Impact of various ß-ketothiolase genes on PHBHHx production in Cupriavidus necator H16 derivatives.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active ß-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important ß-ketothiolases for C6 metabolism in ß-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. KEY POINTS: • Role of six ß-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter.

OCT1 - a yeast mitochondrial thiolase involved in the 3-oxoadipate pathway.

The 3-oxoacyl-CoA thiolases catalyze the last step of the fatty acid ß-oxidation pathway. In yeasts and plants, this pathway takes place exclusively in peroxisomes, whereas in animals it occurs in both peroxisomes and mitochondria. In contrast to baker's yeast Saccharomyces cerevisiae, yeast species from the Debaryomycetaceae family also encode a thiolase with predicted mitochondrial localization. These yeasts are able to utilize a range of hydroxyaromatic compounds via the 3-oxoadipate pathway the last step of which is catalyzed by 3-oxoadipyl-CoA thiolase and presumably occurs in mitochondria. In this work, we studied Oct1p, an ortholog of this enzyme from Candida parapsilosis. We found that the cells grown on a 3-oxoadipate pathway substrate exhibit increased levels of the OCT1 mRNA. Deletion of both OCT1 alleles impairs the growth of C. parapsilosis cells on 3-oxoadipate pathway substrates and this defect can be rescued by expression of the OCT1 gene from a plasmid vector. Subcellular localization experiments and LC-MS/MS analysis of enriched organellar fraction-proteins confirmed the presence of Oct1p in mitochondria. Phylogenetic profiling of Oct1p revealed an intricate evolutionary pattern indicating multiple horizontal gene transfers among different fungal groups.

[Analysis of ACAT1 gene variants in a patient with ß-ketothiolase deficiency].

OBJECTIVE: To explore the genetic etiology of a child suspected for ß-ketothiolase deficiency by neonatal screening. METHODS: All coding exons and flanking sequences of the ACAT1 gene were subjected to targeted capture and high-throughput sequencing. Suspected variants were verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor compound heterozygous variants of the ACAT1 gene, namely c.121-3C>G and c.275G>A (p. Gly92Asp). The c.121-3C>G variant was also detected in his father and two sisters, while the c.275G>A (p. Gly92Asp) was a de novo variant. A c.334+ 172C>G (rs12226047) polymorphism was also detected in his mother and two sisters. Sanger sequencing has verified that the c.275G>A (p. Gly92Asp) and c.334+172C>G (rs12226047) variants are located on the same chromosome. Bioinformatics analysis suggested both c.121-3C>G and c.275G>A (p.G92D) variants to be damaging. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.275G>A variant of the ACAT1 gene was predicted to be pathogenic (PS2+ PM2+ PM3+ PP3+PP4), the c.121-3C>G variant to be likely pathogenic (PM2+ PM3+ PP3+PP4). CONCLUSION: The c.121-3C>G and c.275G>A variants of the ACAT1 gene probably underlay the pathogenesis of the child. Above finding has enriched the variant spectrum of the ACAT1 gene.

Effects of fasting, feeding and exercise on plasma acylcarnitines among subjects with CPT2D, VLCADD and LCHADD/TFPD.

BACKGROUND: The plasma acylcarnitine profile is frequently used as a biochemical assessment for follow-up in diagnosed patients with fatty acid oxidation disorders (FAODs). Disease specific acylcarnitine species are elevated during metabolic decompensation but there is clinical and biochemical heterogeneity among patients and limited data on the utility of an acylcarnitine profile for routine clinical monitoring. METHODS: We evaluated plasma acylcarnitine profiles from 30 diagnosed patients with long-chain FAODs (carnitine palmitoyltransferase-2 (CPT2), very long-chain acyl-CoA dehydrogenase (VLCAD), and long-chain 3-hydroxy acyl-CoA dehydrogenase or mitochondrial trifunctional protein (LCHAD/TFP) deficiencies) collected after an overnight fast, after feeding a controlled low-fat diet, and before and after moderate exercise. Our purpose was to describe the variability in this biomarker and how various physiologic states effect the acylcarnitine concentrations in circulation. RESULTS: Disease specific acylcarnitine species were higher after an overnight fast and decreased by approximately 60% two hours after a controlled breakfast meal. Moderate-intensity exercise increased the acylcarnitine species but it varied by diagnosis. When analyzed for a genotype/phenotype correlation, the presence of the common LCHADD mutation (c.1528G > C) was associated with higher levels of 3-hydroxyacylcarnitines than in patients with other mutations. CONCLUSIONS: We found that feeding consistently suppressed and that moderate intensity exercise increased disease specific acylcarnitine species, but the response to exercise was highly variable across subjects and diagnoses. The clinical utility of routine plasma acylcarnitine analysis for outpatient treatment monitoring remains questionable; however, if acylcarnitine profiles are measured in the clinical setting, standardized procedures are required for sample collection to be of value.

Primrose syndrome: Characterization of the phenotype in 42 patients.

Primrose syndrome (PS; MIM# 259050) is characterized by intellectual disability (ID), macrocephaly, unusual facial features (frontal bossing, deeply set eyes, down-slanting palpebral fissures), calcified external ears, sparse body hair and distal muscle wasting. The syndrome is caused by de novo heterozygous missense variants in ZBTB20. Most of the 29 published patients are adults as characteristics appear more recognizable with age. We present 13 hitherto unpublished individuals and summarize the clinical and molecular findings in all 42 patients. Several signs and symptoms of PS develop during childhood, but the cardinal features, such as calcification of the external ears, cystic bone lesions, muscle wasting, and contractures typically develop between 10 and 16 years of age. Biochemically, anemia and increased alpha-fetoprotein levels are often present. Two adult males with PS developed a testicular tumor. Although PS should be regarded as a progressive entity, there are no indications that cognition becomes more impaired with age. No obvious genotype-phenotype correlation is present. A subgroup of patients with ZBTB20 variants may be associated with mild, nonspecific ID. Metabolic investigations suggest a disturbed mitochondrial fatty acid oxidation. We suggest a regular surveillance in all adult males with PS until it is clear whether or not there is a truly elevated risk of testicular cancer.

The 3-ketoacyl-CoA thiolase: an engineered enzyme for carbon chain elongation of chemical compounds.

Because of their function of catalyzing the rearrangement of the carbon chains, thiolases have attracted increasing attentions over the past decades. The 3-ketoacyl-CoA thiolase (KAT) is a member of the thiolase, which is capable of catalyzing the Claisen condensation reaction between the two acyl-CoAs, thereby achieving carbon chain elongation. In this way, diverse value-added compounds might be synthesized starting from simple small CoA thioesters. However, most KATs are hampered by low stability and poor substrate specificity, which has hindered the development of large-scale biosynthesis. In this review, the common characteristics in the three-dimensional structure of KATs from different sources are summarized. Moreover, structure-guided rational engineering is discussed as a strategy for enhancing the performance of KATs. Finally, we reviewed the metabolic engineering applications of KATs for producing various energy-storage molecules, such as n-butanol, fatty acids, dicarboxylic acids, and polyhydroxyalkanoates. KEY POINTS: • Summarize the structural characteristics and catalyzation mechanisms of KATs. • Review on the rational engineering to enhance the performance of KATs. • Discuss the applications of KATs for producing energy-storage molecules.

ACAA2 and FASN polymorphisms affect the fatty acid profile of Chios sheep milk.

The objective of the research reported in this research communication was the identification and association of single nucleotide polymorphisms (SNP) in the ovine DGAT1, FASN, SCD1 and ACAA2 genes with milk fat percentage and fatty acid (FA) content. Three consecutive monthly milk samplings were obtained from a total of 429 purebred Chios ewes during mid-lactation. Genotypic data were jointly analyzed with 1184 fat content and 37 718 FA percentage records using mixed models. The 3' untranslated region (UTR) of the DGAT1 gene and the 5' and 3'UTRs of the SCD1 gene appeared to be monomorphic. The FASN g.14777C>T SNP on exon 31 was associated with C13:0 and the ACAA2 g.2982T>C SNP on the 3'UTR was associated with C9:0, C11:0, C12:1 cis-9, C13:0 and the ω6/ω3 index, while fat percentage was not affected by the identified SNPs. The results could be useful for breeding programs aiming to improve the quality and nutritional value of ovine milk.

Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency.

Mitochondrial acetoacetyl-CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine-derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2-deficient patients. The 56 disease-associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease-associated missense ACAT1 variants have been measured by expressing them in human SV40-transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild-type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening.

MicroRNA-15a Regulates the Differentiation of Intramuscular Preadipocytes by Targeting ACAA1, ACOX1 and SCP2 in Chickens.

Our previous studies showed that microRNA-15a (miR-15a) was closely related to intramuscular fat (IMF) deposition in chickens; however, its regulatory mechanism remains unclear. Here, we evaluated the expression characteristics of miR-15a and its relationship with the expression of acetyl-CoA acyltransferase 1 (ACAA1), acyl-CoA oxidase 1 (ACOX1) and sterol carrier protein 2 (SCP2) by qPCR analysis in Gushi chicken breast muscle at 6, 14, 22, and 30 weeks old, where we performed transfection tests of miR-15a mimics in intramuscular preadipocytes and verified the target gene of miR-15a in chicken fibroblasts (DF1). The miR-15a expression level at 30 weeks increased 13.5, 4.5, and 2.7-fold compared with the expression levels at 6, 14, and 22 weeks, respectively. After 6 days of induction, miR-15a over-expression significantly promoted intramuscular adipogenic differentiation and increased cholesterol and triglyceride accumulation in adipocytes. Meanwhile, 48 h after transfection with miR-15a mimics, the expression levels of ACAA1, ACOX1 and SCP2 genes decreased by 56.52%, 31.18% and 37.14% at the mRNA level in intramuscular preadipocytes. In addition, the co-transfection of miR-15a mimics and ACAA1, ACOX1 and SCP2 3'UTR (untranslated region) dual-luciferase vector significantly inhibited dual-luciferase activity in DF1 cells. Taken together, our data demonstrate that miR-15a can reduce fatty acid oxidation by targeting ACAA1, ACOX1, and SCP2, which subsequently indirectly promotes the differentiation of chicken intramuscular preadipocytes.

Beta-ketothiolase deficiency in a Malaysian infant.

Methylacetoacetyl-coenzyme A thiolase (MAT) deficiency is an autosomal recessive disease caused by a defect of mitochondrial acetoacetyl-CoA thiolase (T2). There is an error of isoleucine catabolism and ketone body utilization due to mutations in the acetyl-Coenzyme A acetyltransferase 1 (ACAT1) gene. We report a case of a 14 months old Sabahan boy with beta deficiency who presented with severe sepsis and ketoacidosis who subsequently recovered.

The role of yeast m6A methyltransferase in peroxisomal fatty acid oxidation.

The precise and controlled regulation of gene expression at transcriptional and post-transcriptional levels is crucial for the eukaryotic cell survival and functions. In eukaryotes, more than 100 types of post-transcriptional RNA modifications have been identified. The N6-methyladenosine (m6A) modification in mRNA is among the most common post-transcriptional RNA modifications known in eukaryotic organisms, and the m6A RNA modification can regulate gene expression. The role of yeast m6A methyltransferase (Ime4) in meiosis, sporulation, triacylglycerol metabolism, vacuolar morphology, and mitochondrial functions has been reported. Stress triggers triacylglycerol accumulation as lipid droplets. Lipid droplets are physically connected to the different organelles such as endoplasmic reticulum, mitochondria, and peroxisomes. However, the physiological relevance of these physical interactions remains poorly understood. In yeast, peroxisome is the sole site of fatty acid ß-oxidation. The metabolic status of the cell readily governs the number and physiological function of peroxisomes. Under low-glucose or stationary-phase conditions, peroxisome biogenesis and proliferation increase in the cells. Therefore, we hypothesized a possible role of Ime4 in the peroxisomal functions. There is no report on the role of Ime4 in peroxisomal biology. Here, we report that IME4 gene deletion causes peroxisomal dysfunction under stationary-phase conditions in Saccharomyces cerevisiae; besides, the ime4Δ cells showed a significant decrease in the expression of the key genes involved in peroxisomal ß-oxidation compared to the wild-type cells. Therefore, identification and determination of the target genes of Ime4 that are directly involved in the peroxisomal biogenesis, morphology, and functions will pave the way to better understand the role of m6A methylation in peroxisomal biology.

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria.

Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steady-state flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shc-depleted mice in vivo.

Reversal of ß-oxidative pathways for the microbial production of chemicals and polymer building blocks.

ß-Oxidation is the ubiquitous metabolic strategy to break down fatty acids. In the course of this four-step process, two carbon atoms are liberated per cycle from the fatty acid chain in the form of acetyl-CoA. However, typical ß-oxidative strategies are not restricted to monocarboxylic (fatty) acid degradation only, but can also be involved in the utilization of aromatic compounds, amino acids and dicarboxylic acids. Each enzymatic step of a typical ß-oxidation cycle is reversible, offering the possibility to also take advantage of reversed metabolic pathways for applied purposes. In such cases, 3-oxoacyl-CoA thiolases, which catalyze the final chain-shortening step in the catabolic direction, mediate the condensation of an acyl-CoA starter molecule with acetyl-CoA in the anabolic direction. Subsequently, the carbonyl-group at C3 is stepwise reduced and dehydrated yielding a chain-elongated product. In the last years, several ß-oxidation pathways have been studied in detail and reversal of these pathways already proved to be a promising strategy for the production of chemicals and polymer building blocks in several industrially relevant microorganisms. This review covers recent advancements in this field and discusses constraints and bottlenecks of this metabolic strategy in comparison to alternative production pathways.

Clinical presentation and outcome in a series of 32 patients with 2-methylacetoacetyl-coenzyme A thiolase (MAT) deficiency.

2-methylacetoacetyl-coenzyme A thiolase (MAT) deficiency, also known as beta-ketothiolase deficiency, is an inborn error of ketone body utilization and isoleucine catabolism. It is caused by mutations in the ACAT1 gene and may present with metabolic ketoacidosis. In order to obtain a more comprehensive view on this disease, we have collected clinical and biochemical data as well as information on ACAT1 mutations of 32 patients from 12 metabolic centers in five countries. Patients were between 23months and 27years old, more than half of them were offspring of a consanguineous union. 63% of the study participants presented with a metabolic decompensation while most others were identified via newborn screening or family studies. In symptomatic patients, age at manifestation ranged between 5months and 6.8years. Only 7% developed a major mental disability while the vast majority was cognitively normal. More than one third of the identified mutations in ACAT1 are intronic mutations which are expected to disturb splicing. We identified several novel mutations but, in agreement with previous reports, no clear genotype-phenotype correlation could be found. Our study underlines that the prognosis in MAT deficiency is good and MAT deficient individuals may remain asymptomatic, if diagnosed early and preventive measures are applied.

Characterization and outcome of 41 patients with beta-ketothiolase deficiency: 10 years' experience of a medical center in northern Vietnam.

Beta-ketothiolase (T2) deficiency is an inherited disease of isoleucine and ketone body metabolism caused by mutations in the ACAT1 gene. Between 2005 and 2016, a total of 41 patients with T2 deficiency were identified at a medical center in northern Vietnam, with an estimated incidence of one in 190,000 newborns. Most patients manifested ketoacidotic episodes of varying severity between 6 and 18 months of age. Remarkably, 28% of patients showed high blood glucose levels (up to 23.3 mmol/L). Ketoacidotic episodes recurred in 43% of patients. The age of onset, frequency of episodes, and identified genotype did not affect patient outcomes that were generally favorable, with the exception of seven cases (five died and two had neurological sequelae). Custom-tailored acute and follow-up management was critical for a positive clinical outcome. Two null mutations, c.622C>T (p.Arg208*) and c.1006-1G>C (p.Val336fs), accounted for 66% and 19% of all identified ACAT1 mutant alleles, respectively. Most patients showed characteristic biochemical abnormalities. A newborn screening program could be expected to have a high yield in Vietnam. Investigation findings of haplotypes linked to the most common ACAT1 mutation (c.622C>T) are consistent with an ancient common founder of mutation-bearing chromosomes belonging to the Kinh ethnic population. The direct management and long-term follow-up of a large number of T2-deficient patients enabled us to study the natural history of this rare disease.

Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulation in Arxula adeninivorans by stabilization of production.

BACKGROUND: In recent years the production of biobased biodegradable plastics has been of interest of researchers partly due to the accumulation of non-biodegradable plastics in the environment and to the opportunity for new applications. Commonly investigated are the polyhydroxyalkanoates (PHAs) poly(hydroxybutyrate) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-V). The latter has the advantage of being tougher and less brittle. The production of these polymers in bacteria is well established but production in yeast may have some advantages, e.g. the ability to use a broad spectrum of industrial by-products as a carbon sources. RESULTS: In this study we increased the synthesis of PHB-V in the non-conventional yeast Arxula adeninivorans by stabilization of polymer accumulation via genetic modification and optimization of culture conditions. An A. adeninivorans strain with overexpressed PHA pathway genes for ß-ketothiolase, acetoacetyl-CoA reductase, PHAs synthase and the phasin gene was able to accumulate an unexpectedly high level of polymer. It was found that an optimized strain cultivated in a shaking incubator is able to produce up to 52.1% of the DCW of PHB-V (10.8 g L-1) with 12.3%mol of PHV fraction. Although further optimization of cultivation conditions in a fed-batch bioreactor led to lower polymer content (15.3% of the DCW of PHB-V), the PHV fraction and total polymer level increased to 23.1%mol and 11.6 g L-1 respectively. Additionally, analysis of the product revealed that the polymer has a very low average molecular mass and unexpected melting and glass transition temperatures. CONCLUSIONS: This study indicates a potential of use for the non-conventional yeast, A. adeninivorans, as an efficient producer of polyhydroxyalkanoates.

High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells.

Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation.

Oligo(cis-1,4-isoprene) aldehyde-oxidizing dehydrogenases of the rubber-degrading bacterium Gordonia polyisoprenivorans VH2.

The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874-2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via ß-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.

Improved production of adipate with Escherichia coli by reversal of ß-oxidation.

The linear C6 dicarboxylic acid adipic acid is an important bulk chemical in the petrochemical industry as precursor of the polymer nylon-6,6-polyamide. In recent years, efforts were made towards the biotechnological production of adipate from renewable carbon sources using microbial cells. One strategy is to produce adipate via a reversed ß-oxidation pathway. Hitherto, the adipate titers were very low due to limiting enzyme activities for this pathway. In most cases, the CoA intermediates are non-natural substrates for the tested enzymes and were therefore barely converted. We here tested heterologous enzymes in Escherichia coli to overcome these limitations and to improve the production of adipate via a reverse ß-oxidation pathway. We tested in vitro selected enzymes for the efficient reduction of the enoyl-CoA and in the final reaction for the thioester cleavage. The genes encoding the enzymes which showed in vitro the highest activity were then used to construct an expression plasmid for a synthetic adipate pathway. Expression of paaJ, paaH, paaF, dcaA, and tesB in E. coli BL21(DE3) resulted in the production of up to 36 mg/L of adipate after 30 h of cultivation. Beside the activities of the pathway enzymes, the availability of metabolic precursors may limit the synthesis of adipate, providing another key target for further strain engineering towards high-yield production of adipate with E. coli.

Clinical and molecular analysis of 6 Chinese patients with isoleucine metabolism defects: identification of 3 novel mutations in the HSD17B10 and ACAT1 gene.

Hydroxysteroid (17ß) dehydrogenase 10 (HSD10) and mitochondrial acetoacetyl-CoA thiolase (ß-KT) are two adjacent enzymes for the degradation of isoleucine, thus HSD10 and ß-KT deficiencies are confusing at an early stage because of nearly the same elevation of typical metabolites in urine, such as 2-methyl-3-hydroxybutyric acid (2M3HBA) and tiglylglycine (TG). In order to better understand the differences between these two disorders, we described the clinical and molecular characteristics of two HSD10 deficiency patients and four ß-KT deficiency patients. ß-KT deficiency patients had a much more favorable outcome than that of HSD10 deficiency patients, indicating that the multifunction of HSD10, especially neurosteroid metabolic activity, other than only enzymatic degradation of isoleucine, is involved in the pathogenesis of HSD10 deficiency. Two different mutations, a novel mutation p.Ile175Met and a reported mutation p.Arg226Gln, were detected in the HSD17B10 gene of HSD10 deficiency patients. Six different mutations, including four known mutations: p.Ala333Pro, p.Thr297Lys, c.83_84delAT, c.1006-1G > C, and two novel mutations: p.Thr277Pro and c.121-3C > G were identified in the ACAT1 gene of ß-KT deficiency patients. In general, DNA diagnosis played an important role in distinguishing between these two disorders.