Elizabeth Jane Ashforth

Novel lipolytic genes from the microbial metagenomic library of the South China Sea marine sediment

Metagenomic cloning is a powerful tool for the discovery of novel genes and biocatalysts from environmental microorganisms. Based on activity screening of a marine sediment microbial metagenomic library, a total of 19 fosmid clones showing lipolytic activity were identified. After subcloning, 15 different lipolytic genes were obtained; their encoded proteins showed 32-68% amino acid identity with proteins in the database. Multiple sequence alignment and phylogenetic tree analysis demonstrated that most of these predicted proteins are new members of known families of bacterial lipolytic enzymes. However, two proteins, FLS18C and FLS18D, could not be assigned to any known family, thus probably representing a novel family of the bacterial lipolytic enzyme. The activity assay results indicated that most of these lipolytic enzymes showed optimum temperature for hydrolysis at 40-50 degrees C with p-nitrophenol butyrate as a substrate. The lipolytic gene fls18D was overexpressed, and the resulting protein FLS18D was characterized as an alkaline esterase. Furthermore, the whole sequence of fosmid pFL18 containing FLS18C and FLS18D was shotgun sequenced, and a total of 26 ORFs on it were analyzed and annotated.

Bioprospecting microbial natural product libraries from the marine environment for drug discovery

Marine microorganisms are fascinating resources due to their production of novel natural products with antimicrobial activities. Increases in both the number of new chemical entities found and the substantiation of indigenous marine actinobacteria present a fundamental difficulty in the future discovery of novel antimicrobials, namely dereplication of those compounds already discovered. This review will share our experience on the taxonomic-based construction of a highly diversified and low redundant marine microbial natural product library for high-throughput antibiotic screening. We anticipate that libraries such as these can drive the drug discovery process now and in the future.

Bioprospecting for antituberculosis leads from microbial metabolites

Microbial metabolites have been an important source of tuberculosis (TB) therapeutics, but the last truly novel drug that was approved for the treatment of TB was discovered 40 years ago. In light of the growing threat of multi-drug resistance, recent advances have been made to accelerate the discovery rate of novel TB drugs including diversifying strategies for environmental strains, and high-throughput screening assays. This review will discuss the approaches used in biodiversity- and taxonomy-guided microbial natural product library construction, specific cell-based and target-based high-throughput screening assays and early-stage dereplication processes by liquid chromatography-mass spectrometry (LC-MS). New antituberculosis natural products that have been recently discovered are highlighted.

Systematics-guided bioprospecting for bioactive microbial natural products

Advances in the taxonomic characterization of microorganisms have accelerated the rate at which new producers of natural products can be understood in relation to known organisms. Yet for many reasons, chemical efforts to characterize new compounds from new microbes have not kept pace with taxonomic advances. That there exists an ever-widening gap between the biological versus chemical characterization of new microorganisms creates tremendous opportunity for the discovery of novel natural products through the calculated selection and study of organisms from unique, untapped, ecological niches. A systematics-guided bioprospecting, including the construction of high quality libraries of marine microbes and their crude extracts, investigation of bioactive compounds, and increasing the active compounds by precision engineering, has become an efficient approach to drive drug leads discovery. This review outlines the recent advances in these issues and shares our experiences on anti-infectious drug discovery and improvement of avermectins production as well.

Engineering of a genome-reduced host: practical application of synthetic biology in the overproduction of desired secondary metabolites

Synthetic biology aims to design and build new biological systems with desirable properties, providing the foundation for the biosynthesis of secondary metabolites. The most prominent representation of synthetic biology has been used in microbial engineering by recombinant DNA technology. However, there are advantages of using a deleted host, and therefore an increasing number of biotechnology studies follow similar strategies to dissect cellular networks and construct genomereduced microbes. This review will give an overview of the strategies used for constructing and engineering reduced-genome factories by synthetic biology to improve production of secondary metabolites.

Magnetic Field Is the Dominant Factor to Induce the Response of Streptomyces avermitilis in Altered Gravity Simulated by Diamagnetic Levitation

Background Diamagnetic levitation is a technique that uses a strong, spatially varying magnetic field to simulate an altered gravity environment, as in space. In this study, using Streptomyces avermitilis as the test organism, we investigate whether changes in magnetic field and altered gravity induce changes in morphology and secondary metabolism. We find that a strong magnetic field (12T) inhibit the morphological development of S. avermitilis in solid culture, and increase the production of secondary metabolites. Methodology/Principal Findings S. avermitilis on solid medium was levitated at 0 g*, 1 g* and 2 g* in an altered gravity environment simulated by diamagnetic levitation and under a strong magnetic field, denoted by the asterix. The morphology was obtained by electromicroscopy. The production of the secondary metabolite, avermectin, was determined by OD245 nm. The results showed that diamagnetic levitation could induce a physiological response in S. avermitilis. The difference between 1 g* and the control group grown without the strong magnetic field (1 g), showed that the magnetic field was a more dominant factor influencing changes in morphology and secondary metabolite production, than altered gravity. Conclusion/Significance We have discovered that magnetic field, rather than altered gravity, is the dominant factor in altered gravity simulated by diamagnetic levitation, therefore care should to be taken in the interpretation of results when using diamagnetic levitation as a technique to simulate altered gravity. Hence, these results are significant, and timely to researchers considering the use of diamagnetic levitation to explore effects of weightlessness on living organisms and on physical phenomena.

Rational design for over-production of desirable microbial metabolites by precision engineering.

Microbes represent a valuable source of commercially significant natural products that have improved our quality of life. Precision engineering can be used to precisely identify and specifically modify genes responsible for production of natural products and improve this production or modify the genes creating products that would not otherwise be produced. There have been several success stories concerning the manipulation of regulatory genes, pathways, and genomes to increase the productivity of industrial microbes. This review will focus on the strategies and integrated approaches for precisely deciphering regulatory genes by various modern techniques. The applications of precision engineering in rational strain improvement also shed light on the biology of natural microbial systems.

Retraction note: Cryptomycota: the missing link.

The online version of the original article can be found under doi:10.1007/s13238-012-2013-x.

RETRACTED ARTICLE: Cryptomycota: the missing link

Fungi have been well studied for 150 years and it was thought that there was a good understanding of the major evolutionary groups, but new findings by UK researchers from the University of Exeter have radically changed this perception. From a local pond near the Exeter University, UK, they discovered a diverse and deep evolutionary branch of fungi named Cryptomycota (Greek: crypto, hidden; mycota, fungi) that has now doubled our knowledge of fungal diversity. The surprising unique feature of this branch was revealed by co-staining with cell wall markers and demonstrated that Cryptomycota differ from all previously known fungi by lacking a chitin cell wall (Jones et al., 2011). Since our current understanding of fungal evolutionary diversity is based upon species amenable to growth in culture, and these typically yeast or filamentous forms are bound by a rigid cell wall, evolution of this body plan is thought critical for the success of the fungi, enabling them to adapt to heterogeneous habitats and live by osmotrophy. Chitin, the same substance that makes the exoskeleton of arthropods, is the main component of the fungal cell wall. In plants and among protists and algae, the cell wall is made of cellulose whereas in bacteria, the cell wall is made of peptidoglycans. The chitin polymers of the fungal cell wall are also linked to other components including β-1,3 glucan microfibrils with chitosan polymers made of glucosamine, with a surface of antigenic glycoproteins, agglutinans and adhesion compounds playing a major role in tensile strength and structural integrity (Bowman and Free, 2006). The fungal cell walls enclose the cell plasma membrane, and the lower and middle glucans layers and outer glycoprotein layers are interwoven fibrillar polymers held together by covalent bonds. The wall matrix itself contains gel-like mannoproteins, polysaccharides and melanin pigments providing coloration and chitin polymer chains are present throughout the cell wall, helping to maintain its structural morphology. Fungal cell walls are distinctive in three ways. Their synthesis involves the combined action of an exceptionally large number of chitin synthases; they are continuously remodeled to permit active growth; and they surround fungal cells that are actively taking up nutrients (Bulawa, 1993; Munro and Gow, 2001; Ruiz-Herrera et al., 2002). Chitin synthases are widely distributed among opisthokonts and the gene duplications that gave rise to the oldest of the fungal chitin synthases are more ancient than the divergences of the fungi themselves which may explain the complexity and distinctive characteristics of the fungal cell wall. Cryptomycota were first detected as DNA sequences detected by van-Hannen et al. (1999) and showed only a distant match with any of the known sequences at the time. Relatives of the group, named after one of the clones, LKM11, were discovered in many environmental DNA surveys of freshwater aquatic ecosystems as well as terrestrial and marine systems. These observations suggested that LKM11, like fungi in Chytridiomycota (chytrids), reproduce with motile spores, but because no member had ever been isolated, the group remained an enigma (James and Berbee, 2011). The first breakthrough on the placement of the LKM11 clade was the demonstration of a robust phylogenetic relationship to the aquatic genus Rozella (Lara et al., 2010). Rozella is an internal parasite, primarily of water molds (Held, 1981), that diverged to form the primary (basal-most) branch on the fungal tree retaining ancestral protistan characteristics (Lara et al., 2010). Their life cycle comprises a uniflagellate motile stage that allows them to disperse in search of a new host, and a trophic wall-less intracellular stage, which develops inside a host cell (Held, 1981). At this point, the parasite is amoeboid and phagocytoses the organelles of the host (Powell, 1984); no filamentous growth (formation of hyphae/rhizoid) has been observed in this genus. Subsequently, the parasite eventually induces the host to create a cell wall that will surround the parasite’s future sporangium; the para-

Genetic engineering and enzyme research in lignocellulosic ethanol production.

In April this year, Mossi & Ghisolfi Group (Chemtex) commenced construction of a commercial-scale 13million gallons/year (50million liters) cellulosic ethanol production facility in Crescentino, Italy (European biofuels technology platform. http://www.biofuelstp.eu/cell_ethanol.html#ce5. Accessed 8 September 2011). The plant will use Novozymes enzyme technology to convert a range of cellulosic feedstocks to ethanol, and is just one example of the boom the cellulosic bioethanol industry has seen over the last few years. Combining different types of enzymes, and genetically engineering new enzymes, that work together to release both hemicellulosic sugars and cellulosic sugars, can be termed “bioprocessing.” Bioprocessing is a key area of research in the efficient production of ethanol for biofuels; however, the cost of the lignocellulose-hydrolyzing enzymes accounts for a relatively high proportion of total processing costs. One problem in optimizing the activity of the enzymes is the high temperatures under which the enzymes are required to work. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28°C and 37°C, while the activity of cellulolytic enzymes is highest at around 50°C and significantly decreases with a decrease in temperature. Researchers at the South Dakota School of Mines and Technology are investigating the bioconversion of polymeric cellulosic waste materials using thermophiles from local compost facility, hot springs, and the 8000 ft deep Homestake Gold Mine, to source unique enzymes. Several cellulose-degrading Firmicutes including, Brevibacillus, Paenibacillus, Bacillus, and Geobacillus were isolated from enrichment cultures, and isolates were found with optimum temperature for carboxymethyl cellulase (CMCase) of up to 75°C (pH 5.0). One particular isolate retained 26% CMCase activity at 60°C up to a period of 300 h (Rastogi et al., 2009). These thermostable enzymes and robust thermophilic fermentative microbes could facilitate the development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process used to convert lignocellulosic biomass into biofuels. Another major challenge to be faced in commercial production of lignocellulosic bioethanol is the inhibitory compounds generated during the thermo-chemical pretreatment of biomass. These inhibiting compounds, such as weak acids, furans and phenolic compounds, are formed or released during the thermo-chemical pre-treatment step such as acid and steam explosion (Parawira and Tekere, 2011). Inhibition of yeast fermentation by inhibitor compounds in lignocellulosic hydrolysates can be reduced by treating with lignolytic enzymes, for example, laccase, and microorganisms such as Trichoderma reesei, Coniochaeta ligniaria NRRL30616, Trametes versicolor, Pseudomonas putida Fu1, Candida guilliermondii, and Ureibacillus thermosphaericus (see Parawira and Tekere, 2011 for a full review). Genetic engineering is playing a major part in getting the best possible conversion rate in thermotolerant yeasts. A Japanese research group, led by Professor Kondo of the Department of Chemical Science and Engineering, Kobe University, have genetically engineered a strain of Kluyveromyces marxianus, to display T. reesei endoglucanase and Aspergillus aculeatus β-glucosidase on the cell surface, which successfully converts a cellulosic β-glucan to ethanol directly at 48°C, with a theoretical yield of 92.2% (Yanase et al., 2010). Members of the same research group have also recently constructed a diploid Saccharomyces cerevisiae strain to optimize cellulase-expression levels in yeast (Yamada et al., 2011). Glucose and xylose are two major components in the lignocellulosic hydrolysates. While glucose fermentation is well established, xylose fermentation remains a problem in the industrialized lignocellulosic ethanol process (Li et al., 2009). The Komodo group has again used recombinant genetic engineering to construct a recombinant S. cerevisiae that not only hydrolyzed hemicelluloses by codisplaying endoxylanase from T. reesei, β-xylosidase from A. oryzae,