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Phenolic compounds are commonly found in anoxic environments, where they serve as both carbon and energy sources for certain anaerobic bacteria. The anaerobic breakdown of m-cresol, catechol, and certain lignin-derived compounds yields the central intermediate 3-hydroxybenzoate/3-hydroxybenzoyl-CoA. In this study, we have characterized the transcription and regulation of the hbd genes responsible for the anaerobic degradation of 3-hydroxybenzoate in the β-proteobacterium Aromatoleum sp. CIB. The hbd cluster is organized in three catabolic operons and a regulatory hbdR gene that encodes a dimeric transcriptional regulator belonging to the TetR family. HbdR suppresses the activity of the three catabolic promoters (P hbdN , P hbdE and P hbdH ) by binding to a conserved palindromic operator box (ATGAATGAN 4 TCATTCAT). 3-Hydroxybenzoyl-CoA, the initial intermediate of the 3-hydroxybenzoate degradation pathway, along with benzoyl-CoA, serve as effector molecules that bind to HbdR inducing the expression of the hbd genes. Moreover, the hbd genes are subject to additional regulation influenced by the presence of non-aromatic carbon sources (carbon catabolite repression), and their expression is induced in oxygen-deprived conditions by the AcpR transcriptional activator. The prevalence of the hbd cluster among members of the Aromatoleum/Thauera bacterial group, coupled with its association with mobile genetic elements, suggests acquisition through horizontal gene transfer. These findings significantly enhance our understanding of the regulatory mechanisms governing the hbd gene cluster in bacteria, paving the way for further exploration into the anaerobic utilization/valorization of phenolic compounds derived from lignin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

A comprehensive pangenomic approach was employed to analyze the genomes of 75 type II methylotrophs spanning various genera. Our investigation revealed 256 exact core gene families shared by all 75 organisms, emphasizing their crucial role in the survival and adaptability of these organisms. Additionally, we predicted the functionality of 12 hypothetical proteins. The analysis unveiled a diverse array of genes associated with key metabolic pathways, including methane, serine, glyoxylate, and ethylmalonyl-CoA (EMC) metabolic pathways. While all selected organisms possessed essential genes for the serine pathway, Methylooceanibacter marginalis lacked serine hydroxymethyltransferase (SHMT), and Methylobacterium variabile exhibited both isozymes of SHMT, suggesting its potential to utilize a broader range of carbon sources. Notably, Methylobrevis sp. displayed a unique serine-glyoxylate transaminase isozyme not found in other organisms. Only nine organisms featured anaplerotic enzymes (isocitrate lyase and malate synthase) for the glyoxylate pathway, with the rest following the EMC pathway. Methylovirgula sp. 4MZ18 stood out by acquiring genes from both glyoxylate and EMC pathways, and Methylocapsa sp. S129 featured an A-form malate synthase, unlike the G-form found in the remaining organisms. Our findings also revealed distinct phylogenetic relationships and clustering patterns among type II methylotrophs, leading to the proposal of a separate genus for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129. This pangenomic study unveils remarkable metabolic diversity, unique gene characteristics, and distinct clustering patterns of type II methylotrophs, providing valuable insights for future carbon sequestration and biotechnological applications., Importance: Methylotrophs have played a significant role in methane-based product production for many years. However, a comprehensive investigation into the diverse genetic architectures across different genera of methylotrophs has been lacking. This study fills this knowledge gap by enhancing our understanding of core hypothetical proteins and unique enzymes involved in methane oxidation, serine, glyoxylate, and ethylmalonyl-CoA pathways. These findings provide a valuable reference for researchers working with other methylotrophic species. Furthermore, this study not only unveils distinctive gene characteristics and phylogenetic relationships but also suggests a reclassification for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129 into separate genera due to their unique attributes within their respective genus. Leveraging the synergies among various methylotrophic organisms, the scientific community can potentially optimize metabolite production, increasing the yield of desired end products and overall productivity., Competing Interests: The authors declare no conflict of interest.

Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development., (© 2024. The Author(s).)

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 μg/mL - 4.36-fold higher than WT value (48.9 μg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 μg/mL. Overexpression of fatty acid β-oxidation pathway genes in the strain further increased B1a titer to 452.8 μg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 μg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides., (© 2024 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd.)

Lipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders predominantly affecting skeletal muscle by triglyceride accumulation in muscle fibers. Riboflavin therapy has been shown to ameliorate symptoms in some LSM patients who are essentially concerned with multiple acyl-CoA dehydrogenation deficiency (MADD). It is proved that riboflavin responsive LSM caused by MADD is mainly due to ETFDH gene variant (ETFDH-RRMADD). We described here a case with riboflavin responsive LSM and MADD resulting from FLAD1 gene variants (c.1588 C > T p.Arg530Cys and c.1589 G > C p.Arg530Pro, FLAD1-RRMADD). And we compared our patient together with 9 FLAD1-RRMADD cases from literature to 106 ETFDH-RRMADD cases in our neuromuscular center on clinical history, laboratory investigations and pathological features. Furthermore, the transcriptomics study on FLAD1-RRMADD and ETFDH-RRMADD were carried out. On muscle pathology, both FLAD1-RRMADD and ETFDH-RRMADD were proved with lipid storage myopathy in which atypical ragged red fibers were more frequent in ETFDH-RRMADD, while fibers with faint COX staining were more common in FLAD1-RRMADD. Molecular study revealed that the expression of GDF15 gene in muscle and GDF15 protein in both serum and muscle was significantly increased in FLAD1-RRMADD and ETFDH-RRMADD groups. Our data revealed that FLAD1-RRMADD (p.Arg530) has similar clinical, biochemical, and fatty acid metabolism changes to ETFDH-RRMADD except for muscle pathological features., (© 2024. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study. The ATAC sequencing of the CWWSI at the early wax-secretion stage ATAC-seq resulted in 22.75 GB raw data, generated 75,827,225 clean reads and revealed 142,771 peaks. There was one significant peak in the 3 kb upstream regulation regions. The peak sequence is located between -1000 and -670 bp upstream of the far3 transcription start site, spanning a length of 331 bp. This peak sequence served as bait for creating the pAbAi-peak recombinant vector, used in Y1H screenings to identify proteins interacting with far3 gene. The results indicate a successful CWWSI cDNA library construction with a capacity of 1.2 × 10 7 colony forming unit, a 95.8% recombination rate, and insert sizes between 1,000 and 2,000 bp. Self-activation tests established that 100 ng/mL of AbA effectively inhibited bait vector self-activation. Finally, a total of 88 positive clones were selected. After sequencing and removal of duplication, 63 unique clones were obtained from these screened colonies. By aligning the clone sequences with full-length transcriptome and genome of CWWSI, the full-length coding sequences of these clones were obtained. BlastX analysis identified a transcription factor, nuclear transcription factor Y beta, and two co-activators, cAMP-response-element-binding-protein-binding protein and WW domain binding protein 2. Reverse transcription quantitative polymerase chain reaction analysis confirmed that their expression patterns were consistent with the developmental stages preceding wax secretion and matched the wax secretion characteristics during ovulation periods. These results are beneficial for further research into the regulatory mechanisms of wax secretion of CWWSI., (© 2024 Wiley Periodicals LLC.)

Background: Diacylglycerol acyl-CoA acyltransferase 1 (DGAT1) has become a promising candidate gene for milk production traits because of its important role as a key enzyme in catalyzing the final step of triglyceride synthesis. Thus use of bovine DGAT1 gene as milk production markers in cattle is well established. However, there is no report on polymorphism of the DGAT1 gene in Ethiopian cattle breeds. The present study is the first comprehensive report on diversity, evolution, neutrality evaluation and genetic differentiation of DGAT1 gene in Ethiopian cattle population. The aim of this study was to characterize the genetic variability of exon 8 region of DGAT1 gene in Ethiopian cattle breeds., Results: Analysis of the level of genetic variability at the population and sequence levels with genetic distance in the breeds considered revealed that studied breeds had 11, 0.615 and 0.010 haplotypes, haplotype diversity and nucleotide diversity respectively. Boran-Holstein showed low minor allele frequency and heterozygosity, while Horro showed low nucleotide and haplotype diversities. The studied cattle DGAT1 genes were under purifying selection. The neutrality test statistics in most populations were negative and statistically non-significant (p > 0.10) and consistent with a populations in genetic equilibrium or in expansion. Analysis for heterozygosity, polymorphic information content and inbreeding coefficient revealed sufficient genetic variation in DGAT1 gene. The pairwise F ST values indicated significant differentiation among all the breeds (F ST  = 0.13; p ≤ 0.05), besides the rooting from the evolutionary or domestication history of the cattle inferred from the phylogenetic tree based on the neighbourhood joining method. There was four separated cluster among the studied cattle breeds, and they shared a common node from the constructed tree., Conclusion: The cattle populations studied were polymorphic for DGAT1 locus. The DGAT1 gene locus is extremely crucial and may provide baseline information for in-depth understanding, exploitation of milk gene variation and could be used as a marker in selection programmes to enhance the production potential and to accelerate the rate of genetic gain in Ethiopian cattle populations exposed to different agro ecology condition., (© 2022. The Author(s).)

Triacylglycerols (TAGs) are the major component of plant storage lipids such as oils. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and is mainly responsible for plant oil accumulation. We previously found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor bean were introduced into Arabidopsis. All Vernonia DGAT1-expressing lines showed a significantly higher oil content (49% mean increase compared with the wild-type) followed by soybean and castor bean. Most Arabidopsis DGAT1-overexpressing lines did not show a significant increase. In addition to these four DGAT1 genes, sunflower, Jatropha, and sesame DGAT1 genes were introduced into a TAG biosynthesis-defective yeast mutant. In the yeast expression culture, DGAT1s from Arabidopsis, castor bean, and soybean only slightly increased the TAG content; however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame increased TAG content >10-fold more than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions were created by site-directed mutagenesis and substantially increased the TAG content., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

To observe a long-term prognosis in late-onset multiple acyl-coenzyme-A dehydrogenation deficiency (MADD) patients and to determine whether riboflavin should be administrated in the long-term and high-dosage manner, we studied the clinical, pathological and genetic features of 110 patients with late-onset MADD in a single neuromuscular center. The plasma riboflavin levels and a long-term follow-up study were performed. We showed that fluctuating proximal muscle weakness, exercise intolerance and dramatic responsiveness to riboflavin treatment were essential clinical features for all 110 MADD patients. Among them, we identified 106 cases with ETFDH variants, 1 case with FLAD1 variants and 3 cases without causal variants. On muscle pathology, fibers with cracks, atypical ragged red fibers (aRRFs) and diffuse decrease of SDH activity were the distinctive features of these MADD patients. The plasma riboflavin levels before treatment were significantly decreased in these patients as compared to healthy controls. Among 48 MADD patients with a follow-up of 6.1 years on average, 31 patients were free of muscle weakness recurrence, while 17 patients had episodes of slight muscle weakness upon riboflavin withdrawal, but recovered after retaking a small-dose of riboflavin for a short-term. Multivariate Cox regression analysis showed vegetarian diet and masseter weakness were independent risk factors for muscle weakness recurrence. In conclusion, fibers with cracks, aRRFs and diffuse decreased SDH activity could distinguish MADD from other genotypes of lipid storage myopathy. For late-onset MADD, increased fatty acid oxidation and reduced riboflavin levels can induce episodes of muscle symptoms, which can be treated by short-term and small-dose of riboflavin therapy., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Soil salinization is a major factor impacting modern agricultural production, and alkaline soils contain large amounts of NaHCO 3 . Therefore, understanding plant tolerance to high levels of NaHCO 3 is essential. In this study, a transcriptome analysis of shoot and root tissues of wild-type Arabidopsis thaliana was conducted at 0, 4, 12, 24 and 48 h after exposure to a 3 mM NaHCO 3 stress. We focused on differentially expressed genes (DEGs) in roots identified in the early stages (4 h and 12 h) of the NaHCO 3 stress response that were enriched in GO term, carboxylic acid metabolic process, and utilize HCO 3 - . Six genes were identified that exhibited similar expression patterns in both the RNA-seq and qRT-PCR data. We also characterized the phenotypic response of AtMCCA-overexpressing plants to carbonate stress, and found that the ability of AtMCCA-overexpressing plants to tolerate carbonate stress was enhanced by the addition of biotin to the growth medium., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Synthetic biology requires well-characterized biological parts that can be combined into functional modules. One type of biological parts are transcriptional regulators and their cognate operator elements, which enable to either generate an input-specific response or are used as actuator modules. A range of regulators has already been characterized and used for orthogonal gene expression engineering, however, previous efforts have mostly focused on bacterial regulators. This work aims to design and explore the use of an archaeal TetR family regulator, FadR Sa from Sulfolobus acidocaldarius, in a bacterial system, namely Escherichia coli. This is a challenging objective given the fundamental difference between the bacterial and archaeal transcription machinery and the lack of a native TetR-like FadR regulatory system in E. coli. The synthetic σ 70 -dependent bacterial promoter proD was used as a starting point to design hybrid bacterial/archaeal promoter/operator regions, in combination with the mKate2 fluorescent reporter enabling a readout. Four variations of proD containing FadR Sa binding sites were constructed and characterized. While expressional activity of the modified promoter proD was found to be severely diminished for two of the constructs, constructs in which the binding site was introduced adjacent to the -35 promoter element still displayed sufficient basal transcriptional activity and showed up to 7-fold repression upon expression of FadR Sa . Addition of acyl-CoA has been shown to disrupt FadR Sa binding to the DNA in vitro. However, extracellular concentrations of up to 2 mM dodecanoate, subsequently converted to acyl-CoA by the cell, did not have a significant effect on repression in the bacterial system. This work demonstrates that archaeal transcription regulators can be used to generate actuator elements for use in E. coli, although the lack of ligand response underscores the challenge of maintaining biological function when transferring parts to a phylogenetically divergent host., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. In this study, we enabled the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) in Escherichia coli with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Overall, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Mycofactocin (MFT) is a ribosomally synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc 2 155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5' to mftA and found it to be highly conserved in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, and Mycobacterium marinum. To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo, we re-engineered a fluorescent protein reporter system to express an MftA-mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc 2 155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long-chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Polyketides are one of the largest categories of secondary metabolites, and their biosynthesis is initiated by polyketide synthases (PKSs) using coenzyme A esters of short fatty acids (acyl-CoAs) as starter and extender units. In this study, we discover a universal regulatory mechanism in which the starter and extender units, beyond direct precursors of polyketides, function as ligands to coordinate the biosynthesis of antibiotics in actinomycetes. A novel acyl-CoA responsive TetR-like regulator (AcrT) is identified in an erythromycin-producing strain of Saccharopolyspora erythraea. AcrT shows the highest binding affinity to the promoter of the PKS-encoding gene eryAI in the DNA affinity capture assay (DACA) and directly represses the biosynthesis of erythromycin. Propionyl-CoA (P-CoA) and methylmalonyl-CoA (MM-CoA) as the starter and extender units for erythromycin biosynthesis can serve as the ligands to release AcrT from P eryAI , resulting in an improved erythromycin yield. Intriguingly, anabolic pathways of the two acyl-CoAs are also suppressed by AcrT through inhibition of the transcription of acetyl-CoA (A-CoA) and P-CoA carboxylase genes and stimulation of the transcription of citrate synthase genes, which is beneficial to bacterial growth. As P-CoA and MM-CoA accumulate, they act as ligands in turn to release AcrT from those targets, resulting in a redistribution of more A-CoA to P-CoA and MM-CoA against citrate. Furthermore, based on analyses of AcrT homologs in Streptomyces avermitilis and Streptomyces coelicolor, it is believed that polyketide starter and extender units have a prevalent, crucial role as ligands in modulating antibiotic biosynthesis in actinomycetes. IMPORTANCE Numerous antibiotics are derived from polyketides, whose biosynthesis is accurately controlled by transcriptional regulators that respond to diverse physiological or environmental signals. It is generally accepted that antibiotics or biosynthetic intermediates serve as effectors to modulate their production in actinomycetes. Our study unprecedentedly demonstrates that the direct precursors of polyketide, propionyl-CoA and methylmalonyl-CoA, play a role as ligands to modulate erythromycin biosynthesis in Saccharopolyspora erythraea. More importantly, the two acyl-CoAs as ligands could adjust their own supplies by regulating the acetyl-CoA metabolic pathway so as to well settle the relationship between cellular growth and secondary metabolism. Significantly, polyketide starter and extender units have a universal role as ligands to coordinate antibiotic biosynthesis in actinomycetes. These findings not only expand the understanding of ligand-mediated regulation for antibiotic biosynthesis but also provide new insights into the physiological functions of polyketide starter and extender units in actinomycetes.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is a crucial enzyme in the ergosterol biosynthesis pathway. The aim of this study was to obtain, purify, characterize, and overexpress five point mutations in highly conserved regions of the catalytic domain of Candida glabrata HMGR (CgHMGR) to explore the function of key amino acid residues in enzymatic activity. Glutamic acid (Glu) was substituted by glutamine in the E680Q mutant (at the dimerization site), Glu by glutamine in E711Q (at the substrate binding site), aspartic acid by alanine in D805A, and methionine by arginine in M807R (the latter two at the cofactor binding site). A double mutation, E680Q-M807R, was included. Regarding recombinant and wild-type CgHMGR, in vitro enzymatic activity was significantly lower for the former, as was the in silico binding energy of simvastatin, alpha-asarone and the HMG-CoA substrate. E711Q displayed the lowest enzymatic activity and binding energy, suggesting the importance of Glu 711 (in the substrate binding site). The double mutant CgHMGR E680Q-M807R exhibited the second lowest enzymatic activity. Based on the values of the kinetic parameters K M and V max , the mutated amino acids appear to participate in binding. The current findings provide insights into the role of residues in the catalytic site of CgHMGR., (© 2021. The Author(s).)

Background: Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis., Results: Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae)., Conclusions: Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast., Competing Interests: The authors have declared that no competing interests exist.

The cytosolic enzyme ethylmalonyl-CoA decarboxylase (ECHDC1) decarboxylates ethyl- or methyl-malonyl-CoA, two side products of acetyl-CoA carboxylase. These CoA derivatives can be used to synthesize a subset of branched-chain fatty acids (FAs). We previously found that ECHDC1 limits the synthesis of these abnormal FAs in cell lines, but its effects in vivo are unknown. To further evaluate the effects of ECHDC1 deficiency, we generated knockout mice. These mice were viable, fertile, showed normal postnatal growth, and lacked obvious macroscopic and histologic changes. Surprisingly, tissues from wild-type mice already contained methyl-branched FAs due to methylmalonyl-CoA incorporation, but these FAs were only increased in the intraorbital glands of ECHDC1 knockout mice. In contrast, ECHDC1 knockout mice accumulated 16-20-carbon FAs carrying ethyl-branches in all tissues, which were undetectable in wild-type mice. Ethyl-branched FAs were incorporated into different lipids, including acylcarnitines, phosphatidylcholines, plasmanylcholines, and triglycerides. Interestingly, we found a variety of unusual glycine-conjugates in the urine of knockout mice, which included adducts of ethyl-branched compounds in different stages of oxidation. This suggests that the excretion of potentially toxic intermediates of branched-chain FA metabolism might prevent a more dramatic phenotype in these mice. Curiously, ECHDC1 knockout mice also accumulated 2,2-dimethylmalonyl-CoA. This indicates that the broad specificity of ECHDC1 might help eliminate a variety of potentially dangerous branched-chain dicarboxylyl-CoAs. We conclude that ECHDC1 prevents the formation of ethyl-branched FAs and that urinary excretion of glycine-conjugates allows mice to eliminate potentially deleterious intermediates of branched-chain FA metabolism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent studies demonstrate that histones are subjected to a series of short-chain fatty acid modifications that is known as histone acylations. However, the enzymes responsible for histone acylations in vivo are not well characterized. Here, we report that HBO1 is a versatile histone acyltransferase that catalyzes not only histone acetylation but also propionylation, butyrylation and crotonylation both in vivo and in vitro and does so in a JADE or BRPF family scaffold protein-dependent manner. We show that the minimal HBO1/BRPF2 complex can accommodate acetyl-CoA, propionyl-CoA, butyryl-CoA and crotonyl-CoA. Comparison of CBP and HBO1 reveals that they catalyze histone acylations at overlapping as well as distinct sites, with HBO1 being the key enzyme for H3K14 acylations. Genome-wide chromatin immunoprecipitation assay demonstrates that HBO1 is highly enriched at and contributes to bulk histone acylations on the transcriptional start sites of active transcribed genes. HBO1 promoter intensity highly correlates with the level of promoter histone acylation, but has no significant correlation with level of transcription. We also show that HBO1 is associated with a subset of DNA replication origins. Collectively our study establishes HBO1 as a versatile histone acyltransferase that links histone acylations to promoter acylations and selection of DNA replication origins., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Amaranths are recognized by their high nutritive value and their natural tolerance to environmental stresses. In this study, physiological differences in response to water stress were compared between A. hybridus, a wild species considered as weed, and A. hypochondriacus, the most cultivated species for grain production, under the hypothesis that wild species have better adaptation to stress. In both species, photosynthetic parameters, pigments, and gene expression of selected genes were assessed. Biomass, effective quantum efficiency (Φ PSII ), photochemical quenching (q P ), and electron transport rate (ETR) values were reduced only in A. hybridus due to water deficit. Drought stress promoted proline accumulation by twice in A. hybridus but until three times in A. hypochondriacus. In both species, drought stress reduced net assimilation rate (A), transpiration rate (E), stomatal conductance (g s ), and the expression of phosphoenol pyruvate carboxylase (PEPC). While, maximum quantum efficiency (F v /F m ), chlorophyll, betacyanins, and the expression of ribulose1-5, bisphosphate carboxylase/oxygenase large subunit (LSU) did not change when plants were subjected to water stress. Likewise, both species accumulated total phenolic compounds and Oxalyl-CoA gene was up-regulated in response to drought. Our results have shown that A. hypochondriacus, the cultivated species, exhibited better tolerance to drought than A. hybridus, the wild species, probably due to an unconsciously selected trait during the domestication process., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD , but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB , responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics. IMPORTANCE mecA -dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC , increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of β-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome β-lactam resistance.

We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD + . Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD• - in the course of reductive titrations of the depleted EtfAB with sodium dithionite., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Adipic acid is a versatile aliphatic dicarboxylic acid. It is applied mainly in the polymerization of nylon-6,6, which accounts for 50.8% of the global consumption market of adipic acid. The microbial production of adipic acid avoids the usage of petroleum resources and the emission of harmful nitrogen oxides that are generated by traditional chemical synthetic approaches. However, in the fermentation process, the low theoretical yield and the usage of expensive inducers hinders the large-scale industrial production of adipic acid. To overcome these challenges, we established an oxygen-dependent dynamic regulation (ODDR) system to control the expression of key genes ( sucD , pyc , mdh , and frdABCD ) that could be induced to enhance the metabolic flux of the reductive TCA pathway under anaerobic conditions. Coupling of the constitutively expressed adipic acid synthetic pathway not only avoids the use of inducers but also increases the theoretical yield by nearly 50%. After the gene combination and operon structure were optimized, the reaction catalyzed by frdABCD was found to be the rate-limiting step. Further optimizing the relative expression levels of sucD , pyc , and frdABCD improved the titer of adipic acid 41.62-fold compared to the control strain Mad1415, demonstrating the superior performance of our ODDR system.

ATP-binding cassette (ABC) subfamily D transporters are important for the uptake of fatty acids and other beta-oxidation substrates into peroxisomes. Genetic and biochemical evidence indicates that the transporters accept fatty acyl-coenzyme A that is cleaved during the transport cycle and then re-esterified in the peroxisomal lumen. However, it is not known whether free coenzyme A (CoA) is released inside or outside the peroxisome. Here we have used Saccharomyces cerevisiae and isolated peroxisomes to demonstrate that free CoA is released in the peroxisomal lumen. Thus, ABC subfamily D transporter provide an import pathway for free CoA that controls peroxisomal CoA homeostasis and tunes metabolism according to the cell's demands., (© 2020 Federation of European Biochemical Societies.)

Tuberculosis (TB) is the largest infectious disease with 10 million new active-TB patients and1.7 million deaths per year. Active-TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to M. tb. infection. The mechanisms of a switch from latent infection to active disease is not well worked out but a shift in the immune responses is thought to be responsible. Increasingly, the role of gut microbiota has been described as a major influencer of the immune system. And because the gut is the largest immune organ, we aimed to analyze the gut microbiome in active-TB patients in a TB-endemic country, Pakistan. The study revealed that Ruminococcacea, Enetrobactericeae, Erysipelotrichaceae, Bifidobacterium, etc. were the major genera associated with active-TB, also associated with chronic inflammatory disease. Plasma antibody profiles against several M. tb. antigens, as specific biomarkers for active-TB, correlated closely with the patient gut microbial profiles. Besides, bcoA gene copy number, indicative of the level of butyrate production by the gut microbiome was five-fold lower in TB patients compared to healthy individuals. These findings suggest that gut health in TB patients is compromised, with implications for disease morbidity (e.g., severe weight loss) as well as immune impairment., Competing Interests: The authors have declared that no competing interests exist.

Acyl activating enzyme 3 (AAE3) was identified as being involved in the acetylation pathway of oxalate degradation, which regulates the responses to biotic and abiotic stresses in various higher plants. Here, we investigated the role of Glycine soja AAE3 ( GsAAE3 ) in Cadmium (Cd) and Aluminum (Al) tolerances. The recombinant GsAAE3 protein showed high activity toward oxalate, with a K m of 105.10 ± 12.30 μM and V max of 12.64 ± 0.34 μmol min -1 mg -1 protein, suggesting that it functions as an oxalyl-CoA synthetase. The expression of a GsAAE3-green fluorescent protein (GFP) fusion protein in tobacco leaves did not reveal a specific subcellular localization pattern of GsAAE3 . An analysis of the GsAAE3 expression pattern revealed an increase in GsAAE3 expression in response to Cd and Al stresses, and it is mainly expressed in root tips. Furthermore, oxalate accumulation induced by Cd and Al contributes to the inhibition of root growth in wild soybean. Importantly, GsAAE3 overexpression increases Cd and Al tolerances in A. thaliana and soybean hairy roots, which is associated with a decrease in oxalate accumulation. Taken together, our data provide evidence that the GsAAE3 -encoded protein plays an important role in coping with Cd and Al stresses.

Stearoyl-CoA Desaturase-2 (SCD2) is a member of the Stearoyl-CoA Desaturase (SCD) family of enzymes that catalyze the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis. The MUFAs palmitoleoyl-CoA (16:1n7) and oleoyl-CoA (18:1n9) are the major products of SCD2. Palmitoleoyl-CoA and oleoyl-CoA have various roles, from being a source of energy to signaling molecules. Under normal feeding conditions, SCD2 is ubiquitously expressed and is the predominant SCD isoform in the brain. However, obesogenic diets highly induce SCD2 in adipose tissue, lung, and kidney. Here we provide a comprehensive review of SCD2 in mouse development, metabolism, and various diseases, such as obesity, chronic kidney disease, Alzheimer's disease, multiple sclerosis, and Parkinson's disease. In addition, we show that bone mineral density is decreased in SCD2KO mice under high-fat feeding conditions and that SCD2 is not required for preadipocyte differentiation or the expression of PPARγ in vivo despite being required in vitro.

The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl-coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl-CoA to yield acyl 4'-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl-CoA levels as key to ER homeostasis., (© 2020 Becuwe et al.)

The diffusible signal factor (DSF) is a fatty acid signal molecule and is widely conserved in various Gram-negative bacteria. DSF is involved in the regulation of pathogenic virulence in many bacterial pathogens, including Xanthomonas campestris pv. campestris ( Xcc ). Quorum quenching (QQ) is a potential approach for preventing and controlling DSF-mediated bacterial infections by the degradation of the DSF signal. Acinetobacter lactucae strain QL-1 possesses a superb DSF degradation ability and effectively attenuates Xcc virulence through QQ. However, the QQ mechanisms in strain QL-1 are still unknown. In the present study, whole-genome sequencing and comparative genomics analysis were conducted to identify the molecular mechanisms of QQ in strain QL-1. We found that the fadY gene of QL-1 is an ortholog of Xcc rpfB , a known DSF degradation gene, suggesting that strain QL-1 is capable of inactivating DSF by QQ enzymes. The results of site-directed mutagenesis indicated that fadY is required for strain QL-1 to degrade DSF. The determination of FadY activity in vitro revealed that the fatty acyl-CoA synthetase FadY had remarkable catalytic activity. Furthermore, the expression of fadY in transformed Xcc strain XC1 was investigated and shown to significantly attenuate bacterial pathogenicity on host plants, such as Chinese cabbage and radish. This is the first report demonstrating a DSF degradation enzyme from A. lactucae . Taken together, these findings shed light on the QQ mechanisms of A. lactucae strain QL-1, and provide useful enzymes and related genes for the biocontrol of infectious diseases caused by DSF-dependent bacterial pathogens.

Vegetable oils are one of the main agricultural commodities. Demand has been increasing steadily over the last five decades and, with finite land available, it is vital that we increase productivity. My laboratory has focused on the regulation of plant lipid metabolism and, as part of this work, we identified diacylglycerol acyltransferase (DGAT) as important at regulating carbon flux during oil accumulation. This led to collaborations with Randy Weselake's research group when we quantified the importance of DGAT in oilseed rape by using flux control analysis. Later, with David Taylor, we showed that over-expression of DGAT boosted oil accumulation in field-grown crops by around 8%. These studies led to a multitude of experiments with different oil crops, such as oil palm and soybean, as well as many rewarding collaborations with Randy., (© 2020 The Author. Lipids published by Wiley Periodicals LLC. on behalf of American Oil Chemists' Society.)

Haematococcus pluvialis is a green microalga used in the algal biotechnology industry that can accumulate considerable amounts of storage triacylglycerol (TAG) and astaxanthin, which is a high-value carotenoid with strong antioxidant activity, under stress conditions. Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and appears to represent a bottleneck in algal TAG formation. In this study, putative H. pluvialis DGAT2 cDNA (HpDGAT2A, B, D and E) were identified from a transcriptome database and were subjected to sequence-based in silico analyses. The coding sequences of HpDGAT2B, D, and E were then isolated and characterized through heterologous expression in a TAG-deficient Saccharomyces cerevisiae strain H1246. The expression of HpDGAT2D allowed the recovery of TAG biosynthesis in this yeast mutant, and further in vitro enzymatic assays confirmed that the recombinant HpDGAT2D possessed strong DGAT activity. Interestingly, the recombinant HpDGAT2D displayed sigmoidal kinetics in response to increasing acyl-CoA concentrations, which has not been reported in plant or algal DGAT2 in previous studies., (© 2019 AOCS.)

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta., (© 2020 The Authors. Lipids published by Wiley Periodicals, Inc. on behalf of American Oil Chemists' Society.)

The modification of shoot architecture and increased investment into reproductive structures is key for crop improvement and is achieved through coordinated changes in the development and determinacy of different shoot meristems. A fundamental question is how the development of different shoot meristems is genetically coordinated to optimize the balance between vegetative and reproductive organs. Here we identify the MANY NODED DWARF1 ( HvMND1 ) gene as a major regulator of plant architecture in barley ( Hordeum vulgare ). The mnd1.a mutant displayed an extended vegetative program with increased phytomer, leaf, and tiller production but a reduction in the number and size of grains. The induction of vegetative structures continued even after the transition to reproductive growth, resulting in a marked increase in longevity. Using mapping by RNA sequencing, we found that the HvMND1 gene encodes an acyl-CoA N -acyltransferase that is predominately expressed in developing axillary meristems and young inflorescences. Exploration of the expression network modulated by HvMND1 revealed differential expression of the developmental microRNAs miR156 and miR172 and several key cell cycle and developmental genes. Our data suggest that HvMND1 plays a significant role in the coordinated regulation of reproductive phase transitions, thereby promoting reproductive growth and whole plant senescence in barley., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Plant type III polyketide synthases produce diverse bioactive molecules with a great medicinal significance to human diseases. Here, we demonstrated versatility of a stilbene synthase (STS) from Pinus Sylvestris , which can accept various non-physiological substrates to form unnatural polyketide products. Three enzymes (4-coumarate CoA ligase, malonyl-CoA synthetase and engineered benzoate CoA ligase) along with synthetic chemistry was practiced to synthesize starter and extender substrates for STS. Of these, the crystal structures of benzoate CoA ligase (BadA) from Rhodopseudomonas palustris in an apo form or in complex with a 2-chloro-1,3-thiazole-5-carboxyl-AMP or 2-methylthiazole-5-carboxyl-AMP intermediate were determined at resolutions of 1.57 Å, 1.7 Å, and 2.13 Å, respectively, which reinforces its capacity in production of unusual CoA starters. STS exhibits broad substrate promiscuity effectively affording structurally diverse polyketide products. Seven novel products showed desired cytotoxicity against a panel of cancer cell lines (A549, HCT116, Cal27). With the treatment of two selected compounds, the cancer cells underwent cell apoptosis in a dose-dependent manner. The precursor-directed biosynthesis alongside structure-guided enzyme engineering greatly expands the pharmaceutical repertoire of lead compounds with promising/enhanced biological activities.

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by a specific encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. Substrate reduction through inhibition of DHTKD1, an enzyme upstream of the defective glutaryl-CoA dehydrogenase, has been investigated as a potential therapy, but revealed the existence of an alternative enzymatic source of glutaryl-CoA. Here, we show that loss of DHTKD1 in glutaryl-CoA dehydrogenase-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine and demonstrate that oxoglutarate dehydrogenase (OGDH) is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, dihydrolipoyl succinyltransferase and dihydrolipoamide dehydrogenase to form a hybrid 2-oxoglutaric and 2-oxoadipic acid dehydrogenase complex. In summary, 2-oxoadipic acid is a substrate for DHTKD1, but also for OGDH in a cell model system. The classical 2-oxoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards 2-oxoadipic acid., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A propanologenic (i.e., 1-propanol-producing) bacterium Escherichia coli strain was previously derived by activating the genomic sleeping beauty mutase (Sbm) operon. The activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl-CoA node to form propionyl-CoA and its derived metabolites of 1-propanol and propionate. In this study, we targeted several TCA cycle genes encoding enzymes near the succinyl-CoA node for genetic manipulation to identify the individual contribution of the carbon flux into the Sbm pathway from the three TCA metabolic routes, that is, oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. For the control strain CPC-Sbm, in which propionate biosynthesis occurred under relatively anaerobic conditions, the carbon flux into the Sbm pathway was primarily derived from the reductive TCA branch, and both succinate availability and the SucCD-mediated interconversion of succinate/succinyl-CoA were critical for such carbon flux redirection. Although the oxidative TCA cycle normally had a minimal contribution to the carbon flux redirection, the glyoxylate shunt could be an alternative and effective carbon flux contributor under aerobic conditions. With mechanistic understanding of such carbon flux redirection, metabolic strategies based on blocking the oxidative TCA cycle (via ∆sdhA mutation) and deregulating the glyoxylate shunt (via ∆iclR mutation) were developed to enhance the carbon flux redirection and therefore propionate biosynthesis, achieving a high propionate titer of 30.9 g/L with an overall propionate yield of 49.7% upon fed-batch cultivation of the double mutant strain CPC-Sbm∆sdhA∆iclR under aerobic conditions. The results also suggest that the Sbm pathway could be metabolically active under both aerobic and anaerobic conditions., (© 2020 Wiley Periodicals, Inc.)

Background: Methylmalonic acidemia (MMA), which is an autosomal recessive metabolic disorder, is caused by mutations in methylmalonyl-CoA mutase (MUT) gene. As a result, the conversion of methylmalonyl-CoA to succinyl-CoA is impaired in this disorder, leading to a wide range of clinical manifestations varying from no signs or symptoms to severe lethargy and metabolic crisis in newborn infants. Since identification of novel mutations in MUT gene can help discover the exact pathogenesis of MMA and also use these disease-causing mutations in prenatal diagnosis, this study was conducted to uncover the possible mutations in an Iranian couple with a deceased offspring clinically diagnosed as having organic acidemia. Moreover, to prevent the occurrence of the mutation in the next pregnancy, we took the advantage of pre-implantation genetic diagnosis (PGD), which resulted in a successful pregnancy., Case Presentation: The affected individual was a 15-month-old boy who passed away due to aspiration pneumonia. The child presented at the age of 3 months with lethargy, protracted vomiting, hypotonia, and decreased level of consciousness. To find the mutated gene, Next Generation Sequencing (NGS) was performed as carrier testing for the parents and the results revealed a novel (private) heterozygous missense mutation in MUT gene (c.1055A > G, p.Q352R). After performing PGD on three blastomeres, one was identified as being homozygous wild-type that was followed by successful pregnancy., Conclusions: Our study identified a novel, deleterious, heterozygous missense mutation in MUT gene in a couple and helps to consider the genetic counselling and prenatal diagnosis more seriously for this family with clinical phenotypes of organic acidemia.

Protein acylation, exemplified by lysine acetylation, is a type of indispensable and widespread protein posttranslational modification in eukaryotes. Functional annotation of various lysine acetyltransferases (KATs) is critical to understanding their regulatory roles in abundant biological processes. Traditional radiometric and immunosorbent assays have found broad use in KAT study but have intrinsic limitations. Designing acyl-coenzyme A (CoA) reporter molecules bearing chemoselective chemical warhead groups as surrogates of the native cofactor acetyl-CoA for bioorthogonal labeling of KAT substrates has come into a technical innovation in recent years. This chemical biology platform equips molecular biologists with empowering tools in acyltransferase activity detection and substrate profiling. In the bioorthogonal labeling, protein substrates are first enzymatically modified with a functionalized acyl group. Subsequently, the chemical warhead on the acyl chain conjugates with either an imaging chromophore or an affinity handle or any other appropriate probes through an orthogonal chemical ligation. This bioorganic strategy reformats the chemically inert acetylation and acylation marks into a chemically maneuverable functionality and generates measurable signals without recourse to radioisotopes or antibodies. It offers ample opportunities for facile sensitive detection of KAT activity with temporal and spatial resolutions as well as allows for chemoproteomic profiling of protein acetylation pertaining to specific KATs of interest on the global scale. We reviewed here the past and current advances in bioorthogonal protein acylations and highlighted their wide-spectrum applications. We also discussed the design of other related acyl-CoA and CoA-based chemical probes and their deployment in illuminating protein acetylation and acylation biology.

Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l -1 and a yield on glucose of 405.8 µg g -1 . Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.

Polyketides are a diverse class of molecules sought after for their valuable properties, including as potential pharmaceuticals. Previously, we demonstrated that the oleaginous yeast Yarrowia lipolytica is an optimal host for production of the simple polyketide, triacetic acid lactone (TAL). We here expand the capacities of this host by overcoming previous media challenges and enabling production of more complex polyketides. Specifically, we employ a β-oxidation related strategy to improve polyketide production directly from defined media. Beyond TAL production, we establish biosynthesis of the 4-coumaroyl-CoA derived polyketides: naringenin, resveratrol, and bisdemethoxycurcumin, as well as the diketide intermediate, (E)-5-(4-hydroxyphenyl)-3-oxopent-4-enoic acid. In this background, we enable high-level de novo production of naringenin through import of both a heterologous pathway and a mutant Y. lipolytica allele. In doing so, we generated an averaged maximum titer of 898 mg/L naringenin, the highest titer reported to date in any host. These results demonstrate that Y. lipolytica is an ideal polyketide production host for more complex 4-coumaroyl-CoA derived products., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identification of genes that enhance grain size and composition is much desired. Past reports have indicated that Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs) are important in seed development but did not affect seed size. Herein, rice OsACBP2 was demonstrated not only to play a role in seed development and germination, but also to influence grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while β-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos, as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed cis-element, Skn-I-like motif (-1486/-1482, -956/-952, -939/-935, -826/-822, and -766/-762), and the removal of any adversely affected expression in seeds, thereby providing a molecular basis for OsACBP2 expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded in germination, while OsACBP2-OEs performed better than the wild-type and vector-transformed controls, in germination, seedling growth, grain size and grain weight. Transmission electron microscopy of OsACBP2-OE mature seeds revealed an accumulation of oil bodies in the scutellum cells, while confocal laser scanning microscopy indicated oil accumulation in OsACBP2-OE aleurone tissues. Correspondingly, OsACBP2-OE seeds showed gain in triacylglycerols and long-chain fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components, OsACBP2 appears to be a promising candidate for enriching seed nutritional value., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

The pathogenicity of Mycobacterium tuberculosis depends upon its ability to catabolize host cholesterol. Upregulation of the methylcitrate cycle (MCC) is required to assimilate and detoxify propionyl-CoA, a cholesterol degradation product. The transcription of key genes prpC and prpD in MCC is activated by MtPrpR, a member of a family of prokaryotic transcription factors whose structures and modes of action have not been clearly defined. We show that MtPrpR has a novel overall structure and directly binds to CoA or short-chain acyl-CoA derivatives to form a homotetramer that covers the binding cavity and locks CoA tightly inside the protein. The regulation of this process involves a [4Fe4S] cluster located close to the CoA-binding cavity on a neighboring chain. Mutations in the [4Fe4S] cluster binding residues rendered MtPrpR incapable of regulating MCC gene transcription. The structure of MtPrpR without the [4Fe4S] cluster-binding region shows a conformational change that prohibits CoA binding. The stability of this cluster means it is unlikely a redox sensor but may function by sensing ambient iron levels. These results provide mechanistic insights into this family of critical transcription factors who share similar structures and regulate gene transcription using a combination of acyl-CoAs and [4Fe4S] cluster., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Terpene trilactones (TTLs) are the main secondary metabolites of Ginkgo biloba. As one of the rate-limiting enzymes in the mevalonic acid (MVA) pathway of TTL biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the 3-hydroxy-3-methylglutaryl coenzyme A to form MVA. In this study, two cDNA sequences of HMGR genes, namely, GbHMGR2 and GbHMGR3, were cloned from G. biloba. The protein sequences of GbHMGR2 and GbHMGR3, which contain several functional domains, were analyzed. Regulatory elements related to light, hormone, and stress response were detected in the promoter regions of GbHMGR2 and GbHMGR3. The catalytic activity of these genes was verified by a functional complement experiment in yeast. Quantitative real-time PCR (qRT-PCR) showed the distinct expression patterns of the two genes in different organs. The TTL contents in the organs were detected by high-performance liquid chromatography- evaporative light scattering detector. GbHMGR2 and GbHMGR3 were responded to cold, dark, methyl jasmonate (MJ), abscisic acid (ABA), salicylic acid (SA), and ethephon (Eth) treatments. The TTL contents were also regulated by cold, dark, MJ, ABA, SA, and Eth treatment. In conclusion, GbHMGR2 and GbHMGR3 may participate in the MVA pathway of TTL biosynthesis.

The physicochemical properties, biological functions and microbial degradation of inulins differ according to their degree of polymerization. However, the relationship between inulin activities and its effect on gut microbiota remains unknown. In this study, high fat diet with inulin (1 or 5 g/kg·bw), either with short or long chains groups were administered to different groups of mice (n = 10) for 10 weeks in order to investigate the effect of inulin on the microbial diversity of the animals. Litchi pericarp procyanidins (LPPC) were used for comparison purposes. Furthermore, the lipid metabolism and key regulator genes in mice were determined. The results indicated that natural inulin (1 g/kg·bw) ingestion reduced the body weight of fat mice between week 6-9. Glutathione peroxidase (GSH-Px) activity in liver was remarkably higher after adding long chain inulin (5 g/kg·bw) compared to high-fat-diet mice. Moreover, high dose of natural inulin regulated malondialdehyde and advanced glycation end-products levels in mice liver. Likewise, the high dose of short-chain inulin increased sterol response element binding protein 1 (SREBP-1), β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) and ATP-binding cassette transporter A1 (ABCA1) genetic expression. A significant change on the abundance of six genera in gut microbial profile suggested that inulin has the ability to modulate the lipid metabolism regardless of chain length, mainly due to its impact on colon microbiota variety., (Copyright © 2019. Published by Elsevier Ltd.)

The last decade saw major advances in understanding the metabolism of Coenzyme A (CoA) thioesters (acyl-CoAs) and related inborn errors (CoA metabolic diseases, CAMDs). For diagnosis, acylcarnitines and organic acids, both derived from acyl-CoAs, are excellent markers of most CAMDs. Clinically, each CAMD is unique but strikingly, three main patterns emerge: first, systemic decompensations with combinations of acidosis, ketosis, hypoglycemia, hyperammonemia and fatty liver; second, neurological episodes, particularly acute "stroke-like" episodes, often involving the basal ganglia but sometimes cerebral cortex, brainstem or optic nerves and third, especially in CAMDs of long chain fatty acyl-CoA metabolism, lipid myopathy, cardiomyopathy and arrhythmia. Some patients develop signs from more than one category. The pathophysiology of CAMDs is not precisely understood. Available data suggest that signs may result from CoA sequestration, toxicity and redistribution (CASTOR) in the mitochondrial matrix has been suggested to play a role. This predicts that most CAMDs cause deficiency of CoA, limiting mitochondrial energy production, and that toxic effects from the abnormal accumulation of acyl-CoAs and from extramitochondrial functions of acetyl-CoA may also contribute. Recent progress includes the following. (1) Direct measurements of tissue acyl-CoAs in mammalian models of CAMDs have been related to clinical features. (2) Inborn errors of CoA biosynthesis were shown to cause clinical changes similar to those of inborn errors of acyl-CoA degradation. (3) CoA levels in cells can be influenced pharmacologically. (4) Roles for acetyl-CoA are increasingly identified in all cell compartments. (5) Nonenzymatic acyl-CoA-mediated acylation of intracellular proteins occurs in mammalian tissues and is increased in CAMDs., (Copyright © 2019. Published by Elsevier Inc.)

Most fatty acids (FAs) are straight chains and are synthesized by fatty acid synthase (FASN) using acetyl-CoA and malonyl-CoA units. Yet, FASN is known to be promiscuous as it may use methylmalonyl-CoA instead of malonyl-CoA and thereby introduce methyl-branches. We have recently found that the cytosolic enzyme ECHDC1 degrades ethylmalonyl-CoA and methylmalonyl-CoA, which presumably result from promiscuous reactions catalyzed by acetyl-CoA carboxylase on butyryl- and propionyl-CoA. Here, we tested the hypothesis that ECHDC1 is a metabolite repair enzyme that serves to prevent the formation of methyl- or ethyl-branched FAs by FASN. Using the purified enzyme, we found that FASN can incorporate not only methylmalonyl-CoA but also ethylmalonyl-CoA, producing methyl- or ethyl-branched FAs. Using a combination of gas-chromatography and liquid chromatography coupled to mass spectrometry, we observed that inactivation of ECHDC1 in adipocytes led to an increase in several methyl-branched FAs (present in different lipid classes), while its overexpression reduced them below wild-type levels. In contrast, the formation of ethyl-branched FAs was observed almost exclusively in ECHDC1 knockout cells, indicating that ECHDC1 and the low activity of FASN toward ethylmalonyl-CoA efficiently prevent their formation. We conclude that ECHDC1 performs a typical metabolite repair function by destroying methyl- and ethylmalonyl-CoA. This reduces the formation of methyl-branched FAs and prevents the formation of ethyl-branched FAs by FASN. The identification of ECHDC1 as a key modulator of the abundance of methyl-branched FAs opens the way to investigate their function., (© 2019 The Author(s).)

Schizochytrium sp. A-2 is a heterotrophic marine fungus used for the commercial production of docosahexaenoic acid (DHA). However, the pattern of the distribution of DHA and how DHA is channeled into phospholipid (PL) and triacylglycerol (TAG) are unknown. In this study, we systematically analyzed the distribution of DHA in TAG and PL during the growth of the cell. The migration of DHA from PL to TAG was presumed during the fermentation cycle. DHA and docosapentaenoic acid were accumulated in both TAG and phosphatidylcholine (PC), whereas eicosapentaenoic acid was mainly deposited in PC. RNA seq revealed that malic enzyme may provide lipogenic NADPH. In addition, long-chain acyl-CoA synthase and acyl-CoA:lysophosphatidylcholine acyltransferase may participate in the accumulation of DHA in PL. No phosphatidylcholine:diacylglycerol cholinephosphotransferase was identified from the genome sequence. In contrast, phospholipid:diacylglycerol acyltransferase-mediated acyl-CoA-independent TAG synthesis pathway and phospholipase C may contribute to the channeling of DHA from PC to TAG.

The anaerobic degradation of benzoate in bacteria involves the benzoyl-CoA central pathway. Azoarcus /Aromatoleum strains are a major group of anaerobic benzoate degraders, and the transcriptional regulation of the bzd genes was extensively studied in Azoarcus sp. CIB. In this work, we show that the bzdR regulatory gene and the P N promoter can also be identified upstream of the catabolic bzd operon in all benzoate-degrader Azoarcus /Aromatoleum strains whose genome sequences are currently available. All the P N promoters from Azoarcus /Aromatoleum strains described here show a conserved architecture including three operator regions (ORs), i.e., OR1 to OR3, for binding to the BzdR transcriptional repressor. Here, we demonstrate that, whereas OR1 is sufficient for the BzdR-mediated repression of the P N promoter, the presence of OR2 and OR3 is required for de-repression promoted by the benzoyl-CoA inducer molecule. Our results reveal that BzdR binds to the P N promoter in the form of four dimers, two of them binding to OR1. The BzdR/ P N complex formed induces a DNA loop that wraps around the BzdR dimers and generates a superstructure that was observed by atomic force microscopy. This work provides further insights into the existence of a conserved BzdR-dependent mechanism to control the expression of the bzd genes in Azoarcus strains., Competing Interests: Conflicts of interest: The authors declare that there are no competing interests.

Background: Koalas in the Mount Lofty Ranges, South Australia, have a high prevalence of oxalate nephrosis, or calcium oxalate kidney crystals. Gastrointestinal tract oxalate-degrading bacteria, particularly Oxalobacter formigenes, have been identified in other animal species and humans, and their absence or low abundance is postulated to increase the risk of renal oxalate diseases. This study aimed to identify oxalate-degrading bacteria in the gastrointestinal tract of koalas and determine their association with oxalate nephrosis., Methods: Caecal and faecal samples were collected at necropsy from 22 Mount Lofty Ranges koalas that had been euthanased on welfare grounds, with 8 koalas found to have oxalate nephrosis by renal histopathology. Samples were analysed by PCR for the oxc gene, which encodes oxalyl-CoA decarboxylase, and also by Illumina sequencing of the V3-V4 region of the bacterial 16S rRNA gene., Results: The oxc gene was detected in 100% of koala samples, regardless of oxalate nephrosis status. Oxalobacter formigenes was detected in all but one faecal sample, with no difference in abundance between koalas affected and unaffected by oxalate nephrosis. Other species of known oxalate-degrading bacteria were infrequently detected., Conclusion: This is the first study to identify Oxalobacter and other oxalate-degrading bacterial species in koalas, but an association with oxalate nephrosis and absence or low abundance of Oxalobacter was not found. This suggests other mechanisms underlie the risk of oxalate nephrosis in koalas., (© 2019 Australian Veterinary Association.)

N ε -lysine acetylation and succinylation are ubiquitous post-translational modifications in eukaryotes and bacteria. In the present study, we showed a dynamic change in acetylation and succinylation of TufA, the translation elongation factor Tu, from Bacillus subtilis. Increased acetylation of TufA was observed during the exponential growth phase in LB and minimal glucose conditions, and its acetylation level decreased upon entering the stationary phase, while its succinylation increased during the late stationary phase. TufA was also succinylated during vegetative growth under minimal citrate or succinate conditions. Mutational analysis showed that triple succinylation mimic mutations at Lys306, Lys308 and Lys316 in domain-3 of TufA had a negative effect on B. subtilis growth, whereas the non-acylation mimic mutations at these three lysine residues did not. Consistent with the growth phenotypes, the triple succinylation mimic mutant showed 67 % decreased translation activity in vitro, suggesting a possibility that succinylation at the lysine residues in domain-3 decreases the translation activity. TufA, including Lys308, was non-enzymatically succinylated by physiological concentrations of succinyl-CoA. Lys42 in the G-domain was identified as the most frequently modified acetylation site, though its acetylation was likely dispensable for TufA translation activity and growth. Determination of the intracellular levels of acetylating substrates and TufA acetylation revealed that acetyl phosphate was responsible for acetylation at several lysine sites of TufA, but not for Lys42 acetylation. It was speculated that acetyl-CoA was likely responsible for Lys42 acetylation, though AcuA acetyltransferase was not involved. Zn 2+ -dependent AcuC and NAD + -dependent SrtN deacetylases were responsible for deacetylation of TufA, including Lys42. These findings suggest the potential regulatory roles of acetylation and succinylation in controlling TufA function and translation in response to nutrient environments in B. subtilis.

Background: Oleaginous microalgae are promising sources of energy-rich triacylglycerols (TAGs) for direct use for food, feed and industrial applications. Lobosphaera incisa is a fresh water unicellular alga, which in response to nutrient stress accumulates a high amount of TAGs with a high proportion of arachidonic acid (ARA). The final committed step of de novo TAG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferases (DGATs), which add a fatty acid (FA) to the final sn-3 position of diacylglycerol (DAG)., Results: Genome analysis revealed the presence of five putative DGAT isoforms in L. incisa, including one DGAT of type 1, three DGATs of type 2 and a single isoform of a type 3 DGAT. For LiDGAT1, LiDGAT2.1, LiDGAT2.2 and LiDGAT2.3 enzyme activity was confirmed by expressing them in the TAG-deficient yeast strain H1246. Feeding experiments of yeast transformants with fatty acids suggest a broad substrate specificity spectrum for LiDGAT1. A significant TAG production in response to exogenous ARA was found for LiDGAT2.2. Cellular localization of the four type 1 and type 2 DGATs expressed in yeast revealed that they all localize to distinct ER domains. A prominent association of LiDGAT1 with ER domains in close proximity to forming lipid droplets (LDs) was also observed., Conclusions: The data revealed a distinct molecular, functional and cellular nature of type 1 and type 2 DGATs from L. incisa, with LiDGAT1 being a major contributor to the TAG pool. LiDGATs of type 2 might be in turn involved in the incorporation of unusual fatty acids into TAG and thus regulate the composition of TAG. This report provides a valuable resource for the further research of microalgae DGATs oriented towards production of fresh-water strains with higher oil content of valuable composition, not only for oil industry but also for human and animal nutrition.