Expression of fungal biosynthetic gene clusters in S. cerevisiae for natural product discovery

Fungi are well known for production of antibiotics and other bioactive secondary metabolites, that can be served as pharmaceuticals, therapeutic agents and industrially useful compounds. However, compared with the characterization of prokaryotic biosynthetic gene clusters (BGCs), less attention has been paid to evaluate fungal BGCs. This is partially because heterologous expression of eukaryotic gene constructs often requires replacement of original promoters and terminators, as well as removal of intron sequences, and this substantially slow down the workflow in natural product discovery. It is therefore of interest to investigate the possibility and effectiveness of heterologous expression and library screening of intact BGCs without refactoring in industrial friendly microbial cell factories, such as the yeast Saccharomyces cerevisiae. Here, we discuss the importance of developing new research directions on library screening of fungal BGCs in yeast without refactoring, followed by outlooking prominent opportunities and challenges for future advancement

Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters

Since the discovery of penicillin, natural products and their derivatives have been a valuable resource for drug discovery. With recent development of genome mining approaches in the post-genome era, a great number of natural product biosynthetic gene clusters (BGCs) have been identified and these can potentially be exploited for the discovery of novel natural products that can find application as pharmaceuticals. Since many BGCs are silent or do not express in native hosts under laboratory conditions, heterologous expression of BGCs in genetically tractable hosts becomes an attractive route to activate these BGCs to discover the corresponding products. Here, we highlight recent achievements in cloning and discovery of natural product biosynthetic pathways via intact BGC capturing, and discuss the prospects of high-throughput and multiplexed cloning of rational-designed gene clusters in the future

Characterization of cross-species transcription and splicing from Penicillium to Saccharomyces cerevisiae

Heterologous expression of eukaryotic gene clusters in yeast has been widely used for producing high-value chemicals and bioactive secondary metabolites. However, eukaryotic transcription cis-elements are still undercharacterized, and the cross-species expression mechanism remains poorly understood. Here we used the whole expression unit (including original promoter, terminator, and open reading frame with introns) of orotidine 5\u27-monophosphate decarboxylases from 14 Penicillium species as a showcase, and analyzed their cross-species expression in Saccharomyces cerevisiae. We found that pyrG promoters from the Penicillium species could drive URA3 expression in yeast, and that inefficient cross-species splicing of Penicillium introns might result in weak cross-species expression. Thus, this study demonstrates cross-species expression from Penicillium to yeast, and sheds light on the opportunities and challenges of cross-species expression of fungi expression units and gene clusters in yeast without refactoring for novel natural product discovery

An Experimental Investigation of the Scaling of Columnar Joints

Columnar jointing is a fracture pattern common in igneous rocks in which cracks self-organize into a roughly hexagonal arrangement, leaving behind an ordered colonnade. We report observations of columnar jointing in a laboratory analog system, desiccated corn starch slurries. Using measurements of moisture density, evaporation rates, and fracture advance rates as evidence, we suggest an advective-diffusive system is responsible for the rough scaling behavior of columnar joints. This theory explains the order of magnitude difference in scales between jointing in lavas and in starches. We investigated the scaling of average columnar cross-sectional areas due to the evaporation rate, the analog of the cooling rate of igneous columnar joints. We measured column areas in experiments where the evaporation rate depended on lamp height and time, in experiments where the evaporation rate was fixed using feedback methods, and in experiments where gelatin was added to vary the rheology of the starch. Our results suggest that the column area at a particular depth is related to both the current conditions, and hysteretically to the geometry of the pattern at previous depths. We argue that there exists a range of stable column scales allowed for any particular evaporation rate.Comment: 12 pages, 11 figures, for supporting online movies, go to http://www.physics.utoronto.ca/nonlinear/movies/starch_movies.htm

Aggregate size distributions in migration driven growth models

The kinetics of aggregate growth through reversible migrations between any two aggregates is studied. We propose a simple model with the symmetrical migration rate kernel $K(k;j)\propto (kj)^\upsilon$ at which the monomers migrate from the aggregates of size k to those of size j. The results show that for the $\upsilon \leq 3/2$ case, the aggregate size distribution approaches a conventional scaling form; moreover, the typical aggregate size grows as $t^{1 / (3 - 2\upsilon )}$ in the $\upsilon < 3/2$ case and as $\exp(C_1 t)$ in the $\upsilon = 3/2$ case. We also investigate another simple model with the asymmetrical rate kernel $K(k;j)\propto k^\mu j^\nu$ ($\mu \neq \nu$), which exhibits some scaling properties quite different from the symmetrical one. The aggregate size distribution satisfies the conventional scaling form only in the case of $\mu < \nu$ and $\mu+\nu<2$, and the typical aggregate size grows as $t^{2-\mu-\nu}$

Layer-correlated motion estimation and motion vector coding for 3D wavelet video coding

This paper proposes an efficient layer-correlated scheme to reduce the bit cost for motion vectors in the 3D wavelet coding. Previous works show that incorporating motion alignment into the lifting structure enables the 3D wavelet coding to provide a competitive performance to the state-of-the-art JVT standard. In general, the temporal wavelet decomposition consists of multiple layers, while each layer adopts one set of motion vectors to achieve high coding efficiency and temporal scalability. Since the current schemes code these MVs independently, this greatly increases the bit cost for coding MV. In order to reduce the motion cost, the proposed scheme performs motion estimation considering the MV correlation among layers. Several modes are proposed to describe the different local correlations at the macroblock level. By an R-D optimized mode selection engine, the proposed scheme can save up to 33 % bits of MVs at the similar texture quality. 1

Advanced motion threading for 3D wavelet video coding

This paper presents an advanced motion threading technique for improved performance in 3D wavelet coding. First, we extend an original motion threading idea of ours to a lifting- based implementation. Methods for enabling fractional-pixel alignment in motion threading and for processing many-to-one pixel mapping and non-referred pixels are proposed to reduce the wavelet boundary effects. Second, we devise an advanced motion threading technique, in which one set of motion vectors is generated for each temporal layer of wavelet coefficients for temporal scalability. In order to reduce the motion overhead information, especially at lowbit rates, several correlated motion prediction modes at the macroblock level are defined to exploit the intra/inter layer correlation in motion vector coding. Finally, rate-distortion optimization is utilized in motion estimation to select the best motion prediction mode for each macroblock. With the newmotion threading technique, we are able to achieve 1.5–6.0 dB gain in average PSNR in 3D wavelet coding over our previous implementation of motion threading

Header for SPIE use Advanced Lifting-Based Motion-Threading (MTh) Technique for the 3D Wavelet Video Coding

This paper proposes an advanced motion-threading technique to improve the coding efficiency of the 3D wavelet coding. We extend the original motion-threading technique to the lifting wavelet structure. This extension solves the artificial motion thread truncation problem in long support temporal wavelet filtering, and enables the accuracy of motion alignment to be fractional-pixel with guaranteed perfect reconstruction. Furthermore, the mismatch problem in the motion-threading caused by occlusion or scene-change is considered. In general, the temporal wavelet decomposition consists of multiple layers. Unlike the original motion-threading scheme, in the proposed scheme each layer owns one set of motion vectors so as to achieve both high coding efficiency and temporal scalability. To reduce the motion cost, direct mode is used to exploit the motion vector correlation. An R-D optimized technique is introduced to estimate motion vectors and select proper prediction modes for each macroblock. The proposed advanced motion-threading scheme can outperform the original motionthreading scheme up to 1.5~5.0 dB. The experimental results also demonstrate that the 3D wavelet coding scheme can be competitive with the start-of-the-art JVT video standard on coding efficiency

Motion Compensated Lifting Wavelet And Its Application In Video Coding

A motion compensated lifting (MCLIFT) framework is proposed for the 3D wavelet video coder. By using bi-directional motion compensation in each lifting step of the temporal direction, the video frames are effectively de-correlated. With proper entropy coding and bitstream packaging schemes, the MCLIFT wavelet video coder can be scalable in frame rate and quality level. Experimental results show that the MCLIFT video coder outperforms the 3D wavelet video coder with the same entropy coding scheme by an average of 1.1-1.6dB, and outperforms MPEG-4 coder by an average of 0.9-1.4dB