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

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 Rhodospirillum rubrum, 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. In R. rubrum, the AACT enzyme is encoded within the phaABC1 operon, which is responsible for PHA biosynthesis, in addition there are two phaC1–like genes in the genome, called phaC2 and phaC3. Furthermore, R. rubrum contains one phaJ 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 pha–operon pathway for PHA biosynthesis, and that PhaC2 is the major PHA polymerase for synthesizing PHA in vivo. 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 in vivo. Moreover, monitoring the growth and analyzing the PHA content of R. rubrum mutants indicate that eliminating PHA affects R. rubrum growth. Genomic analyses revealed two AACT genes in the Arabidopsis genome, At5g47720 (AACT1) and At5g48230 (AACT2). These two genes code for proteins that share 78.4% sequence identity. Complementation of yeast AACT knock–out mutant Δerg10 shows that both Arabidopsis AACTs are functional. To study the physiological function of each AACT–coding genes, two T–DNA insertion alleles at AACT1 and one T–DNA insertion allele at AACT2 gene have been characterized. These characterizations indicate that although both genes are expressed (as evidenced by western analysis); mutation in AACT2 is embryo lethal whereas null alleles of AACT1 are viable and show no apparent growth phenotypes. Furthermore, segregation analysis and genetic complementation demonstrate that mutations in AACT2 affect male transmission, and in vivo pollen germination and elongation. Promoter::GUS fusion experiments indicate that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. AACT2–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 AACT2–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 aact1 mutant. These results indicate that AACT2 is essential in plant growth and development and cannot be replaced by AACT1. 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

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C5 and C6 monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer

The 3-hydroxyacyl-ACP dehydratase component of the plant mitochondrial fatty acid synthase system

We report the characterization of the Arabidopsis 3-hydroxyacyl-acyl carrier protein (ACP) dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein (GFP) transgenesis experiment, and by in vivo complementation and in vitro enzymatic assays. RNAi knockdown lines with reduced mtHD expression exhibit traits typically associated with mtFAS mutants, namely a miniaturized morphological appearance, reduced lipoylation of lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO2 atmosphere, indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition, both mthd-rnai and mtkas mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO2 atmosphere, demonstrating two novel mtFAS functions that are independent of photorespiration. Finally, RNA-Seq analysis revealed that mthd-rnai and mtkas mutants are near equivalent to each other in altering transcriptome, and these analyses further identified genes whose expression is affected by a functional mtFAS system, but independent of photorespiratory deficiency. These data demonstrate the non-redundant nature of the mtFAS system, which contributes unique lipid components needed to support plant cell structure and metabolism

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

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 Rhodospirillum rubrum, 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. In R. rubrum, the AACT enzyme is encoded within the phaABC1 operon, which is responsible for PHA biosynthesis, in addition there are two phaC1–like genes in the genome, called phaC2 and phaC3. Furthermore, R. rubrum contains one phaJ 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 pha–operon pathway for PHA biosynthesis, and that PhaC2 is the major PHA polymerase for synthesizing PHA in vivo. 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 in vivo. Moreover, monitoring the growth and analyzing the PHA content of R. rubrum mutants indicate that eliminating PHA affects R. rubrum growth. Genomic analyses revealed two AACT genes in the Arabidopsis genome, At5g47720 (AACT1) and At5g48230 (AACT2). These two genes code for proteins that share 78.4% sequence identity. Complementation of yeast AACT knock–out mutant Δerg10 shows that both Arabidopsis AACTs are functional. To study the physiological function of each AACT–coding genes, two T–DNA insertion alleles at AACT1 and one T–DNA insertion allele at AACT2 gene have been characterized. These characterizations indicate that although both genes are expressed (as evidenced by western analysis); mutation in AACT2 is embryo lethal whereas null alleles of AACT1 are viable and show no apparent growth phenotypes. Furthermore, segregation analysis and genetic complementation demonstrate that mutations in AACT2 affect male transmission, and in vivo pollen germination and elongation. Promoter::GUS fusion experiments indicate that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. AACT2–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 AACT2–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 aact1 mutant. These results indicate that AACT2 is essential in plant growth and development and cannot be replaced by AACT1. 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

Evaluating PHA Productivity of Bioengineered Rhodosprillum rubrum

This study explored the potential of using Rhodosprillum rubrum as the biological vehicle to convert chemically simple carbon precursors to a value-added bio-based product, the biopolymer PHA. R. rubrum strains were bioengineered to overexpress individually or in various combinations, six PHA biosynthetic genes (phaC1, phaA, phaB, phaC2, phaC3, and phaJ), and the resulting nine over-expressing strains were evaluated to assess the effect on PHA content, and the effect on growth. These experiments were designed to genetically evaluate: 1) the role of each apparently redundant PHA polymerase in determining PHA productivity; 2) identify the key gene(s) within the pha biosynthetic operon that determines PHA productivity; and 3) the role of phaJ to support PHA productivity. The result of overexpressing each PHA polymerase-encoding gene indicates that phaC1 and phaC2 are significant contributors to PHA productivity, whereas phaC3 has little effect. Similarly, over-expressing individually or in combination the three PHA biosynthesis genes located in the pha operon indicates that phaB is the key determinant of PHA productivity. Finally, analogous experiments indicate that phaJ does not contribute significantly to PHA productivity. These bioengineering strains achieved PHA productivity of up to 30% of dry biomass, which is approximately 2.5-fold higher than the non-engineered control strain, indicating the feasibility of using this approach to produce value added bio-based products.This is an article from PLoS ONE 9 (2014): 1, doi:10.1371/journal.pone.0096621. Posted with permission.</p

Study on the Degree of Rural Empty Nesters' Satisfaction with Life Quality Based on Ordered Logit－ISM Model

It is an ultimate objective to solve the rural supporting problems to improve life satisfaction of empty nesters in rural areas. Based on data of 546 empty nesters in rural areas, the factors influencing life quality satisfaction of empty nesters in rural areas are firstly analyzed using ordered logit model, and then the relationship between various influencing factors is analyzed using ISM. Study shows that health condition, degree of participation in cultural and sports activities, spouse status, family harmony degree, annual family income, degree of worry about life, degree of government and social care and pension mode, have a significant impact on the life quality satisfaction of empty nesters in rural areas. The degree of worry about life, degree of government and social care are surface direct factors; degree of participation in cultural and sports activities, family harmony degree, pension mode are intermediate indirect factors; health condition, spouse status and annual family income are deep root factors

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C5 and C6 monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.This is an article from Journal of Bacteriology 194 (2012): 5522, doi:10.1128/JB.01111-12. Posted with permission.</p

Study on the Degree of Rural Empty Nesters' Satisfaction with Life Quality Based on Ordered Logit－ISM Model

It is an ultimate objective to solve the rural supporting problems to improve life satisfaction of empty nesters in rural areas. Based on data of 546 empty nesters in rural areas, the factors influencing life quality satisfaction of empty nesters in rural areas are firstly analyzed using ordered logit model, and then the relationship between various influencing factors is analyzed using ISM. Study shows that health condition, degree of participation in cultural and sports activities, spouse status, family harmony degree, annual family income, degree of worry about life, degree of government and social care and pension mode, have a significant impact on the life quality satisfaction of empty nesters in rural areas. The degree of worry about life, degree of government and social care are surface direct factors; degree of participation in cultural and sports activities, family harmony degree, pension mode are intermediate indirect factors; health condition, spouse status and annual family income are deep root factors

Evaluating PHA Productivity of Bioengineered <i>Rhodosprillum rubrum</i>

<div><p>This study explored the potential of using <i>Rhodosprillum rubrum</i> as the biological vehicle to convert chemically simple carbon precursors to a value-added bio-based product, the biopolymer PHA. <i>R. rubrum</i> strains were bioengineered to overexpress individually or in various combinations, six PHA biosynthetic genes (<i>phaC1</i>, <i>phaA</i>, <i>phaB</i>, <i>phaC2</i>, <i>phaC3, and phaJ</i>), and the resulting nine over-expressing strains were evaluated to assess the effect on PHA content, and the effect on growth. These experiments were designed to genetically evaluate: 1) the role of each apparently redundant PHA polymerase in determining PHA productivity; 2) identify the key gene(s) within the <i>pha</i> biosynthetic operon that determines PHA productivity; and 3) the role of <i>phaJ</i> to support PHA productivity. The result of overexpressing each PHA polymerase-encoding gene indicates that <i>phaC1</i> and <i>phaC2</i> are significant contributors to PHA productivity, whereas <i>phaC3</i> has little effect. Similarly, over-expressing individually or in combination the three PHA biosynthesis genes located in the <i>pha</i> operon indicates that <i>phaB</i> is the key determinant of PHA productivity. Finally, analogous experiments indicate that <i>phaJ</i> does not contribute significantly to PHA productivity. These bioengineering strains achieved PHA productivity of up to 30% of dry biomass, which is approximately 2.5-fold higher than the non-engineered control strain, indicating the feasibility of using this approach to produce value added bio-based products.</p></div

Recombination based integration of the CO-inducible <i>P_CooF</i> promoter for bioengineering the overexpression of <i>pha</i> biosynthetic genes.

<p>Construction of the suicide plasmids pUX19-pha(gene) is described in the Material and Methods. Recombination at either the <i>P_CooF</i> locus or the <i>pha</i> structural gene provides a mechanism for generating different strains listed in Table I that carry alleles for overexpressing the <i>pha</i> biosynthetic genes.</p