Used to look for changes in a quantitative trait This result suggests that with the TTBc Development of a Genetic Transformation System for Microalgae

right method to screen for mutants with the desired properties, mutagenesis could rnsuldlirc tmsroalgae with altered lipid compositions. However, this project was very

DtagRhaSvst twilil(hBCl<SreICanopuoadi^i?SofCí||iRlS^s Vlln})iSe?l^i^nreífng}•has become routine in a number of animal, bacterial, fungal, and plant systems. However, before the research was done at NREL, very little work in this area had been done with microalgae. In fact, the only species for which there was a reproducible transformation system was the single-celled, flagellated green alga C. reinhardtii, which is studied extensively in laboratories as a model photosynthetic cell. The focus of the research in the ASP during the early 1990s was to develop genetic transformation methods for microalgae with potential for biodiesel production. Based on the collection and screening efforts of the 1980s, this approach was considered to have the highest potential to produce organisms with high constitutive lipid levels, and to use genetic manipulation to understand the molecular regulation of lipid synthesis in the oleaginous algae. Studies on the biochemistry and molecular biology of lipid production in C. cryptica had identified

§gv6raco)Aocca£tsDxyi^easi a cuss edgratoey mowing1 n lipid ^oftes? r§poft°nhat were ms in orypgirou^thohp . oflLncf1íiiisin^^1peoifiiiviwyis)ft(0hesenuymi^ai!iilbhe puxmofixedind mariboer ^ereipthat were reported to function in other eukaryotic systems. Various methods were also tried to get DNA into the cell, initially focusing on enzymatically removing the cell wall or perturbating the cell membrane using electroporation.

Unsuccessful experiments represented a "Catch 22" scenario, as negative results could mean either the DNA was not getting into the cells, or the DNA entered but could not be expressed ,at detectable levels. Subsequent experiments were designed The projects that will be discussed here include a basic study on the DNA

to increase the understanding of the processes involved in DNA uptake and composition of microalgal strains, with implications for the choice of reporter or expression and to.increase the probability of obtaining transformants by developing marker genes used to monitor gene expression in transgenic algae. Other aspects of mh||)1^05(esi :iffofrhieeU^i^¡etLirig^ cl?^5t:iiiiir5ie=i(^^LmrrHi L tDhinAaiPi£PUa£riPe£p?§feon in

Chlorella protoplasts,

? attempts to develop heterologous and homologous genetic markers for algal transformation,

? the development of methods to introduce DNA into algal cells through the cell wall, and

? the successful development of a stable genetic transformation system for diatoms.

Once the methods were available to obtain genetic transformants, efforts were made to use the transformation system to manipulate lipid content in the algae by overexpressing or downregulating key genes. In addition, the transformation system was used to introduce a reporter gene under the control of various regulatory sequences, to better understand the regulation of gene expression in microalgae.

Analysis of Microalgal DNA Composition:

Several oleaginous microalgal strains had been identified as potential candidates for biodiesel fuel production. These organisms became the target of genetic engineering efforts to manipulate the lipid biosynthetic pathways. Before the work on genetic transformation of algae at NREL, very little information was available on the molecular biology of these organisms. One of the first steps was to develop techniques to isolate and purify DNA from these organisms. A desirable protocol would disrupt the cell wall using methods gentle enough to prevent shearing of the genomic DNA. This was not trivial for some species, such as Monoraphidium, which has a very resistant wall that contains sporopollenin. A method that worked for most species tested (described in Jarvis et al. 1992) was developed based on a protocol used to isolate yeast DNA (Hoffman and Winston 1987). The cells were suspended in buffer that contained 2% Triton X100 and 1% SDS, then added to a tube thai contained glass beads and an equal volume of phenol:cholorform:isoamyl alcohol NREL researcher Eric Jarvis .theorized that poor digestion of the „DNA J?y .some (PCLJ] The ceils were agitated for 1 minute using a vortex mixer. The DNA in the enzymes could be attributable to characteristics of the DNA. All DNA is composed of aqueous phase was purified by re-extraction with PCI, ethanol precipitation, and four , nucleosides; ■ deoxycytidine, i dloxyguanosine,i, deoxythymidine and treated wiin KNase A. For some species, the DNA had to be purififd further Dy using deoxyadenosine, (abbreviated dC, dG, dT, dA); in double stranded DNA, dC is always precipitation with hexadecyltrimethylammonium bromide (CTAB; Murray and paired with dG, and dT with dA. The percentage of each nucleoside (often Thompson 1980) to remove contaminating carboHydrates or by purifying the DNA on represented as %GCJ is variable between rSpecilSv Restriction enzymes cut DNA at CsCl gradients. This procedure produced DNA that digested well with many common specific nucleotide .sequences, _gfneraI1y recegniziftgiA4-6 bp motifs. Therefore, ithf restriction endonucleases, but even highly purified DNA would not digest well with all frequency of cutting by a particular enzyme will be affected by the total nucleotide restriction enzymes? J r J J

composition of the DNA (i.e., an enzyme that recognizes CCGG would cut infrequently in an organism with a low %GC). The GC content is also reflected in the codon usage by each organism, as DNA with a high GC content would show a bias toward codons ending with G or C in the variable third position. DNA can also contain unusual

modified nucleosides, including 5hydroxymethy1deoxycytidine (hm dC) and

5-hydroxymethyldeoxyuridine (hm dU), although the biological significance is unclear. Another common modification is the presence of methylated nucleosides, in

particular 5-methyldeoxycytidine (m dC) and 6-methyldeoxyadenosine (m dA). The degree of methylation has been associated with levels of gene expression. In addition, some microorganisms use DNA methylation as a defense mechanism, in that methylated DNA sequences are often not recognized by endonucleases from invading pathogens. Although the presence of methylated nucleosides is characteristic for some species, the degree of methylation can vary on a short time scale with changing environmental conditions' In contrast, the %GC and presence of modified nucleosides are characteristic for a particular organism. These characteristics only on an evolutionary time scale.

DNA was isolated from microalgae strains, including 10 species from 5 classes. The nucleoside composition was analyzed by reverse-phase HPLC and by digestion with restriction endonucleases. The results of the HPLC analysis are summarized in Table I.B.4-1. Although the diatoms showed a GC content typical for most eukaryotes (42%-48% GC), the GC content of the green algae (excepting Stichococcus) was significantly higher. In particular, Monoraphidium DNA contains 71% GC. The table

also shows the presence of m dC in the algal DNA. All species tested contained some level of this modified base, although once again Monoraphidium stands out with These data provided 5a good background for developing genetic transformation

. e hm5 (fee rnfleste^iiMh

G(t r . t o be true for the green alga Chlamydomonas; successful transformation of this organism was achieved only by the use of homologous selectable markers (discussed in more detail later). Also, GC content should be considered when designing synthetic DNA probes based on protein sequences, i.e.,

WMBon • o f DNSa 1iunteo!iiY1ep(SomPTCsttcdTTi o0{ DNAh fnleMYcíif§TiíciaT? fct the a^OiCSyftocd0fOinrUdUEitCstyííb(faifttlsiaaCl2tOPc•333 agCi -Ms ¿eisiIna:9 9h() uire the use of bacterial host strains that are insensitive to DNA methylation.

V.A. SERI/NREL/DOE REPORTS AND PUBLICATIONS V.B. ADDITIONAL REFERENCES V.C. PATENTS

Laboratory Studies

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