научная статья по теме GENETIC ENGINEERING OF ALGAL CHLOROPLASTS: PROGRESS AND PROSPECTS Биология

Текст научной статьи на тему «GENETIC ENGINEERING OF ALGAL CHLOROPLASTS: PROGRESS AND PROSPECTS»

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Genetic Engineering of Algal Chloroplasts: Progress and Prospects1 © 2013 S. Purton, J. B. Szaub, T. W&nnathong, R. Young, C. K. Economou

Algal Biotechnology Group, Institute of Structural and Molecular Biology, University College London, London, United Kingdom

Received October 29, 2012

The last few years has seen an ever-increasing interest in the exploitation of microalgae as recombinant platforms for the synthesis of novel bioproducts. These could be biofuel molecules, speciality enzymes, nutraceu-ticals, or therapeutic proteins, such as antibodies, hormones, and vaccines. This exploitation requires the development of new genetic engineering technologies for those fast-growing, robust species suited for intensive commercial cultivation in bioreactor systems. In particular, there is a need for routine methods for the genetic manipulation of the chloroplast genome, for two reasons: firstly, the chloroplast genetic system is well-suited to the targeted insertion into the genome and high-level expression of foreign genes; secondly, the organelle is the site of numerous biosynthetic pathways and therefore represents the obvious "chassis," on which to bolt new metabolic pathways that divert the carbon fixed by photosynthesis into novel hydrocarbons, pigments, etc. Stable transformation of the algal chloroplast was first demonstrated in 1988, using the model chlorophyte, Chlamydomonas reinhardtii. Since that time, tremendous advances have been made in the development of sophisticated tools for engineering this particular species, and efforts to transfer this technology to other commercially attractive species are starting to bear fruit. In this article, we review the current field of algal chloroplast transgenics and consider the prospects for the future.

Keywords: Chlamydomonas reinhardtii - algae - chloroplast - genetic engineering - transformation - transplas-tomics

DOI: 10.7868/S0015330313040155

INTRODUCTION

The chloroplast organelle of plant and algal cells contains its own genetic system - a legacy from the ancestral cyanobacterium that originally gave rise to the organelle through a process of endosymbiosis that occurred over 1 billion years ago [1]. The chloroplast genetic system therefore reflects its prokaryotic origins with a polyploid circular genome (termed the "plas-tome") containing some 100-200 genes, together with a eubacterial-type transcription-translation system. The genes encode primarily the core components of the photosynthetic apparatus, the subunits of the RNA polymerase, and approximately half of the 70S riboso-mal subunits, together with the ribosomal and transfer RNAs [2]. Related genes are often organized into co-transcribed operons, and in actively growing cells the level of chloroplast gene expression is high. This is the case particularly for core photosynthetic genes such as psbA that encode high-turnover proteins, or rbcL that

1 This text is published in original.

Abbreviations'. GOI - gene-of-interest; IPTG - isopropyl-ß-D-thiolgalactopyranoside; UTR - untranslated region. Corresponding author. Saul Purton. Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom. Fax. +44 (0) 20 7679-7193; e-mail. s.purton@ucl.ac.uk

encodes the large subunit of ribulose bisphosphate carboxylase/oxygenase - the most abundant protein in nature. However, chloroplast gene expression is not an autonomous process: rather, the control of expression is mediated by numerous nuclear-encoded factors that are targeted into the chloroplast and act primarily at post-transcriptional steps, such as RNA processing, RNA stability, and translation [3].

The exploitation of the chloroplast as a biotechnology platform for the synthesis of recombinant products is particularly attractive for a number of reasons. These include the relative simplicity of the genome compared to the nuclear genome, the high level of gene expression, the opportunity for multiple gene expression through the introduction of whole transgenic operons, and the precise targeting of foreign DNA to any specific loci within the plastome [4, 5]. Furthermore, the chloroplast is the site of numerous biochemical pathways, including the synthesis of proteins, lipids, carbohydrates, tetrapyrroles, and iso-prenoids [6]. The chloroplast of unicellular microalgae is particularly appealing, since such algae typically contain only one chloroplast per cell, making chloroplast genetic engineering that much easier [7]. Furthermore, the algal chloroplast functions both as a site of synthesis and of storage, the organelle being capable of accumulating remarkable levels of starch, lip-

Fig. 1. Introducing a gene-of-interest (GOI) into the chloroplast genome.

The coding sequence of the GOI is fused to a promoter and UTR elements to ensure successful expression. This "gene cassette" is then flanked with left and right DNA arms that ensure the targeted integration of the cassette into the genome via two homologous recombination events, giving a transformed genome. As discussed in the text, various selection strategies can be used to select for cells with a transformed chloroplast, and ensure that all copies of the genome contain the cassette [7].

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ids, or pigments when exposed to stress conditions [8]. When combined with the high growth rates and low cost of cultivation of certain algal species, then algal chloroplast engineering offers commercial opportunities for light-driven synthesis of hydrocarbons for use as biofuels; valuable nutraceuticals, such as long-chain polyunsaturated fatty acids; carotenoid-based antioxidants and vitamins; and therapeutic proteins, such as vaccines, hormone, and antibodies [9, 10].

The first demonstration of stable transformation of a chloroplast genome was reported by Boynton et al. [11] who showed that a photosynthetic mutant of the green unicellular alga Chlamydomonas reinhardtii carrying a deletion in the chloroplast gene atpB, could be rescued to photoautotrophic growth by introduction of cloned DNA carrying a wild-type copy of atpB. The DNA was introduced into the chloroplast compartment by high velocity bombardment of an algal lawn with DNA-coated tungsten microparticles. This DNA delivery technique subsequently became known as "biolistics" [12], and the method of genetically engineering the plastome has been termed "transplastom-ics". An important observation from this initial experiment was that transforming DNA integrated into the plastome via a mechanism of homologous recombina-

tion that resulted in the replacement of the mutant atpB locus with the wild-type copy on the introduced DNA. Subsequent work (e.g. [13]] showed that any foreign DNA could be targeted to specific loci by flanking the elements with homologous sequences as shown in Fig. 1. Expression of the foreign DNA was achieved by fusing coding sequence to chloroplast DNA elements containing promoter and untranslated regions (UTRs). This strategy led to the development of dominant selectable markers based on antibiotic-resistance genes [14, 15] and allowed the genetic manipulation of wild-type plastomes by linking the desired change (be it, site-directed change within the plastome; disruption of a chloroplast gene, or insertion of foreign genes) to the marker on the transforming plasmid. This ability to create specific, designed changes to the plastome was termed "chloroplast DNA surgery" by Rochaix [16] and led to the adoption of C. reinhardtii as a model organism for reverse-genetic studies of chloroplast biology; in particular, the expression and regulation of photosynthetic genes, and the role of their gene products in light energy transduction and electron transfer [17].

The pioneering work on plastome engineering carried out using C. reinhardtii quickly led to the develop-

ment of equivalent technologies for higher plants, with reports to-date of over 25 plant species, in which chloroplast transformation has been achieved [18]. Amongst these species, it is tobacco (Nicotiana tabacum) where most progress in transplastomics has been made. Many hundreds of foreign genes encoding metabolic enzymes, therapeutic proteins, insecticidal proteins, etc. have been expressed successfully in the tobacco chloroplast, with remarkable levels of recombinant protein reported [19]. However, the use of plants as recombinant platforms presents a number of issues. These include: the many months needed to create stable transplastomic lines since this involves several rounds of plant regeneration from tissue culture; the time required for cultivation of the plants in a greenhouse or field; the presence of toxic secondary metabolites in Solanaceae plants, such as tobacco and potato; the concerns of containment of field-grown genetically modified (GM) plants and problems of ingression and contamination of crops grown for food; and the problem of meeting strict regulatory standards for the consistent production of therapeutic proteins given significant plant-to-plant and batch-to-batch variations in quality.

In contrast, the use of single-cell algae, such as C. reinhardtii avoids or minimizes many of these issues. The time taken to generate stable transplastomic strains of C. reinhardtii is measured in weeks rather than months, and the scale-up from lab flask to industrial-scale cultivation can similarly be achieved in a few weeks [9]. The production of these microalgae in closed fermentor or photobioreactor systems ensures a much tighter containment regime, limits contamination or infection with viral or microbial pathogens, and allows the precise control of growth parameters and therefore batch-to-batch consistency. Furthermore, green algae, such as C. reinhardtii, Chlorella sp., H

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