High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolytica NRRL-143

<p>Abstract</p> <p>Background</p> <p>The 3,4-dihydroxy phenyl L-alanine (L-dopa) is a drug of choice for Parkinson's disease, controlling changes in energy metabolism enzymes of the myocardium following neurogenic injury. <it>Aspergillus oryzae </it>is commonly used for L-dopa production; however, potential improvements in ease of handling, growth rate and environmental impact have led to an interest in exploiting alternative yeasts. The two important elements required for L-dopa production are intracellular tyrosinases (thus pre-grown yeast cells are required for the transformation of L-tyrosine to L-dopa) and L-ascorbate, which acts as a reducing agent.</p> <p>Results</p> <p>Pre-grown cells of <it>Yarrowia lipolytica </it>NRRL-143 were used for the microbiological transformation of L-tyrosine to L-dopa. Different diatomite concentrations (0.5–3.0 mg/ml) were added to the acidic (pH 3.5) reaction mixture. Maximum L-dopa biosynthesis (2.96 mg/ml L-dopa from 2.68 mg/ml L-tyrosine) was obtained when 2.0 mg/ml diatomite was added 15 min after the start of the reaction. After optimizing reaction time (30 min), and yeast cell concentration (2.5 mg/ml), an overall 12.5 fold higher L-dopa production rate was observed when compared to the control. Significant enhancements in Y_p/s, Q_sand q_sover the control were observed.</p> <p>Conclusion</p> <p>Diatomite (2.0 mg/ml) addition 15 min after reaction commencement improved microbiological transformation of L-tyrosine to L-dopa (3.48 mg/ml; p ≤ 0.05) by <it>Y. lipolytica </it>NRRL-143. A 35% higher substrate conversion rate was achieved when compared to the control.</p

A sequence based synteny map between soybean and Arabidopsis thaliana

BACKGROUND: Soybean (Glycine max, L. Merr.) is one of the world's most important crops, however, its complete genomic sequence has yet to be determined. Nonetheless, a large body of sequence information exists, particularly in the form of expressed sequence tags (ESTs). Herein, we report the use of the model organism Arabidopsis thaliana (thale cress) for which the entire genomic sequence is available as a framework to align thousands of short soybean sequences. RESULTS: A series of JAVA-based programs were created that processed and compared 341,619 soybean DNA sequences against A. thaliana chromosomal DNA. A. thaliana DNA was probed for short, exact matches (15 bp) to each soybean sequence, and then checked for the number of additional 7 bp matches in the adjacent 400 bp region. The position of these matches was used to order soybean sequences in relation to the A. thaliana genome. CONCLUSION: Reported associations between soybean sequences and A. thaliana were within a 95% confidence interval of e(-30 )– e(-100). In addition, the clustering of soybean expressed sequence tags (ESTs) based on A. thaliana sequence was accurate enough to identify potential single nucleotide polymorphisms (SNPs) within the soybean sequence clusters. An EST, bacterial artificial chromosome (BAC) end sequence and marker amplicon sequence synteny map of soybean and A. thaliana is presented. In addition, all JAVA programs used to create this map are available upon request and on the WEB

Development of a physical map of the soybean pathogen Fusarium virguliforme based on synteny with Fusarium graminearum genomic DNA

<p>Abstract</p> <p>Background</p> <p>Reference genome sequences within the major taxa can be used to assist the development of genomic tools for related organisms. A major constraint in the use of these sequenced and annotated genomes is divergent evolution. Divergence of organisms from a common ancestor may have occurred millions of years ago, leading to apparently un-related and un-syntenic genomes when sequence alignment is attempted.</p> <p>Results</p> <p>A series of programs were written to prepare 36 Mbp of <it>Fusarium graminearum </it>sequence in 19 scaffolds as a reference genome. Exactly 4,152 Bacterial artificial chromosome (BAC) end sequences from 2,178 large-insert <it>Fusarium virguliforme </it>clones were tested against this sequence. A total of 94 maps of <it>F. graminearum </it>sequence scaffolds, annotated exonic fragments and associated <it>F. virguliforme </it>sequences resulted.</p> <p>Conclusion</p> <p>Developed here was a technique that allowed the comparison of genomes based on small, 15 bp regions of shared identity. The main power of this method lay in its ability to align <it>diverged </it>sequences. This work is unique in that discontinuous sequences were used for the analysis and information not readily apparent, such as match direction, are presented. The 94 maps and JAVA programs are freely available on the Web and by request.</p

Development of a pooled probe method for locating small gene families in a physical map of soybean using stress related paralogues and a BAC minimum tile path

BACKGROUND: Genome analysis of soybean (Glycine max L.) has been complicated by its paleo-autopolyploid nature and conserved homeologous regions. Landmarks of expressed sequence tags (ESTs) located within a minimum tile path (MTP) of contiguous (contig) bacterial artificial chromosome (BAC) clones or radiation hybrid set can identify stress and defense related gene rich regions in the genome. A physical map of about 2,800 contigs and MTPs of 8,064 BAC clones encompass the soybean genome. That genome is being sequenced by whole genome shotgun methods so that reliable estimates of gene family size and gene locations will provide a useful tool for finishing. The aims here were to develop methods to anchor plant defense- and stress-related gene paralogues on the MTP derived from the soybean physical map, to identify gene rich regions and to correlate those with QTL for disease resistance. RESULTS: The probes included 143 ESTs from a root library selected by subtractive hybridization from a multiply disease resistant soybean cultivar 'Forrest' 14 days after inoculation with Fusarium solani f. sp. glycines (F. virguliforme). Another 166 probes were chosen from a root EST library (Gm-r1021) prepared from a non-inoculated soybean cultivar 'Williams 82' based on their homology to the known defense and stress related genes. Twelve and thirteen pooled EST probes were hybridized to high-density colony arrays of MTP BAC clones from the cv. 'Forrest' genome. The EST pools located 613 paralogues for 201 of the 309 probes used (range 1–13 per functional probe). One hundred BAC clones contained more than one kind of paralogue. Many more BACs (246) contained a single paralogue of one of the 201 probes detectable gene families. ESTs were anchored on soybean linkage groups A1, B1, C2, E, D1a+Q, G, I, M, H, and O. CONCLUSION: Estimates of gene family sizes were more similar to those made by Southern hybridization than by bioinformatics inferences from EST collections. When compared to Arabidopsis thaliana there were more 2 and 4 member paralogue families reflecting the diploidized-tetraploid nature of the soybean genome. However there were fewer families with 5 or more genes and the same number of single genes. Therefore the method can identify evolutionary patterns such as massively extensive selective gene loss or rapid divergence to regenerate the unique genes in some families

The Soybean Genome Database (SoyGD): a browser for display of duplicated, polyploid, regions and sequence tagged sites on the integrated physical and genetic maps of Glycine max

Genomes that have been highly conserved following increases in ploidy (by duplication or hybridization) like Glycine max (soybean) present challenges during genome analysis. At the Soybean Genome Database (SoyGD) genome browser has, since 2002, integrated and served the publicly available soybean physical map, bacterial artificial chromosome (BAC) fingerprint database and genetic map associated genomic data. The browser shows both build 3 and build 4 contiguous sets of clones (contigs) of the soybean physical map. Build 4 consisted of 2854 contigs that encompassed 1.05 Gb and 404 high-quality DNA markers that anchored 742 contigs. Many DNA markers anchored sets of 2–8 different contigs. Each contig in the set represented a homologous region of related sequences. GBrowse was adapted to show sets of homologous contigs at all potential anchor points, spread laterally and prevented from overlapping. About 8064 minimum tiling path (MTP2) clones provided 13 473 BAC end sequences (BES) to decorate the physical map. Analyses of BES placed 2111 gene models, 40 marker anchors and 1053 new microsatellite markers on the map. Estimated sequence tag probes from 201 low-copy gene families located 613 paralogs. The genome browser portal showed each data type as a separate track. Tetraploid, octoploid, diploid and homologous regions are shown clearly in relation to an integrated genetic and physical map

A Genome-Wide Analysis of FRT-Like Sequences in the Human Genome

Efficient and precise genome manipulations can be achieved by the Flp/FRT system of site-specific DNA recombination. Applications of this system are limited, however, to cases when target sites for Flp recombinase, FRT sites, are pre-introduced into a genome locale of interest. To expand use of the Flp/FRT system in genome engineering, variants of Flp recombinase can be evolved to recognize pre-existing genomic sequences that resemble FRT and thus can serve as recombination sites. To understand the distribution and sequence properties of genomic FRT-like sites, we performed a genome-wide analysis of FRT-like sites in the human genome using the experimentally-derived parameters. Out of 642,151 identified FRT-like sequences, 581,157 sequences were unique and 12,452 sequences had at least one exact duplicate. Duplicated FRT-like sequences are located mostly within LINE1, but also within LTRs of endogenous retroviruses, Alu repeats and other repetitive DNA sequences. The unique FRT-like sequences were classified based on the number of matches to FRT within the first four proximal bases pairs of the Flp binding elements of FRT and the nature of mismatched base pairs in the same region. The data obtained will be useful for the emerging field of genome engineering