No abstract

Sildenafil improves erectile function by inhibiting the cGMP-catalytic activity of phosphodiesterase type V (PDE5). We used rapid amplification of cDNA Ends-polymerase chain reaction (RACE-PCR) to isolate three PDE5 isoforms from human corpus cavernosum. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis on eight human cavernous tissue samples showed that all samples expressed the PDE5A1 at a lower level than the PDE5A2 isoform. Five samples expressed the PDE5A3 isoform at various levels while the other three did not. Analysis on non-penile tissues showed that all tissues expressed the A1 and A2 isoforms while only those that have substantial amounts of smooth muscle expressed the A3 isoform. Cloning and sequencing of the PDE5A gene showed that the isoform-specific 5 0 -ends of the PDE5 mRNAs are encoded from three alternative first exons arranged in the order of A1 -A3 -A2. Promoter activities were detected upstream from the A1-specific exon and in the intron preceding the A2-specific exon. The upstream PDE5A promoter is expected to direct the expression of all three PDE5 isoforms while the intronic PDE5A2 promoter only the A2 isoform. Both promoters were upregulated by increasing concentrations of either cAMP or cGMP. Several transcription factor AP2 and Sp1-binding sequences identified in the promoters are likely to be the mediators of cAMP=cGMP-responsiveness.

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K(cat)) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5-fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions.

No abstract

Lipases are ubiquitous enzymes of great physiological significance that have been used extensively in multiple industries. Environmental microorganisms are a major source for the discovery of novel lipases with high catalytic efficiency and selectivity. However, current plate-based screening of lipase-producing strains is time consuming, labour intensive and inefficient. In this study, we developed an ultra-high throughput screening pipeline for lipase-producing strains based on fluorescence-activated droplet sorting (FADS) using a compact optical system that could be easily set up in an alignment-free manner. The pipeline includes droplet generation, droplet incubation, picoinjection of the fluorescence probe, and sorting of droplets with a throughput of 2 × 10 drops per h. We applied the pipeline to screen samples collected from different locations, including sediments from a hot spring in Tibet, soils from the Zoige wetland, contaminated soils from an abandoned oilfield, and a Chinese Daqu starter. In total, we obtained 47 lipase-producing bacterial strains belonging to seven genera, including Staphylococcus, Bacillus, Enterobacter, Serratia, Prolinoborus, Acinetobacter, and Leclercia. We believe that this FADS-based pipeline could be extended to screen various enzymes from the environment, and may find wide applications in breeding of industrial microorganisms.

Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs. Previously reported flow cytometry-based screening methods can only screen spores or protoplasts released from mycelium, which do not represent the filamentous stationary phase Streptomyces used in industrial cultivation. Here we show a droplet-based microfluidic platform to facilitate more relevant, reliable and rapid screening of Streptomyces mycelium, and achieved an enrichment ratio of up to 334.2. Using this platform, we rapidly characterized a series of native and heterologous constitutive promoters in Streptomyces lividans 66 in droplets, and efficiently screened out a set of engineered promoter variants with desired strengths from two synthetic promoter libraries. We also successfully screened out several hyperproducers of cellulases from a random S. lividans 66 mutant library, which had 69.2–111.4% greater cellulase production than the wild type. Our method provides a fast, simple, and powerful solution for the industrial engineering and screening of Streptomyces in more industry-relevant conditions.

We use a droplet-microfluidics-based platform to rapidly identify and isolate individual primary cells that secrete desired antibodies. We then retrieve the antibody-encoding sequences and create recombinant antibodies that bind the target protein.

Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and β-galactosidase. Further advancements of flow cytometrybased screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance. (Journal of Biomolecular Screening 2011;16:285-294)