Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae.

Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1mM).

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants.

AIMS: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. METHODS AND RESULTS: Streptococcus bovis strains were tested for production of bacteriocin-like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin-positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. CONCLUSIONS: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin(+) trait is maintained in animals at the same location. The HC5-like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. SIGNIFICANCE AND IMPACT OF THE STUDY: The HC5-like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.

A synthetic xylanase as a novel reporter in plants.

Transient gene expression assays are often used to screen promoters before stable transformation. Current transient quantification methods have several problems, including a lack of reporter gene stability and expense. Here we report a synthetic, codon-optimised xylanase gene ( sXynA) as a reporter gene for quantitative transient analyses in plants. Azurine-crosslinked xylan (AZCL-xylan) was used as a substrate for assaying xylanase activity. The enzymatic nature of the protein allows for sensitive assays at the low levels of transgene protein found in transiently transformed tissue extracts. The xylanase (XYN) protein is stable, activity slopes are linear over long time periods and assays are cost-effective. Coupled with the GUS Plus reporter gene, the XYN reporter allows sensitive and accurate quantification of gene control sequences in transient expression systems.

Selectable marker-free transgenic barley producing a high level of cellulase (1,4-beta-glucanase) in developing grains.

The use of barley grains as bioreactors for high-level production of cellulase (1,4-beta-glucanase) was investigated. A hybrid cellulase gene, cel-hyb1, driven by the rice GluB-1 promoter was expressed specifically in developing endosperm. Codon usage optimisation of cel-hyb1 increased its expression in barley grains 527-fold and led to cellulase production of up to 1.5% of total grain protein. CEL-HYB1 enzyme in barley grains was highly stable during post-harvest storage. Selectable marker gene ( hph) was subsequently eliminated from transgenic lines through segregation of hph from synthetic cel-hyb1 ( syn.cel-hyb1) in T1 progeny, using a binary plasmid containing hph and syn.cel-hyb1 in separate T-DNAs. These data suggest that barley grains can potentially be used for the commercial production of cellulase.