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[SANET-MG] background on GM chlamydomonas



June 24, 2005
Prof. Joe Cummins
“Producing human pharmaceutical proteins in a green alga, Chlamydomonas reinhardtii” Two proposals have recently been made to produce a number of pharmaceutical proteins using a green alga, Chlamydomonas in the state of Hawaii. Both of those proposals were couched in broad general terms and made scant effort to critically evaluate the impact of the recombinant algae on humans and the environment (1). The production of human pharmaceutical proteins has been reported in journals but to appreciate the actual process it is necessary to consider the basics of the alga and its molecular genetic makeup. Chlamydomonas has three genomes, nuclear, chloroplast and mitchondria. The nuclear genome has an unusually high GC content (62%) with a DNA size about 100 mega base pair. The high GC content of the nuclear genome creates a codin bias that makes it difficult for foreign genes to be expressed until their codons are adjusted. There are 17 nuclear chromosomes. There are two mating types (+ the maternal gamete and – the paternal gamete). The chloroplast genome consists of a circular DNA molecule of about 200 kilo base pairs (2). Prior to zygote formation the chloroplast DNA of the + (maternal) gamete is methylated to protect it from degradation, the -(paternal) DNA is degraded , thus chloroplast inheritance is maternal(3). The chloroplast DNA has a GC content 34%. Foreign genes more readily transform the chloroplast than the nuclear genome but in many instances codon bias adjustments are needed(2).The Chlamydomonas mitochondrial genome is tiny linear 15.8 kilobase DNA molecule the mitochondrial ribosome genes are peculiar in that they synthesized as fragments whose transcripts are joined to form a larger more typical ribosomal RNA. The mitochondrial genome is transformed with great difficulty. Finally, peculiarly, the mitochondrial genes are inherited paternally, through the minus mating type (2). The Chlamydomonas chloroplast has proved to be the most effective system for expressing foreign transgenes, however, the transformation of the chloroplast chromosome fundamentally differs from the transformation of nuclear genes of algae and higher plants. Transformation of nuclear genes in algae and higher plants is achieved by illegitimate recombination involving DNA double strand breaks and insertion of the transgenes at the the break site. Chloroplast transformation resembles transformation in bacteria or yeast , it is achieved using homologous ( legitimate) recombination. Such transformations requires sequence tails at the ends of the gene to be inserted, such sequence tails are homologous to a chloroplast gene(4). Insertion of the transgene leads disruption of the gene into which the transgene has been inserted. The transgene inserted is targeted to particular gene so that it location is established at a defined insertion site. The inserted transgene includes an appropriate promoter and sequences at both the 5’ and 3’ untranslated regions of the the transgenes. Such sequences govern RNA editing in which some messenger RNA cytosine molecules are converted to uridine. RNA editing is commonplace in chloroplasts and must be accounted for(5). It has been noted that RNA editing sites appear more readily in genes (such as the transgene), whose loss can be tolerated (6). Along with RNA editing some chloroplast genes contain introns and some of those may have alternate splicing such as has been observed in a chloroplast heat shock protein (7). An example of the use of Chlamydomonas chloroplast to produce a mammalian vaccine is the production for a foot and mouth disease vaccine protein fused with a cholera toxin B subunit. The cholera toxin B subunit a mucosal adjuvant that binds to intestinal epithelial surfaces. The foot and mouth disease antigen gene fused to the choleraB subunit gene was driven by the constitutive ATP stynthetase (atpA) promoter and transcription was terminated by the rubisco (rbcL) terminator. A spectomycin selective marker (aadA) was also inserted downstream from the antigen-toxin fusion protein gene. At the 5’ (start) end of the gene insert there was a short sequence from the Chlamydomonas chloroplast chlorophyl L (chlL) and another short sequence from the chlorophyll L (chlL) gene at the 3’ end of the insert, these short sequences target the insert to the chloroplast chlL gene , disrupting its function. The disrupted strain had a “yellow in the dark” phenotype in contrast to the “green in the dark” phenotype of the wild type alga.The foot and mouth antigen-cholera toxin B fusion protein produced in Chlamydomonas made up about 3% of the soluble protein in the algal cell(8). The algal system was satisfactory for producing a mucosal vaccine source. The problems that may be encountered include the predictable release of modified Chlamyomonas to the environment. Chlamydomonas is commonly associated with the soil and aquatic environment and contamination of the environment would lead to dispersal of the gene for spectinomycin resistance , the antibiotic is currently employed in treating a number of infections. Pollution of the aquatic and soil environment with the modified alga would, also, create oral tolerance in farm animals leading to tolerance to the pathogen rendering the vaccine ineffective. Finally, the potential impact of RNA editing on the vaccine construction was not discussed even though it is a potential source of dangerous variability in a vaccine. A good deal more thought should be given to the environmental hazards associated with the production of recombinant algae bearing vaccines and other pharmaceuticals.
References
1.Cummins,J. Algal recombinant pharmaceuticals in Hawaii 18 June 2005 Brief to Hearing,Plant Quarentine Honolulu Office 2.Rochaix,J. The three genomes of Chlamydomonas Photosynthesis Research 2002,73,285-93 3. Nishiyama R, Wada Y, Mibu M, Yamaguchi Y, Shimogawara K and Sano H. Role of a nonselective de novo DNA methyltransferase in maternal inheritance of chloroplast genes in the green alga, Chlamydomonas reinhardtii Genetics. 2004 Oct;168(2):809-16 4. Staub,J. Expression of eukaryotic peptides in plant plastids United States Patent 6,812,379 2004 5. Hefitietz,P. and Tuttle,A. Protein expression in plastids Current Opinion in Plant Biology 2001,4,157-61 6. Fiebig A, Stegemann S and Bock R. Rapid evolution of RNA editing sites in a small non-essential plastid gene Nucleic Acids Res. 2004 Jul 7;32(12):3615-22 7. Schroda M, Vallon O, Whitelegge JP, Beck CF and Wollman FA. The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing. Plant Cell. 2001 Dec;13(12):2823-39 8. Sun M, Qian K, Su N, Chang H, Liu J and Shen G. Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett. 2003 Jul;25(13):1087-92.

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