Christopher Collet

Microalgae as bioreactors

Microalgae already serve as a major natural source of valuable macromolecules including carotenoids, long-chain polyunsaturated fatty acids and phycocolloids. As photoautotrophs, their simple growth requirements make these primitive plants potentially attractive bioreactor systems for the production of high-value heterologous proteins. The difficulty of producing stable transformants has meant that the field of transgenic microalgae is still in its infancy. Nonetheless, several species can now be routinely transformed and algal biotechnology companies have begun to explore the possibilities of synthesizing recombinant therapeutic proteins in microalgae and the engineering of metabolic pathways to produce increased levels of desirable compounds. In this review, we compare the current commercially viable bioreactor systems, outline recent progress in microalgal biotechnology and transformation, and discuss the potential of microalgae as bioreactors for the production of heterologous proteins.

THE PHYLOGENY OF THE INSULIN-LIKE GROWTH FACTORS

The insulin-like growth factors are major regulators of growth and development in mammals and their presence in lower vertebrates suggests that they played a similarly fundamental role throughout vertebrate evolution. While originally perceived simply as mediators of growth hormone, on-going research in mammals has revealed several hierarchical layers of complexity in the regulation of ligand bioavailability and signal transduction. Our understanding of the biological role and mechanisms of action of these important growth factors in mammals patently requires further elucidation of the IGF hormone system in the simple model systems that can be found in lower vertebrates and protochordates. This review contrasts our knowledge of the IGF hormone system in mammalian and nonmammalian models through comparison of tissue and developmental distributions and gene structures of IGF system components in different taxa. We also discuss the evolutionary origins of the system components and their possible evolutionary pathways.

ALGAL TRANSGENICS IN THE GENOMIC ERA

The last few years have witnessed significant advances in the field of algal genomics. Complete genome sequences from the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana have been published, the genomes for two more algae (Chlamydomonas reinhardtii and Ostreococcus tauri) are nearing completion, and several others are in progress or at the planning stage. In addition, large‐scale cDNA sequencing projects are being carried out for numerous algal species. This wealth of genome data is serving as a powerful catalyst for the development and application of recombinant techniques for these species. The data provide a rich resource of DNA elements such as promoters that can be used for transgene expression as well as an inventory of genes that are possible targets for genetic engineering programs aimed at manipulating algal metabolism. It is not surprising therefore that significant progress in the genetic engineering of eukaryotic algae is being made. Nuclear transformation of various microalgal species is now routine, and progress is being made on the transformation of macroalgae. Chloroplast transformation has been achieved for green, red, and euglenoid algae, and further success in organelle transformation is likely as the number of sequenced plastid, mitochondrial, and nucleomorph genomes continues to grow. Importantly, the commercial application of algal transgenics is beginning to be realized, and algal biotechnology companies are being established. Recent work has shown that recombinant proteins of therapeutic value can be produced in microalgal species, and it is now realistic to envisage the genetic engineering of commercially important species to improve production of valuable algal products. In this article we review the recent progress in algal transgenics and consider possible future developments now that phycology has entered the genomic era.

How many insulin-like growth factor binding proteins?

Mac25, connective tissue growth factor, the nov-oncogene and cyr61 have been proposed as insulin-like growth factor binding proteins (IGFBPs) although they bind the ligand with very low affinity. Sequence similarity between the candidate proteins and the recognised IGFBPs is restricted to a single cysteine-rich N-terminal domain. The cysteine-rich domain (CRD) can be found in other vertebrate and invertebrate proteins that are associated with the extracellular matrix but otherwise have vastly different functions. Characteristically, the proteins with the CRD have a modular architecture suggesting that exon shuffling has played a significant role in their evolution. Although the proposed candidate proteins may not be IGFBPs, linkage relationships of the latter suggest that two other IGFBPs may indeed exist.

TaNF-YB3 is involved in the regulation of photosynthesis genes in Triticum aestivum

Nuclear factor Y (NF-Y) transcription factor is a heterotrimer comprised of three subunits: NF-YA, NF-YB and NF-YC. Each of the three subunits in plants is encoded by multiple genes with differential expression profiles, implying the functional specialisation of NF-Y subunit members in plants. In this study, we investigated the roles of NF-YB members in the light-mediated regulation of photosynthesis genes. We identified two NF-YB members from Triticum aestivum (TaNF-YB3 & 7) which were markedly upregulated by light in the leaves and seedling shoots using quantitative RT-PCR. A genome-wide coexpression analysis of multiple Affymetrix Wheat Genome Array datasets revealed that TaNF-YB3-coexpressed transcripts were highly enriched with the Gene Ontology term photosynthesis. Transgenic wheat lines constitutively overexpressing TaNF-YB3 had a significant increase in the leaf chlorophyll content, photosynthesis rate and early growth rate. Quantitative RT-PCR analysis showed that the expression levels of a number of TaNF-YB3-coexpressed transcripts were elevated in the transgenic wheat lines. The mRNA level of TaGluTR encoding glutamyl-tRNA reductase, which catalyses the rate-limiting step of the chlorophyll biosynthesis pathway, was significantly increased in the leaves of the transgenic wheat. Significant increases in the expression level in the transgenic plant leaves were also observed for four photosynthetic apparatus genes encoding chlorophyll a/b-binding proteins (Lhca4 and Lhcb4) and photosystem I reaction centre subunits (subunit K and subunit N), as well as for a gene coding for chloroplast ATP synthase γ subunit. These results indicate that TaNF-YB3 is involved in the positive regulation of a number of photosynthesis genes in wheat.

ORGANIZATION, SEQUENCE, AND EXPRESSION OF THE GENE ENCODING IGFII FROM BARRAMUNDI (TELEOSTEII; LATES CALCARIFER)

We have characterized the gene encoding IGFII from the teleost fish species barramundi, Lates calcarifer. The barramundi gene spans 5.5 kb of DNA and comprises four exons and three introns. The barramundi and salmon IGFII genes share >85% sequence similarity across all the exons and also share some regions of high sequence identity within the promoter regions and the introns. The mature barramundi IGFII peptide comprises 70 amino acid residues and shares 84 and 96% similarity with salmonid and seabream IGFII, respectively, with the majority of replacements located in regions cleaved from the mature peptide. IGFII mRNA transcripts were detected in liver, muscle, intestine, gill, heart, and brain from juvenile barramundi. This distribution mirrors that seen in the rainbow trout and seabream and extends the tissue types which synthesize IGFII in fish to include the intestine.

TaNF-YC11 , one of the light-upregulated NF-YC members in Triticum aestivum , is co-regulated with photosynthesis-related genes

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor complex. Each of the NF-Y subunits (NF-YA, NF-YB and NF-YC) in plants is encoded by multiple genes. Quantitative RT-PCR analysis revealed that five wheat NF-YC members (TaNF-YC5, 8, 9, 11 and 12) were upregulated by light in both the leaf and seedling shoot. Co-expression analysis of Affymetrix wheat genome array datasets revealed that transcript levels of a large number of genes were consistently correlated with those of the TaNF-YC11 and TaNF-YC8 genes in three to four separate Affymetrix array datasets. TaNF-YC11-correlated transcripts were significantly enriched with the Gene Ontology term photosynthesis. Sequence analysis in the promoters of TaNF-YC11-correlated genes revealed the presence of putative NF-Y complex binding sites (CCAAT motifs). Quantitative RT-PCR analysis of a subset of potential TaNF-YC11 target genes showed that ten out of the 13 genes were also light-upregulated in both the leaf and seedling shoot and had significantly correlated expression profiles with TaNF-YC11. The potential target genes for TaNF-YC11 include subunit members from all four thylakoid membrane-bound complexes required for the conversion of solar energy into chemical energy and rate-limiting enzymes in the Calvin cycle. These data indicate that TaNF-YC11 is potentially involved in regulation of photosynthesis-related genes.

Lysozyme and alpha-lactalbumin from the milk of a marsupial, the common brush-tailed possum (Trichosurus vulpecula).

Lysozyme and alpha-lactalbumin have been identified using N-terminal sequence analysis of whey proteins from the common brush-tailed possum, Trichosurus vulpecula after separation by two-dimensional denaturing electrophoresis. Both proteins were purified from pooled possum milk using ion exchange chromatography and gave mass values of 14,896 and 13,985 Da respectively by MALDI-TOF mass spectrometry. Clones containing the full coding sequences of the genes for both proteins were isolated from a possum mammary cDNA library and the DNA sequence of the coding region determined. The inferred protein sequences were used in phylogenetic analysis of both protein classes. These showed that the T. vulpecula alpha-lactalbumin, along with other marsupial alpha-lactalbumins, formed a family distinct from the eutherian alpha-lactalbumins and the alpha-lactalbumin of a monotreme, the platypus, consistent with the separate evolution of the marsupials. By contrast the T. vulpecula lysozyme was shown to be similar to the ruminant stomach lysozymes and primate lysozymes and quite distinct from the Ca2+-binding lysozymes found in the milk of the echidna and horse.

Towards the development of a nuclear transformation system for Dunaliella tertiolecta

A nuclear transformation system for the microalga Dunaliella tertiolecta was explored using electroporation. Plasmids incorporating the D. tertiolecta RbcS1 5′ and 3′ untranslated regions flanking the Streptoalloteichus hindustanus gene encoding bleomycin resistance (ble) were introduced into D. tertiolecta cells both transiently and stably. Southern hybridisation was used to examine the fate of the plasmid following electroporation and revealed that the DNA was entering the cells but was quickly degraded. Using the same procedure one stably transformed line was recovered.

Employability Skills: Perspectives from a Knowledge-Intensive Industry.

Purpose: While the global education debate remains focused on graduate skills and employability, the absence of a shared language between student, academic and industry stakeholder groups means that defining industry skills requirements is both essential and difficult. The aim of this study was to assess graduate skills requirements in a knowledge intensive industry from a demand perspective as distinct from a curriculum (supply) viewpoint. Design/methodology/approach: Skills items were derived from a breadth of disciplines across academic, policy and industry literature. CEOs and senior managers in the innovation and commercialisation industry were surveyed regarding perceptions of skills in graduates and skills in demand by the firm. Two rounds of exploratory factor analyses were undertaken to examine employers’ perceptions of the skills gap. Findings: First order analysis resolved 10 broad constructs that represent cognitive, interpersonal and intrapersonal skills domains as applied in this industry. Knowledge, leadership and interprofessional collaboration feature as prominent skills. Second order analysis revealed employers’ perceptions of graduate skills specifically centre on organisational fit and organisational success. An over-arching theme relates to performance of the individual in organisations. Research limitations/implications: Our findings suggest that the discourse on employability and the design of curriculum need to shift from instilling lists of skills towards enabling graduates to perform in a diversity of workplace contexts and expectations centred on organisational purpose. Originality/value: In contrast to the heterogeneous nature of industry surveys, we targeted a homogenous sector that is representative of knowledge intensive industries. This study contributes to the broader stakeholder dialogue of the value and application of graduate skills in this and other industry sectors.