Metabolic engineering of the model photoautotrophic cyanobacterium synechocystis for ethanol production: optimization strategies and challenges

peer-reviewedPhotoautotrophic ethanol production using model cyanobacteria is an attractive technology that offers potential for sustainable ethanol production as a biofuel. Model strains of Synechocystis PCC6803 have been metabolically engineered to convert central metabolic intermediates such as pyruvate to acetaldehyde via cloned heterologous pyruvate decarboxylase and from acetaldehyde to ethanol via cloned homologous or heterologous alcohol dehydrogenase. While the technology is now proven, strategies are required to increase the ethanol levels through metabolic and genetic engineering and in addition, production and process strategies are required to make the process sustainable. Here we discuss both genetic and molecular strategies in combination with do wnstream strategies that are being applied while also discussing challenges to future application

The Potential of the Photoautotroph Synechocystis for Metal Bioremediation

The photoautotrophic cyanobacterium Synechocystis PCC6803 has received much attention as a model photosynthetic cell factory for the production of a range of important biotech products. The biomass remaining from this activity may then have further utility in processes such as metal bioremediation. In addition Synechocystis being an inhabitant of many natural aquatic environments is seen as an environmentally friendly alternative to using chemical precipitation methodologies for metal remediation. Synechocystis produces a range of extracellular polysaccharide substances (EPS) that can undergo modification as a function of culture age and growth nutrients which have been implicated in metal biosorption. Many studies have demonstrated that high levels of charged groups present in EPS are important in forming polymeric matrices with metallic ions allowing their biosorption. Genetic studies has revealed genes involved in such metal binding indicating that EPS can be modified for potential enhancement of binding or modification of the types of metals bound. The utility of metal binding to live and dead biomass of Synechocystis has been demonstrated for a range of metals including Cr(VI), Cd(II), Cu(II), Pb(II), Sb, Ni(II), Mn(II), Mn(IV), As(III), As(V), Cs and Hg. The potential of using Synechocystis as a biosorption platform is discussed

New and emerging SXT/R391 integrative conjugative elements as vehicles for stable mobile element transfer and spread of antibiotic resistance in both human and animals.

peer-reviewedThe integrative conjugative elements, ICE s SXT and R391 are the prototypes of a group of gram negative integrative elements known as the SXT/R391 group. R391 was identified in a clinical isolate of Providencia in the late 1960 s in South Africa, while SXT was initially isolated in 1992 in a clinical isolate of Vibrio cholerae O139 and variants have since been isolated in pandemic strains throughout the world. Subsequent sequencing of both elements demonstrated a high degree of structural similarity leading to the group being classified as the SXT/R391 group. The SXT/R391 ICE elements are characterised as integrating into a specific chromosomal site within gram ve hosts, being extremely stable and promiscuous and possessing a number of element hotspots for integration of heterologous DNA including increasingly, antibiotic resistance determinants. This makes such ICE s highly adapted for antibiotic spread. New evidence emerging indicates that SXT/R391-like ICE s are increasingly being identified worldwide particularly in Asia not only from Vibrio species, where they have been found widely in human clinical isolates, but from other gram -ve associated infections of domestic animals and fish. Evidence of more such elements may emerge in the future as a new trapping vector pIceCap has been developed to capture them in a circular form, aiding characterisation. The types of the novel ICE s now emerging, their comparison with prototype elements and the antibiotic resistances associated with them are important given their promiscuous nature and stability. PUBLISHEDPeer reviewe

Ultrasonic intensification as a tool for enhanced microbial biofuel yields

peer-reviewedUltrasonication has recently received attention as a novel bioprocessing tool for process intensification in many areas of downstream processing. Ultrasonic intensification (periodic ultrasonic treatment during the fermentation process) can result in a more effective homogenization of biomass and faster energy and mass transfer to biomass over short time periods which can result in enhanced microbial growth. Ultrasonic intensification can allow the rapid selective extraction of specific biomass components and can enhance product yields which can be of economic benefit. This review focuses on the role of ultrasonication in the extraction and yield enhancement of compounds from various microbial sources, specifically algal and cyanobacterial biomass with a focus on the production of biofuels. The operating principles associated with the process of ultrasonication and the influence of various operating conditions including ultrasonic frequency, power intensity, ultrasonic duration, reactor designs and kinetics applied for ultrasonic intensification are also described

Functional genomic analysis of the UV-inducible ‘cytotoxic’ gene from the ICE R391, an archetypal member of the SXT/R391 family of integrative conjugative elements

The enterobacterial mobile element, R391, is a prototype member of the SXT/R391 family of Integrative Conjugative Elements (ICEs). SXT/R391-like ICEs are sitespecific genome-integrating mobile elements, frequently isolated from human pathogens. They encode a range of known adaptive traits, including multiple antibiotic resistance determinants and DNA repair mechanisms. The functionality of a large percentage of SXT/R391-like ICEs genes is unknown (31% of predicted ORFs of R391). These currently cryptic genes may encode as yet uncharacterised adaptive traits. Unusually, SXT/R391-like ICEs have also been found to exhibit an apparently disadvantageous recA-dependent UV-inducible cell sensitising phenotype. The identification and characterisation of the R391 ICE gene(s) responsible for this unusual cell sensitising, cytotoxic effect was investigated. A comparative bioinformatic analysis of SXT/R391-like ICEs was performed, which predicted that 49 putative core genes were shared amongst all ICE members. Construction of a complete R391 gene deletion library implicated three of these core genes, orf90, orf91 and orf43, in the sensitising phenotype. Cloning of orfs90/91 and orf43, precise deletion mutant construction and RT-PCR analysis determined that orf90, orf91 and orf43 are UV-inducible and that orfs90/91 putatively encode a transcriptional activator complex which regulates orf43 transcription. Functional analysis utilising recombinant expression, TEM, laser scanning confocal microscopy and site-directed mutagenesis of orf43 which encodes a putative TraV homolog (TraVR391), functioning as an outer membrane-associated pore-forming protein, determined that expression of orf43 (TraVR391) was cytotoxic to host cells by destabilising cell integrity. Results suggested that orf43 (TraVR391) expression alone was responsible for the cell sensitisation phenotype and that UV was a facilitator of this overexpression. A hypothesis was proposed and supporting evidence presented suggesting that orf43 may function as a stress inducible ICE ‘trap door’ escape mechanism for SXT/R391- like ICEs. Hence conservation of this UV-inducible ‘cytotoxic’ gene may aid element survival and be advantageous under stress conditions

Chapter The Potential of the Photoautotroph Synechocystis for Metal Bioremediation

Pollution & threats to the environmen

Utilising the native plasmid, pCA2.4, from the cyanobacterium synechocystis sp. strain PCC6803 as a cloning site for enhanced product production

BackgroundThe use of photosynthetic autotrophs and in particular the model organism Synechocystis PCC6803 is receiving much attention for the production of sustainable biofuels and other economically useful products through metabolic engineering. Optimisation of metabolic-engineered organisms for high-level sustained production of product is a key element in the manipulation of this organism. A limitation to the utilisation of metabolically-engineered Synechocystis PCC6803 is the availability of strong controllable promoters and stable gene dosage methods for maximising gene expression and subsequent product formation following genetic manipulation.ResultsA native Synechocystis PCC6803 small plasmid, pCA2.4, is consistently maintained at a copy level of up to 7 times that of the polyploid chromosome. As this plasmid is stable during cell division, it is potentially an ideal candidate for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expression vector generated from the native plasmid, pCA2.4. To investigate the feasibility of this new expression system, a yellow fluorescent protein (YFP) encoding gene was cloned downstream of the strong Ptrc promoter and integrated into a predicted neutral site within the pCA2.4 plasmid. The stability of the integrated construct was monitored over time compared to a control strain containing an identical YFP-expressing construct integrated at a known neutral site in a chromosomal location.ConclusionsA significantly higher fluorescence level of the yellow fluorescent protein was observed when its encoded gene was integrated into the pCA2.4 native plasmid when compared to the isogenic chromosomally integrated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integration into the native plasmid. Fluorescence was also monitored as a function of culture time and demonstrated to be stable over multiple sub-cultures even after the removal of selective pressure. Therefore, the native small plasmid, pCA2.4 may be utilised to stably increase gene expression levels in Synechocystis PCC6803. With the complementary utilisation of an inducible promoter system, rapid generation of commodity-producing SynechocystisPCC6803 strains having high level, controlled expression may be more achievable