Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering.

BACKGROUND: Classical directed evolution is a powerful approach for engineering biomolecules with improved or novel functions. However, it traditionally relies on labour- and time-intensive iterative cycles, due in part to the need for multiple molecular biology steps, including DNA transformation, and limited screening throughput. RESULTS: In this study, we present an ultrahigh throughput in vivo continuous directed evolution system with thermosensitive inducible tunability, which is based on error-prone DNA polymerase expression modulated by engineered thermal-responsive repressor cI857, and genomic MutS mutant with temperature-sensitive defect for fixation of mutations in Escherichia coli. We demonstrated the success of the in vivo evolution platform with ß-lactamase as a model, with an approximately 600-fold increase in the targeted mutation rate. Furthermore, the platform was combined with ultrahigh-throughput screening methods and employed to evolve α-amylase and the resveratrol biosynthetic pathway. After iterative rounds of enrichment, a mutant with a 48.3% improvement in α-amylase activity was identified via microfluidic droplet screening. In addition, when coupled with an in vivo biosensor in the resveratrol biosynthetic pathway, a variant with 1.7-fold higher resveratrol production was selected by fluorescence-activated cell sorting. CONCLUSIONS: In this study, thermal-responsive targeted mutagenesis coupled with ultrahigh-throughput screening was developed for the rapid evolution of enzymes and biosynthetic pathways.

Copy number alterations in pancreatic cancer identify recurrent PAK4 amplification.

Pancreatic cancer is one of the most lethal of all cancers. The median survival is six months and less than 5% of those diagnosed survive five years. Recurrent genetic deletions and amplifications in 72 pancreatic adenocarcinomas, the largest sample set analyzed to date for pancreatic cancer, were defined using comparative genomic hybridization The recurrent genetic alterations identified target a number of previously well-characterized genes, as well as regions that contain possible new oncogenes and tumor suppressor genes. We have focused on chromosome 19q13, a region frequently found amplified in pancreatic cancer and demonstrate how boundaries of common regions of mutation can be mapped and how a gene, in this case PAK4 amplified on chromosome19q13, can be functionally validated. We show that although the PAK4 gene is not activated by mutation in cell lines with gene amplification, an oncogenic form of the KRAS2 gene is present in these cells and oncogenic KRAS2 can activate PAK4. In fact in the three samples we identified with PAK4 gene amplification, the KRAS2 gene was activated and genomically amplified. The kinase activity of the PAK4 protein is significantly higher in cells with genomic amplification as compared to cells without amplification. Our study demonstrates the utility of analyzing copy number data in a large set of neoplasms to identify genes involved in cancer. We have generated a useful dataset which will be particularly useful for the pancreatic cancer community as efforts are undertaken to sequence the pancreatic cancer genome.

Alterations in gene expression in rat skin exposed to 56Fe ions and dietary vitamin A acetate.

The purpose of the present work was to examine gene expression patterns in rat skin exposed to a beam of (56)Fe ions, a surrogate for the high-energy, heavy-ion galactic radiation background, as a basis for obtaining a better understanding of the possible mechanism(s) behind the radioprotective activity of vitamin A. A 2 x 4-cm rectangle of dorsal rat skin was exposed to 1.01 GeV/nucleon (56)Fe ions generated by the Alternating Gradient Synchrotron at Brookhaven National Laboratory. Gene expression patterns were monitored in either the presence or absence of a 250-ppm dietary supplement of vitamin A acetate in powdered lab chow. Although vitamin A and other retinoids show anti-carcinogenic activity in several animal models, the underlying changes in gene expression have not been examined extensively. At either 1 or 7 day after irradiation, a 1-cm square of irradiated and control rat skin was excised and analyzed using the Affymetrix rat microarray (RG_U34A) system. Microarray responses were displayed and processed by GeneSpring 7.0 and GOTree software. At 1 day after 3 Gy of (56)Fe-ion irradiation, the expression of 110 genes was significantly up-regulated (P < = 0.05) in comparison to levels in control rat skin, while no genes were altered by the vitamin A acetate supplement alone. Combined with (56)Fe-ion radiation, the vitamin A acetate supplement blocked the expression of 88 (80%) of the 110 genes and eliminated 16 of 18 gene categories that were significantly altered (all increased) by the (56)Fe-ion radiation. Categories with large numbers of genes eliminated by the retinoid included response to stress, 33 genes; response to biotic stimulus, 38 genes; signal transduction, 35 genes; and regulation of cellular/physiological process, 40 genes. Even for immune response and response to biotic stimulus, the only two categories that remained significantly altered in the presence of the vitamin, the combined number of altered genes was reduced from 74 to 13. No significant alterations in gene expression were found at 7 days relative to the numbers in controls. The results indicate that at 1 day dietary vitamin A acetate strongly interfered with (56)Fe-ion-induced gene expression within the broad categories of stimulus- and stress-related genes, implying that the latter gene categories likely play a role in the radioprotective action of the vitamin.

The action of a dietary retinoid on gene expression and cancer induction in electron-irradiated rat skin.

Current models of radiation carcinogenesis generally assume that the DNA is damaged in a variety of ways by the radiation and that subsequent cell divisions contribute to the conversion of the damage to heritable mutations. Cancer may seem complex and intractable, but its complexity provides multiple opportunities for preventive interventions. Mitotic inhibitors are among the strongest cancer preventive agents, not only slowing the growth rate of preneoplasias but also increasing the fidelity of DNA repair processes. Ionizing radiation, including electrons, is a strong inducer of cancer in rat skin, and dietary retinoids have shown potent cancer preventive activity in the same system. A non-toxic dietary dose of retinyl acetate altered gene expression levels 24 hours after electron irradiation of rat skin. Of the 8740 genes on an Affymetrix rat expression array, the radiation significantly (5 fold or higher) altered 188, while the retinoid altered 231, including 16 radiation-altered genes that were reversely altered. While radiation strongly affected the expression of stress response, immune/inflammation and nucleic acid metabolism genes, the retinoid most strongly affected proliferation-related genes, including some significant reversals, such as, keratin 14, retinol binding protein, and calcium binding proteins. These results point to reversal of proliferation-relevant genes as a likely basis for the anti-radiogenic effects of dietary retinyl acetate.