There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H2 yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H2 production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.

Genetic Engineering of Algae for Enhanced Biofuel Production

Oxygenic photosynthesis, the principal converter of sunlight into chemical energy on earth, is catalyzed by four multi-subunit membrane-protein complexes: photosystem I (PSI), photosystem II (PSII), the cytochrome b6f complex, and F-ATPase PSI generates the most negative redox potential in nature and largely determines the global amount of enthalpy in living systems PSII generates an oxidant whose redox potential is high enough to enable it to oxidize H2O, a substrate so abundant that it assures a practically unlimited electron source for life on earth During the last century, the sophisticated techniques of spectroscopy, molecular genetics, and biochemistry were used to reveal the structure and function of the two photosystems The new structures of PSI and PSII from cyanobacteria, algae, and plants has shed light not only on the architecture and mechanism of action of these intricate membrane complexes, but also on the evolutionary forces that shaped oxygenic photosynthesis

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Structure and function of photosystems i and ii

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

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Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin

The unicellular green alga Chlamydomonas offers a simple life cycle, easy isolation of mutants, and a growing array of tools and techniques for molecular genetic studies. Among the principal areas of current investigation using this model system are flagellar structure and function, genetics of basal bodies (centrioles), chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. A genome project has begun with compilation of expressed sequence tag data and gene expression studies and will lead to a complete genome sequence. Resources available to the research community include wild-type and mutant strains, plasmid constructs for transformation studies, and a comprehensive on-line database.

Chlamydomonas as a model organism

We demonstrate that two rhodopsins, identified from cDNA sequences, function as low- and high-light-intensity phototaxis receptors in the eukaryotic alga Chlamydomonas reinhardtii. Each of the receptors consists of an ≈300-residue seven-transmembrane helix domain with a retinal-binding pocket homologous to that of archaeal rhodopsins, followed by ≈400 residues of additional membrane-associated portion. The function of the two rhodopsins, Chlamydomonas sensory rhodopsins A and B (CSRA and CSRB), as phototaxis receptors is demonstrated by in vivo analysis of photoreceptor electrical currents and motility responses in transformants with RNA interference (RNAi) directed against each of the rhodopsin genes. The kinetics, fluence dependencies, and action spectra of the photoreceptor currents differ greatly in transformants in accord with the relative amounts of photoreceptor pigments expressed. The data show that CSRA has an absorption maximum near 510 nm and mediates a fast photoreceptor current that saturates at high light intensity. In contrast, CSRB absorbs maximally at 470 nm and generates a slow photoreceptor current saturating at low light intensity. The relative wavelength dependence of CSRA and CSRB activity in producing phototaxis responses matches precisely the wavelength dependence of the CSRA- and CSRB-generated currents, demonstrating that each receptor mediates phototaxis. The saturation of the two photoreceptor currents at different light fluence levels extends the range of light intensity to which the organism can respond. Further, at intensities where both operate, their light signals are integrated at the level of membrane depolarization caused by the two photoreceptor currents.

https://www.pnas.org/content/pnas/99/13/8689.full.pdf

Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii.

Summary
Heterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 5′ and 3′ sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation-independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high-level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.

Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5′ and 3′ untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg+ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (PmR) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the PmR transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a PmR transformant to wild-type strains. Direct selection of PmR transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.

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The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas.

Although eukaryotic algae are of value both as organisms for fundamental biological research and as a resource for the biotechnology industry, their importance in these areas would be enhanced significantly by the availability of genetic engineering methodology. Algae are used as model systems to study many different aspects of cell biology, biochemistry, and physiology (Fogg 1989). Major advances can be made in these studies through the use of recombinant DNA techniques to manipulate individual genes within the organism or to introduce novel genes into the genome. Within the biotechnology industry, there is increasing interest in the exploitation of algal species as sources of valuable biochemicals and pharmaceuticals and in processes such as fuel and polysaccharide production, bioremediation, and aquaculture (Cannell 1990, Renn 1997). These industrial applications would benefit tremendously from genetic engineering technology that allowed the manipulation of biochemical pathways leading to higher product yields and improved strains. The development of DNA transformation proceures for the many important algae species is still in its infancy. However, significant progress has been made in the nine years or so since Craig et al. (1988) reviewed the subject. In particular, the development of nuclear and organellar transformation systems for the green alga Chlamydomonas reinhardtii has led to a wealth of studies on gene function and expression in this organism. Success with Chlamydomonas has also acted as a catalyst in the development of transformation systems for other algae, most notably the related green alga Volvox carteri and several diatom species. In this article, we discuss the requirements for successful transformation of a given algal species, review the progress made in the manipulation of green algae, diatoms, and red algae, and consider future prospects for the engineering of biologically and commercially important algae. Special emphasis is given to Chlamydomonas, since research with this organism has now progressed from the development of transformation methodology to its application as a research tool. We have not included in this review the large body of work on the genetic manipulation of macroalgae and microalgae through conventional mutagenesis or the creation of new hybrid strains by protoplast fusion. Additionally, the

Genetic engineering of eukarygtic algae: progress and prospects

The bacterial phleomycin resistance gene

To investigate the environment of the phylloquinone secondary electron acceptor A1 within the photosystem I reaction center, we have carried out site-directed mutagenesis of two tryptophan residues (W693 and W702) in the PsaA subunit of Chlamydomonas reinhardtii. One of these conserved tryptophans (W693) is predicted to be close to the phylloquinone and has been implicated in the interaction of A1 with an aromatic residue through π−π stacking. We find that replacement of W702 with either histidine or leucine has no effect on the electronic structure of A1•- or on forward electron transfer from A1•- to the iron−sulfur center Fx. In contrast, the same mutations of W693 alter the electronic structure of the photoaccumulated A1•- and slow forward electron transfer as measured by the decay of the electron spin-polarized signal arising from the P700•+/A1•- radical pair. These results provide support for the hypothesis that W693 has a role in poising the redox potential of A1/A1•- so it can reduce Fx, and they i...

Site-directed mutagenesis of PsaA residue W693 affects phylloquinone binding and function in the photosystem I reaction center of Chlamydomonas reinhardtii.

David R. Stevens

Papers

Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron

The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas.

Genetic engineering of eukarygtic algae: progress and prospects

The bacterial phleomycin resistance gene

Site-directed mutagenesis of PsaA residue W693 affects phylloquinone binding and function in the photosystem I reaction center of Chlamydomonas reinhardtii.