Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Differential expression of hydroxyacyl-CoA dehydrogenase in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding hydroxyacyl-CoA dehydrogenase, HADH, when comparing primary tumors of the breast to the tissue of origin, the normal breast. HADH was also differentially expressed in lymph node metastasis in human breast cancer. HADH mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of HADH in primary tumors of the breast was correlated with distant metastasis-free survival in patients with basal-like subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. HADH may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Molecular characterization and immune protection of the 3-hydroxyacyl-CoA dehydrogenase gene in Echinococcus granulosus

Background Cystic echinococcosis (CE) is a serious parasitic zoonosis caused by the larvae of the tapeworm Echinococcus granulosus. The development of an effective vaccine is one of the most promising strategies for controlling CE. Methods The E. granulosus 3-hydroxyacyl-CoA dehydrogenase (EgHCDH) gene was cloned and expressed in Escherichia coli. The distribution of EgHCDH in protoscoleces (PSCs) and adult worms was analyzed using immunofluorescence. The transcript levels of EgHCDH in PSCs and adult worms were analyzed using quantitative real-time reverse transcription PCR (RT-qPCR). The immune protective effects of the rEgHCDH were evaluated. Results The 924-bp open reading frame sequence of EgHCDH, which encodes a protein of approximately 34 kDa, was obtained. RT-qPCR analysis revealed that EgHCDH was expressed in both the PSCs and adult worms of E. granulosus. Immunofluorescence analysis showed that EgHCDH was mainly localized in the tegument of PSCs and adult worms. Western blot analysis showed that the recombinant protein was recognized by E. granulosus-infected dog sera. Animal challenge experiments demonstrated that dogs immunized with recombinant (r)EgHCDH had significantly higher serum IgG, interferon gamma and interleukin-4 concentrations than the phosphate-buffered saline (PBS) control group. The rEgHCDH vaccine was able to significantly reduce the number of E. granulosus and inhibit the segmental development of E. granulosus compared to the PBS control group. Conclusions The results suggest that rEgHCDH can induce partial immune protection against infection with E. granulosus and could be an effective candidate for the development of new vaccines. Graphical abstract

Impact of Newborn Screening and Early Dietary Management on Clinical Outcome of Patients with Long Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency and Medium Chain Acyl-CoA Dehydrogenase Deficiency—A Retrospective Nationwide Study

Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD/MTPD) and medium chain acyl-CoA dehydrogenase deficiency (MCADD) were included in the expanded neonatal screening program (ENBS) in Czechia in 2009, allowing for the presymptomatic diagnosis and nutritional management of these patients. The aim of our study was to assess the nationwide impact of ENBS on clinical outcome. This retrospective study analysed acute events and chronic complications and their severity in pre-ENBS and post-ENBS cohorts. In total, 28 children (12 before, 16 after ENBS) were diagnosed with LCHADD/MTPD (incidence 0.8/100,000 before and 1.2/100,000 after ENBS). In the subgroup detected by ENBS, a significantly longer interval from birth to first acute encephalopathy was observed. In addition, improvement in neuropathy and cardiomyopathy (although statistically non-significant) was demonstrated in the post-ENBS subgroup. In the MCADD cohort, we included 69 patients (15 before, 54 after ENBS). The estimated incidence rose from 0.7/100,000 before to 4.3/100,000 after ENBS. We confirmed a significant decrease in the number of episodes of acute encephalopathy and lower proportion of intellectual disability after ENBS (p < 0.0001). The genotype–phenotype correlations suggest a new association between homozygosity for the c.1528C > G variant and more severe heart involvement in LCHADD patients.

Improving the Production of 9,21-di​hydroxy-​20-​methyl-pregna-​4-​en-​3-​one from Phytosterols in Mycobacterium Neoaurum by Modifying Multiple Genes and Improving the Intracellular Environment

BackgroundSteroid drugs are particularly important for disease prevention and clinical treatment. However, traditional chemical methods are rarely implemented during the whole synthetic process to generate steroid intermediates due to the intricate steroid structure. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9-OH-4-HP is a potential steroid drug precursor for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain.ResultsA Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5 encoding an acyl-COA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains could accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 double-deficient strain was 11.9% higher than that of the Hsd4A -deficient strain and 40.4% higher than that of the strain with FadA5 deficiency, and its selectivity reached 94.9%. Subsequently, the catalase katE from Mycobacterium and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the selectivity of 9-OH-4-HP improved to 97%.Conclusionhsd4A and fadA5 are key enzymes in the C19 pathway for phytosterol side chain degradation. Deletion of hsd4A and fadA5 could almost entirely block the C19 pathway. Improving the intracellular environment of Mycobacterium during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Burkholderia Pseudomallei Short-Chain Dehydrogenase/ Oxidoreductase: Potential Urine Biomarker Candidate for Acute Melioidosis

INTRODUCTION: In the current climate of urgency in identifying biomarkers for the development of rapid diagnostic kits, the use of urine samples to diagnose acute melioidosis was evaluated, comparing urine samples from Burkholderia pseudomallei culture-positive and culture-negative patients, and comparing pneumonic and septicemic melioidosis. MATERIAL AND METHODS: Eleven urine samples from clinically suspected melioidosis patients from a tertiary referral center, Hospital Tengku Ampuan Afzan, Pahang was used. An in-solution method for the detection of bacterial proteins using liquid chromatography-mass spectrometry quadrupole time-of-flight (LCMS QTOF) was used. RESULTS: Three bacterial proteins were consistently detected among all the culture[1]positive and PCR-positive cases tested, namely SDR family NAD(P)-dependent oxidoreductase protein (32kDa), 3-hydroxyacyl-CoA dehydrogenase Burkholderia sp. (32kDa), and NAD(P)-dependent dehydrogenase (short-subunit alcohol dehydrogenase family) Burkholderia sp. (33kDa). CONCLUSIONS: Short-chain dehydrogenase (SDO) proteins could potentially be a urine biomarker candidate as these have shown to aid in the ability of Burkholderia spp. to invade host cells as this action is important for the initial intracellular survival of the organism.

Molecular Characterization and Expression Analysis of the Gene Encoding 3-Hydroxyacyl-CoA Dehydrogenase (EGR-03347) from Echinococcus Granulosus and the Evaluation of the Immune Protection of the Definitive Hosts (dogs)

Background: Cystic echinococcosis, a serious parasitic zoonosis, is caused by tapeworm (Echinococcus granulosus) larvae. The development of an effective vaccine is a promising strategy to control echinococcosis. E. granulosus has a complete tricarboxylic acid cycle pathway, in which 3-hydroxyacyl-CoA dehydrogenase (EGR-03347) is a key enzyme.Methods: In the present study, the cDNA encoding EGR-03347 in Echinococcus granulosus (rEGR-03347) was successfully cloned and the molecular and biochemical characterizations carried out. The immunoreactivity of recombinant EGR-03347 (rEGR-03347) was investigated using western blotting. The immunolocalization of EGR-03347 in different life stages of E. granulosus was determined using specific polyclonal antibody, quantitative real-time reverse transcription polymerase chain reaction was used to analyze their transcript levels in PSCs and 28-day strobilated worms stages. In addition, recombinant protein rEGR-03347 was mixed with the adjuvant Quil A for vaccinating dogs, after three vaccine injections, all the dogs were orally challenge-infected with 100000 protoscoleces of E. granulosus. After 28 days of infection, all the dogs were euthanized and necropsied for collecting and counting E. granulosus worms, post-infection the antibody and cytokine were measured for the immunogenicity analysis of this protein.Results: rEGR-03347 is a highly conserved protein, consisting of 308 amino acids. Recombinant EGR-03347 could be identifed in the sera of patients with CE and in mouse anti-rEGR-03347 sera. Immunofluorescence analysis showed that EGR-03347 mainly localized in the tegument of protoscoleces and adults, and their transcript levels were high in the 28-day strobilated worms. Furthermore, enzyme-linked immunosorbent assays post‑infection showed that IgG gradually increased after the first immunization with rEGR-03347 compared with the control group, rEGR-03347 changed the interferon gamma and interleukin 4 levels. We observed an 87.2 % reduction in E. granulosus numbers and 66.7 % inhibition of the segmental development of E. granulosus in the rEGR‑03347‑vaccinated dogs compared with the nonvaccinated controls.Conclusions: This is the first report characterizing a 3-hydroxyacyl-CoA dehydrogenase from the tapeworm E. granulosus. We have characterized the sequence, structure and location of EGR‑03347 and investigated the immunoreactivity, immunogenicity and serodiagnostic potential of rEGR‑03347 . The results demonstrated rEGR-03347 as a potential vaccine against E. granulosus infection in dogs.

An application experience of a specialized product based on 100% medium-chain triglyceride oil in diet therapy of a child with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD) is a hereditary disease referred to the group of disorders of mitochondrial β-oxidation of fatty acids with autosomal recessive inheritance. The main symptoms include hypoglycemia, hepatic steatosis, cardiomyopathy, cardiac arrhythmias, progressive muscle hypotension. We present a case of successful diagnosis and treatment of a long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD) with the use of 100% medium chain triglycerides’ oil product. The importance of the possibly earliest verification of the diagnosis and initiation of diet therapy using medium-chain triglyceride oils is emphasized, which allows to reduce the disease manifestations and determines the need to include diseases of mitochondrial fatty acids β-oxidation into the neonatal screening program.

Differentially expressed genes PCCA, ECHS1, and HADH are potential prognostic biomarkers for gastric cancer

Gastric cancer (GC) is one of the most common malignant tumors in the world. As far as we know, no biomarker has been widely accepted for early diagnosis and prognosis prediction of GC. The purpose of this study is to find potential biomarkers to predict the prognosis of GC. The differentially expressed gene (DEG) was analyzed from GSE93774. Enrichr was used to analyze the gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, the enrichment of transcription factors (TF), miRNA, and kinase. GO analysis showed DEGs was enriched in the process of amino acid metabolism. Pathway results showed DEGs was mainly enriched in cell cycle. Propionyl CoA carboxylase alpha (PCCA), Enoyl coenzyme A hydratase short chain 1 (ECHS1), and 3-hydroxyacyl-CoA dehydrogenase (HADH) have prognostic value in patients with GC. ECHS1 and HADH genes were significantly associated with disease-free survival. There was a significant correlation between PCCA and overall survival rate. The results of this study suggest that PCCA, ECHS1, and HADH may be new biomarkers for predicting the prognosis of GC.