Genetic, biochemical and physiological studies of acetyl-CoA metabolism via acyl-condensation

dc.contributor.advisor Basil J. Nikolau Jin, Huanan
dc.contributor.department Biochemistry, Biophysics and Molecular Biology 2018-08-12T03:08:01.000 2020-06-30T02:34:47Z 2020-06-30T02:34:47Z Fri Jan 01 00:00:00 UTC 2010 2013-06-05 2010-01-01
dc.description.abstract <p>Acetyl–CoA is metabolized via one of three mechanisms: carboxylation, acetylation and condensation. Acetoacetyl–CoA thiolase (AACT) catalyzes the condensation of two acetyl–CoA molecules to form acetoacetyl–CoA. The metabolic fate of acetoacetyl–CoA depends on the biological context in which it is generated. In microbes, such as <i>Rhodospirillum rubrum</i>, acetoacetyl–CoA is the precursor of the storage polymer polyhydroxyalkanoate (PHA). In the cytosol of plant cells, it is the precursor of mevalonate–derived isoprenoids.</p> <p>In <i>R. rubrum</i>, the AACT enzyme is encoded within the <i>phaABC1</i> operon, which is responsible for PHA biosynthesis, in addition there are two <i>phaC1</i>–like genes in the genome, called <i>phaC2</i> and <i>phaC3</i>. Furthermore, <i>R. rubrum</i> contains one <i>phaJ</i> gene, encoding (R)–specific 2–enoyl–CoA hydratase. To characterize the roles of these genes in PHA biosynthesis, I generated over–expressing and deletion mutants of PHA genes. Characterization of these mutants show that PhaB is the key enzyme of the <i>pha</i>–operon pathway for PHA biosynthesis, and that PhaC2 is the major PHA polymerase for synthesizing PHA <i>in vivo</i>. These studies further demonstrated that PhaC2 is responsible for integrating 3–hydroxybutyrate, 3–hydroxyvalerate and 3–hydroxyhexanoate monomers into the PHA–polymer, whereas PhaC1 and PhaC3 are specific for integrating 3–hydroxybutyrate. These data also indicate that PhaC1 and PhaC3 may interact with each other. We also demonstrated that PhaJ is responsible for converting trans–2,3–enoylacyl–CoA to (R)–3–hydroxyacyl–CoA <i>in vivo</i>. Moreover, monitoring the growth and analyzing the PHA content of <i>R. rubrum</i> mutants indicate that eliminating PHA affects <i>R. rubrum</i> growth.</p> <p>Genomic analyses revealed two AACT genes in the <i>Arabidopsis</i> genome, At5g47720 (<i>AACT1</i>) and At5g48230 (<i>AACT2</i>). These two genes code for proteins that share 78.4% sequence identity. Complementation of yeast <i>AACT</i> knock–out mutant <i>Δerg10</i> shows that both <i>Arabidopsis</i> AACTs are functional. To study the physiological function of each AACT–coding genes, two T–DNA insertion alleles at <i>AACT1</i> and one T–DNA insertion allele at <i>AACT2</i> gene have been characterized. These characterizations indicate that although both genes are expressed (as evidenced by western analysis); mutation in <i>AACT2</i> is embryo lethal whereas null alleles of <i>AACT1</i> are viable and show no apparent growth phenotypes. Furthermore, segregation analysis and genetic complementation demonstrate that mutations in <i>AACT2</i> affect male transmission, and <i>in vivo</i> pollen germination and elongation. Promoter::GUS fusion experiments indicate that <i>AACT1</i> is primarily expressed in the vascular system and <i>AACT2</i> is highly expressed in root tips, young leaves, top stems and anthers. <i>AACT2</i>–RNAi lines show pleiotropic phenotypes, including elongated life–span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that dwarfing is caused by smaller cell size and reduced cell numbers and loss of pollen coat resulted in male–sterility, probably due to the faster degeneration of tapetum cells during pollen development. These phenotypes were rescued when mutant plants were grown in the presence of mevalonate. Phytosterol analysis of <i>AACT2</i>–RNAi plants shows reduced sterol content and altered composition in the seedling roots. The accumulation of these sterols was restored to wild type levels when the plants were feed with mevalonate. In contrast, no significant phytosterol changes were detected in the <i>aact1</i> mutant. These results indicate that <i>AACT2</i> is essential in plant growth and development and cannot be replaced by <i>AACT1</i>.</p> <p>In combination, the product of acetyl–CoA condensation, acetoacetyl–CoA, is used for producing different biomolecules in different organisms. Correspondingly, knock–out of this pathway results in different consequences in different organisms. Deleting acetyl–CoA condensation pathway affects growth in microbes, whereas it leads to lethality in plants.</p>
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dc.identifier.articleid 2231
dc.identifier.contextkey 2807429
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath etd/11309
dc.language.iso en
dc.source.bitstream archive/|||Fri Jan 14 18:47:30 UTC 2022
dc.subject.disciplines Biochemistry, Biophysics, and Structural Biology
dc.subject.keywords Acetoacetyl-CoA thiolase
dc.subject.keywords Arabidopsis thaliana
dc.subject.keywords Polyhydroxyalkanoate
dc.subject.keywords Rhodospirillum rubrum
dc.title Genetic, biochemical and physiological studies of acetyl-CoA metabolism via acyl-condensation
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication faf0a6cb-16ca-421c-8f48-9fbbd7bc3747 dissertation Doctor of Philosophy
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