondary structure. The K_{m (RS)-mevalonate} of purified mutant MK decreased by 74% compared with the K_{m (RS)-mevalonate} of the wild-type MK, and the K_{cat (RS)-mevalonate} was improved by 26% compared with wild type. Fermentation experiments revealed that lycopene production of the mutant MK increased 2.4-fold compared with wild-type MK.

Lycopene is a terpenoid pigment that has diverse applications in the fields of food and medicine.     

保加利亚乳酸杆菌分解多种尿毒素的定向诱变

目的 选育一株能分解多种尿毒素的益生菌为慢性肾衰的肠道细菌疗法提供新菌种.方法 原始保加利亚乳酸杆菌先经尿毒症患者血清诱导培养,后用物理（紫外线）和化学（硫酸二乙酯）双重多次诱变方法对其进行诱变,以肌酐分解率为观察指标,筛选出具有高效分解肌酐的乳酸菌,测定其遗传稳定性并观察其对尿素氮、尿酸、血磷、甲状旁腺素、同型半胱氨酸的分解能力.结果 从大量突变株中选育出一株高效分解尿毒素的菌株DUC3-17,其肌酐、尿素氦、尿酸、血磷、甲状旁腺素、同型半胱氨酸的分解率为17.23％,36.02％,9.84％,15.73％,78.26％,12.69％.经5次传代其分解力仍稳定.原始菌株不具有分解上述毒素的能力.结论 运用尿毒症患者血清定向诱导后经硫酸二乙酯-紫外线复合诱变的方法,成功获得一株高效分解多种尿毒素的保加利亚乳酸工程菌,其尿毒素分解力能够稳定遗传.     

Directed evolution of retroviruses activatable by tumour-associated matrix metalloproteases

Protease-activatable retroviral vectors offer the possibility of targeted gene transfer into cancer cells expressing a unique set of proteases as, for example, the matrix metalloproteases (MMPs). However, it is difficult to predict which substrate sequence will be optimally cleaved by a given tumour cell type. Therefore, we developed a novel approach that allows the selection of MMP-activatable retroviruses from libraries of viruses displaying combinatorially diversified protease substrates. Starting from a virus harbouring a standard MMP-2 substrate motif, after only two consecutive cycles of diversification and in vivo selection, MMP-activatable viruses were recovered. Biochemical characterization of the selected viruses revealed that their linker peptides showed a considerably increased sensitivity for MMP-2 cleavage, and interestingly also improved the particle incorporation rate of the Env protein. Owing to the optimized linker peptide, the selected viruses exhibited a greatly enhanced spreading efficiency through human fibrosarcoma cells, while having retained the dependency on MMP activation. Moreover, cell entry efficiency and virus titres were considerably improved as compared to the parental virus displaying the standard MMP-2 substrate. The results presented imply that retroviral protease substrate libraries allow the definition of MMP substrate specificities under in vivo conditions as well as the generation of optimally adapted tumour-specific viruses.     

Weak mutators can drive the evolution of fluoroquinolone resistance in Escherichia coli

Weak mutators are common among clinical isolates of Escherichia coli. We show that the relative mutation rate and the "evolvability of fluoroquinolone resistance" are related by a power law slope of 1.2 over 3 orders of magnitude. Thus, even weak mutators can drive the evolution of fluoroquinolone resistance under selection pressure.     

Correction: Photodynamic antimicrobial chemotherapy with cationic phthalocyanines against Escherichia coli planktonic and biofilm cultures

Correction for ‘Photodynamic antimicrobial chemotherapy with cationic phthalocyanines against Escherichia coli planktonic and biofilm cultures’ by Min Li et al., RSC Adv., 2017, 7, 40734–40744, https://doi.org/10.1039/C7RA06073D.

The authors regret that incorrect versions of Fig. 7F (Control) and Fig. 8A (Light-alone) were included in the original article. The corrected versions are shown below. The correction does not change any results or conclusions of the original paper.Open in a separate windowFig. 7Membrane integrity detected by PI staining. (F) Images taken by fluorescence microscope of E. coli treated with 5 μM ZnPc2 in different groups.Open in a separate windowFig. 8SEM images of PACT-subjected E. coli biofilms. (A) Images of E. coli treated with 20 μM ZnPc1-PACT in different groups.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

人线粒体肌酸激酶广泛型亚型在大肠埃希菌中的表达及抗体制备

目的　获得人线粒体肌酸激酶广泛型亚型 (uMtCK)的全长基因 ,在大肠埃希菌中表达 ,研制抗uMtCK多克隆抗体。方法　采用逆转录PCR从培养的肿瘤细胞 (Hela细胞 )中成功扩增出 10 6 2bp的uMtCK的基因片段 ,插入到质粒pMD18 T中 ,经酶切和测序证实为uMtCK的基因编码序列 ,再插入到质粒pQE30 ,转化大肠埃希菌 ,诱导重组质粒pQE30 uMtCK表达酶融合蛋白。经亲和层析纯化 ,并进行SDS PAGE、westernblot鉴定及酶活性测定。纯化的uMtCK蛋白免疫家兔制备抗uMtCK多抗。结果　RT PCR扩增出的片段经酶切鉴定和测序证实为uMtCK的基因 ;在大肠埃希菌M15中实现了高效、可溶性的融合表达 ,表达量约 17%。重组uMtCK具有CK样催化活性 ,表明原核表达的uMtCK具有类似于天然蛋白质的生物学活性。经免疫印迹分析 ,制备的抗uMtCK多抗适合作uMtCK的进一步分析之用。结论　人uMtCK在大肠埃希菌中实现了高效表达 ,制备了针对uMtCK的抗体。     

Escherichia coli: rapid identification by chromogenic tests.

A system has been assessed for the identification of Esch. coli using a rapid triple chromogenic test which relies on the ability of the organism to produce a beta-galactosidase, a beta-glucuronidase, and indole. Coliforms which had been fully identified were tested by this system. Of 512 non-Esch. coli strains there were no false positives, whereas of 514 Esch. coli strains 486 (94.5%) were found to give positive results. Two hundred and twenty-one coliforms that had been isolated from blood cultures were also tested using the colistrip in advance of, or without knowledge of the API 20E result. The test was found to be 100% specific and 94% sensitive for the 105 Esch. coli strains. The test was rapid, simple to perform and economical.     

Directed evolution of novel adeno-associated viruses for therapeutic gene delivery

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution-the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest-offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties.     

Uptake of minocycline by Escherichia coli.

Uptake of tetracyclines into Escherichia coli was assessed with a strain carrying a tetA-lacZ translational fusion, in which expression of the enzyme is controlled by the pSC101 tetR repressor gene, by examining beta-galactosidase induction. The ability of tetracycline analogues to induce beta-galactosidase synthesis was correlated with their hydrophobicity, such that hydrophobic analogues were poor enzyme inducers. Treatment of E. coli with polymyxin B nonapeptide (PMBN) rendered cells more permeable to minocycline, but not to tetracycline.     

Inhibition of Escherichia coli by thioglycerol.

Thioglycerol inhibits the growth of various Escherichia coli strains and other microorganisms, both gram positive and gram negative. The susceptibility of organisms varies. The bactericidal action of this substance is not continuous and stops after an initial burst. At subbactericidal concentrations synthesis of ribonucleic acid is the most strongly affected. This is not due to interference with nucleoside biosynthesis or to direct inhibition of ribonucleic acid polymerase by thioglycerol.     

Inhibition of Transpeptidase Activity in Escherichia coli by Thienamycin

Thienamycin was shown to be a more potent inhibitor than ampicillin of the enzyme peptidoglycan transpeptidase from Escherichia coli.     

Microbial Degradation of Cephalothin by Cephalothin-Susceptible Escherichia coli

Cephalothin (CET)-susceptible Escherichia coli, which can degrade CET after prolonged incubation in broth containing a concentration of the drug greater than the minimum inhibitory concentration, was found in a clinical specimen. The substrate specificity of the partially purified enzyme to cephalosporin analogs strongly indicated the occurrence of CET-specific degradation. Nuclear magnetic resonance analysis of the degradation reaction demonstrated the appearance of two new signals attributed to deacetyl CET. This suggests the possibility of the presence of acylesterase.     

Morphological Changes of Escherichia coli Induced by Bicyclomycin

Electron microscopic studies with Escherichia coli revealed that bicyclomycin inhibits septum formation and converts the cells to filamentous forms. The antibiotic induced high undulation and numerous blebs of the outer membrane. Sometimes cytoplasmic contents leaked into the lumen of the bleb through a disrupted region of the membrane. Breakage of the outer membrane or blebs led to cell lysis. Electron-dense masses of amorphous material and vesicles were found in the cytoplasm.     

Directed HIV-1 evolution of protease inhibitor resistance by second-generation short hairpin RNAs

Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PI-resistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches.