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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.)

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.)

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.)

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.)

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.

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.

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.)

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.

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 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.

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.

The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters. IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide access to this chemodiversity for the discovery and synthesis of molecules with new bioactivities. The identification and successful cloning of the previously elusive hirsutene synthase from the S. hirsutum provide important insights and strategies for biosynthetic gene discovery in Basidiomycota. The finding of a terpene synthase-HMGS fusion, the discovery of other sesquiterpenoid biosynthetic gene clusters with dedicated HMGS genes, and HMGS gene duplications in fungal genomes give new importance to the role of HMGS as a key regulatory enzyme in isoprenoid and sterol biosynthesis that should be exploited for metabolic engineering., (Copyright © 2018 American Society for Microbiology.)

Ocimum kilimandscharicum is unique in possessing terpenoids whereas other Ocimum species are renowned for phenylpropanoids as major constituents of essential oil. The key enzyme of MVA/terpenoid metabolic pathway viz 3-hydroxy-3-methylglutaryl Co-A reductase (OkHMGR) of 1.7-Kb ORF encoding ~60-kDa protein was cloned from O. kilimandscharicum and its kinetic characteristics revealed the availability of HMG-CoA as a control point of MVA-pathway. Transcript profiling of the OkHMGR elucidated tissue-specific functions of the gene in flower and leaf tissues in accumulation of terpenoidal essential oil. OkHMGR was differentially regulated in response to exposure to methyl-jasmonate, salicylic-acid, and stress conditions such-as salt and temperature stress, demonstrating its key role in managing signaling and stress-responses. To elucidate its functional role, OkHMGR was transiently over-expressed in homologous and heterologous plants such as O. sanctum, O. basilicum, O. gratissimum, Withania somnifera and Artemisia annua. The over-expression and inhibition dual strategy revealed that the additional OkHMGR in-planta could afford endogenous flow of isoprenoid units towards synthesis of terpenoids. The present study provides in-depth insight of OkHMGR in regulation of biosynthesis of non-plastidal isoprenoids. This is first report on any gene of MVA/isoprenoid pathway from under-explored Camphor Tulsi belonging to genus Ocimum. Studies also suggested that OkHMGR could be a potential tool for attempting metabolic engineering for enhancing medicinally important terpenoidal metabolites in plants.

Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in β-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8-fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The existence of genetic traits might explain the susceptibility to develop hypercholesterolemia and the inter-individual differences in statin response. This study was performed to evaluate whether individuals' polymorphisms in HMG-CoA and KIF6 genes are independently associated with hypercholesterolemia, other lipid-associated traits, and statin response in unselected individuals enrolled in the Brisighella heart study (Survey 2012). A total of 1622 individuals, of which 183 under statin medication, were genotyped for a total of five polymorphisms (KIF6 rs20455, rs9471077, rs9462535; HMG-CoA rs3761740, rs3846662). The relationships between the five loci and clinical characteristics were analyzed. The principal basic parameters calculated on 12 h fasting blood included total cholesterol (TC), High Density Lipoprotein Cholesterol (HDL-C), Low-Density Lipoprotein Cholesterol (LDL-C), and triglycerides (TG). Hypercholesterolemia was defined as a TC >200 mg/dL or use of lipid-lowering medication. 965 individuals were characterized by hypercholesterolemia; these subjects were significantly older ( p < 0.001), with body mass index (BMI) and waist circumference significantly higher ( p < 0.001) compared to the others. HMG-CoA rs3846662 GG genotype was significantly over-represented in the hypercholesterolemic group ( p = 0.030). HMG-CoA rs3846662 genotype was associated with the level of TC and LDL-C. Furthermore, in the same subset of untreated subjects, we observed a significant correlation between the KIF6 rs20455 and HDL-C. KIF6 variants were associated with a significantly lower (rs20455) or higher (rs9471077 and rs9462535) risk of obesity, in males only. No association between responsiveness to statins and the polymorphisms under investigation were observed. Our results showed associations between HMG-CoA rs3846662 and KIF6 rs20455 and lipid phenotypes, which may have an influence on dyslipidemia-related events. Moreover, this represents the first study implicating KIF6 variants with obesity in men, and point to the possible involvement of this genetic locus in the known gender-related differences in coronary artery disease., Competing Interests: The authors declare no conflict of interest.

The heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, encoded by genes within the intracellular growth ( igr ) operon of Mycobacterium tuberculosis , catalyze the dehydrogenation of the cholesterol metabolite 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA), with a 3-carbon side chain, and subsequent hydration of the product 3-oxo-4,17-pregnadiene-20-carboxyl-CoA (3-OPDC-CoA) to form 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). The gene downstream of chsH2 , i.e., ltp2 , was expressed in recombinant Rhodococcus jostii RHA1 in combination with other genes within the igr operon. His-tagged Ltp2 copurified with untagged ChsH1-ChsH2, ChsH2, or the C-terminal domain of ChsH2, which contains a domain of unknown function (DUF35). Ltp2 in association with ChsH1-ChsH2 or just the DUF35 domain of ChsH2 was shown to catalyze the retroaldol cleavage of 17-HOPC-CoA to form androst-4-ene-3,17-dione and propionyl-CoA. Steady-state kinetic analysis using the Ltp2-DUF35 complex showed that the aldolase had optimal activity at pH 7.5, with a K m of 6.54 ± 0.90 μM and a k cat of 159 ± 8.50 s -1 ChsH1-ChsH2 could hydrate only about 30% of 3-OPDC-CoA, but this unfavorable equilibrium could be overcome when the aldolase was present to remove the hydrated product, providing a rationale for the close association of the aldolase with the hydratase. Homologs of ChsH1, ChsH2, and Ltp2 are found in steroid-degrading Gram-positive and Gram-negative bacteria, suggesting that side chains of diverse steroids may be cleaved by aldolases in the bacteria. IMPORTANCE The C-C bond cleavage of the D-ring side chain of cholesterol was shown to be catalyzed by an aldolase. The aldolase associates with the hydratase that catalyzes the preceding reaction in the cholesterol side chain degradation pathway. These enzymes are encoded by genes within the intracellular growth ( igr ) operon of M. tuberculosis , and the operon was demonstrated previously to be linked to the pathogenicity and persistence of the bacteria in macrophages and in mice., (Copyright © 2017 American Society for Microbiology.)

Histones are highly covalently modified, but the functions of many of these modifications remain unknown. In particular, it is unclear how histone marks are coupled to cellular metabolism and how this coupling affects chromatin architecture. We identified histone H3 Lys14 (H3K14) as a site of propionylation and butyrylation in vivo and carried out the first systematic characterization of histone propionylation. We found that H3K14pr and H3K14bu are deposited by histone acetyltransferases, are preferentially enriched at promoters of active genes and are recognized by acylation-state-specific reader proteins. In agreement with these findings, propionyl-CoA was able to stimulate transcription in an in vitro transcription system. Notably, genome-wide H3 acylation profiles were redefined following changes to the metabolic state, and deletion of the metabolic enzyme propionyl-CoA carboxylase altered global histone propionylation levels. We propose that histone propionylation, acetylation and butyrylation may act in combination to promote high transcriptional output and to couple cellular metabolism with chromatin structure and function.

Indole, an important signaling molecule as well as a typical N-heterocyclic aromatic pollutant, is widespread in nature. However, the biotransformation mechanisms of indole are still poorly studied. Here, we sought to unlock the genetic determinants of indole biotransformation in strain Cupriavidus sp. SHE based on genomics, proteomics and functional studies. A total of 177 proteins were notably altered (118 up- and 59 downregulated) in cells grown in indole mineral salt medium when compared with that in sodium citrate medium. RT-qPCR and gene knockout assays demonstrated that an indole oxygenase gene cluster was responsible for the indole upstream metabolism. A functional indole oxygenase, termed IndA, was identified in the cluster, and its catalytic efficiency was higher than those of previously reported indole oxidation enzymes. Furthermore, the indole downstream metabolism was found to proceed via the atypical CoA-thioester pathway rather than conventional gentisate and salicylate pathways. This unusual pathway was catalyzed by a conserved 2-aminobenzoyl-CoA gene cluster, among which the 2-aminobenzoyl-CoA ligase initiated anthranilate transformation. This study unveils the genetic determinants of indole biotransformation and will provide new insights into our understanding of indole biodegradation in natural environments and its functional studies., (© 2017 John Wiley & Sons Ltd.)

Microorganisms able to degrade aromatic contaminants constitute potential valuable biocatalysts to deal with a significant reusable carbon fraction suitable for eco-efficient valorization processes. Metabolic engineering of anaerobic pathways for degradation and recycling of aromatic compounds is an almost unexplored field. In this work, we present the construction of a functional bzd cassette encoding the benzoyl-CoA central pathway for the anaerobic degradation of benzoate. The bzd cassette has been used to expand the ability of some denitrifying bacteria to use benzoate as sole carbon source under anaerobic conditions, and it paves the way for future pathway engineering of efficient anaerobic biodegraders of aromatic compounds whose degradation generates benzoyl-CoA as central intermediate. Moreover, a recombinant Azoarcus sp. CIB strain harbouring the bzd cassette was shown to behave as a valuable biocatalyst for anaerobic toluene valorization towards the synthesis of poly-3-hydroxybutyrate (PHB), a biodegradable and biocompatible polyester of increasing biotechnological interest as a sustainable alternative to classical oil-derived polymers., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Background: 3-Hydroxypropionic acid (3-HP) is an important platform chemical, serving as a precursor for a wide range of industrial applications such as the production of acrylic acid and 1,3-propanediol. Although Escherichia coli or Saccharomyces cerevisiae are the primary industrial microbes for the production of 3-HP, alternative engineered hosts have the potential to generate 3-HP from other carbon feedstocks. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating 3-HP from one-carbon feedstock methanol., Results: Here we constructed a malonyl-CoA pathway by heterologously overexpressing the mcr gene to convert methanol into 3-HP in M. extorquens AM1. The engineered strains demonstrated 3-HP production with initial titer of 6.8 mg/l in shake flask cultivation, which was further improved to 69.8 mg/l by increasing the strength of promoter and mcr gene copy number. In vivo metabolic analysis showed a significant decrease of the acetyl-CoA pool size in the strain with the highest 3-HP titer, suggesting the supply of acetyl-CoA is a potential bottleneck for further improvement. Notably, 3-HP was rapidly degraded after the transition from exponential phase to stationary phase. Metabolomics analysis showed the accumulation of intracellular 3-hydroxypropionyl-CoA at stationary phase with the addition of 3-HP into the cultured medium, indicating 3-HP was first converted to its CoA derivatives. In vitro enzymatic assay and β-alanine pathway dependent 13 C-labeling further demonstrated that a reductive route sequentially converted 3-HP-CoA to acrylyl-CoA and propionyl-CoA, with the latter being reassimilated into the ethylmalonyl-CoA pathway. The deletion of the gene META1&#95;4251 encoding a putative acrylyl-CoA reductase led to reduced degradation rate of 3-HP in late stationary phase., Conclusions: We demonstrated the feasibility of constructing the malonyl-CoA pathway in M. extorquens AM1 to generate 3-HP. Furthermore, we showed that a reductive route coupled with the ethylmalonyl-CoA pathway was the major channel responsible for degradation of the 3-HP during the growth transition. Engineered M. extorquens AM1 represents a good platform for 3-HP production from methanol.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is one of the rate-limiting enzymes in the mevalonate pathway as it catalyzes the condensation of acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA. In this study, A HMGS gene (designated as GbHMGS1 ) was cloned from Ginkgo biloba for the first time. GbHMGS1 contained a 1422-bp open-reading frame encoding 474 amino acids. Comparative and bioinformatics analysis revealed that GbHMGS1 was extensively homologous to HMGSs from other plant species. Phylogenetic analysis indicated that the GbHMGS1 belonged to the plant HMGS superfamily, sharing a common evolutionary ancestor with other HMGSs, and had a further relationship with other gymnosperm species. The yeast complement assay of GbHMGS1 in HMGS -deficient Saccharomyces cerevisiae strain YSC6274 demonstrated that GbHMGS1 gene encodes a functional HMGS enzyme. The recombinant protein of GbHMGS1 was successfully expressed in E. coli . The in vitro enzyme activity assay showed that the k cat and K m values of GbHMGS1 were 195.4 min -1 and 689 μM, respectively. GbHMGS1 was constitutively expressed in all tested tissues, including the roots, stems, leaves, female flowers, male flowers and fruits. The transcript accumulation for GbHMGS1 was highest in the leaves. Expression profiling analyses revealed that GbHMGS1 expression was induced by abiotic stresses (ultraviolet B and cold) and hormone treatments (salicylic acid, methyl jasmonate, and ethephon) in G. biloba , indicating that GbHMGS1 gene was involved in the response to environmental stresses and plant hormones., Competing Interests: The authors declare no conflict of interest.

Background: Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant and antifungal activity. As the lower production in bio-fermentation, global metabolic analysis is required to further explore its biosynthetic network and determine the key limiting steps for rationally engineering. To achieve this goal, an engineering approach guided by a metabolic network model was implemented to better understand ascomycin biosynthesis and improve its production., Results: The metabolic conservation of Streptomyces species was first investigated by comparing the metabolic enzymes of Streptomyces coelicolor A3(2) with those of 31 Streptomyces strains, the results showed that more than 72% of the examined proteins had high sequence similarity with counterparts in every surveyed strain. And it was found that metabolic reactions are more highly conserved than the enzymes themselves because of its lower diversity of metabolic functions than that of genes. The main source of the observed metabolic differences was from the diversity of secondary metabolism. According to the high conservation of primary metabolic reactions in Streptomyces species, the metabolic network model of Streptomyces hygroscopicus var. ascomyceticus was constructed based on the latest reported metabolic model of S. coelicolor A3(2) and validated experimentally. By coupling with flux balance analysis and using minimization of metabolic adjustment algorithm, potential targets for ascomycin overproduction were predicted. Since several of the preferred targets were highly associated with ethylmalonyl-CoA biosynthesis, two target genes hcd (encoding 3-hydroxybutyryl-CoA dehydrogenase) and ccr (encoding crotonyl-CoA carboxylase/reductase) were selected for overexpression in S. hygroscopicus var. ascomyceticus FS35. Both the mutants HA-Hcd and HA-Ccr showed higher ascomycin titer, which was consistent with the model predictions. Furthermore, the combined effects of the two genes were evaluated and the strain HA-Hcd-Ccr with hcd and ccr overexpression exhibited the highest ascomycin production (up to 438.95 mg/L), 1.43-folds improvement than that of the parent strain FS35 (305.56 mg/L)., Conclusions: The successful constructing and experimental validation of the metabolic model of S. hygroscopicus var. ascomyceticus showed that the general metabolic network model of Streptomyces species could be used to analyze the intracellular metabolism and predict the potential key limiting steps for target metabolites overproduction. The corresponding overexpression strains of the two identified genes (hcd and ccr) using the constructed model all displayed higher ascomycin titer. The strategy for yield improvement developed here could also be extended to the improvement of other secondary metabolites in Streptomyces species.

An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization, fast growth, abundant precursors and energy supply, and a pronounced gene expression. Streptomyces albus BK3-25 is a high-yield industrial strain producing type-I polyketide salinomycin, with a unique ability of bean oil utilization. Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein. Firstly, introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and salinomycin, and subsequent deletion of the salinomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors, including malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA. Secondly, the energy and reducing force were measured, and the improved accumulation of ATP and NADPH was observed in the mutant. Furthermore, the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases, and a strong constitutive promoter was identified, providing a useful tool for further engineered gene expression. Finally, the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor, and the heterologous production of actinorhodin was obtained. This work clearly indicated the potential of the high-yield salinomycin producer as a surrogate host for heterologous production of polyketides, although more genetic manipulation should be conducted to streamline its performance.

Genetic research enables the evolution of novel biochemical reactions for the production of valuable chemicals from environmentally-friendly raw materials. However, the choice of appropriate microorganisms to support these reactions, which must have strong robustness and be capable of a significant product output, is a major difficulty. In the present study, the complete genome of the Clostridium tyrobutyricum strain CCTCC W428, a hydrogen- and butyric acid-producing bacterium with increased oxidative tolerance was analyzed. A total length of 3,011,209 bp of the C. tyrobutyricum genome with a GC content of 31.04% was assembled, and 3038 genes were discovered. Furthermore, a comparative clustering of proteins from C. tyrobutyricum CCTCC W428, C. acetobutylicum ATCC 824, and C. butyricum KNU-L09 was conducted. The results of genomic analysis indicate that butyric acid is produced by CCTCC W428 from butyryl-CoA through acetate reassimilation via CoA transferase, instead of the well-established phosphotransbutyrylase-butyrate kinase pathway. In addition, we identified ten proteins putatively involved in hydrogen production and 21 proteins associated with CRISPR systems, together with 358 ORFs related to ABC transporters and transcriptional regulators. Enzymes, such as oxidoreductases, HNH endonucleases, and catalase, were also found in this species. The genome sequence illustrates that C. tyrobutyricum has several desirable traits, and is expected to be suitable as a platform for the high-level production of bulk chemicals as well as bioenergy.

From various screens, we found that Kaposi's sarcoma-associated herpesvirus (KSHV) viral microRNAs (miRNAs) target several enzymes in the mevalonate/cholesterol pathway. 3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR [a rate-limiting step in the mevalonate pathway]), and farnesyl-diphosphate farnesyltransferase 1 (FDFT1 [a committed step in the cholesterol branch]) are repressed by multiple KSHV miRNAs. Transfection of viral miRNA mimics in primary endothelial cells (human umbilical vein endothelial cells [HUVECs]) is sufficient to reduce intracellular cholesterol levels; however, small interfering RNAs (siRNAs) targeting only HMGCS1 did not reduce cholesterol levels. This suggests that multiple targets are needed to perturb this tightly regulated pathway. We also report here that cholesterol levels were decreased in de novo -infected HUVECs after 7 days. This reduction is at least partially due to viral miRNAs, since the mutant form of KSHV lacking 10 of the 12 miRNA genes had increased cholesterol compared to wild-type infections. We hypothesized that KSHV is downregulating cholesterol to suppress the antiviral response by a modified form of cholesterol, 25-hydroxycholesterol (25HC). We found that the cholesterol 25-hydroxylase (CH25H) gene, which is responsible for generating 25HC, had increased expression in de novo -infected HUVECs but was strongly suppressed in long-term latently infected cell lines. We found that 25HC inhibits KSHV infection when added exogenously prior to de novo infection. In conclusion, we found that multiple KSHV viral miRNAs target enzymes in the mevalonate pathway to modulate cholesterol in infected cells during latency. This repression of cholesterol levels could potentially be beneficial to viral infection by decreasing the levels of 25HC. IMPORTANCE A subset of viruses express unique microRNAs (miRNAs), which act like cellular miRNAs to generally repress host gene expression. A cancer virus, Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8 [HHV-8]), encodes multiple miRNAs that repress gene expression of multiple enzymes that are important for cholesterol synthesis. In cells with these viral miRNAs or with natural infection, cholesterol levels are reduced, indicating these viral miRNAs decrease cholesterol levels. A modified form of cholesterol, 25-hydroxycholesterol, is generated directly from cholesterol. Addition of 25-hydroxycholesterol to primary cells inhibited KSHV infection of cells, suggesting that viral miRNAs may decrease cholesterol levels to decrease the concentration of 25-hydroxycholesterol and to promote infection. These results suggest a new virus-host relationship and indicate a previously unidentified viral strategy to lower cholesterol levels., (Copyright © 2017 Serquiña et al.)

Lambs ( = 18; 40.1 ± 7.4 kg BW) were used to assess supplementation of glycerol or fructose via drinking water on growth, tissue glycogen levels, postmortem glycolysis, and lipogenesis. Lambs were blocked by BW and allocated to alfalfa paddocks (2 lambs/paddock and 3 paddocks/treatment). Each paddock within a block was assigned randomly to drinking water treatments for 30 d: 1) control (CON), 2) 120 g fructose/L of drinking water (FRU), or 3) 120 g glycerol/L of drinking water (GLY). Lambs grazed alfalfa with free access to water treatments for 28 d and then were fasted in indoor pens for a final 2 d with access to only water treatments. Data were analyzed using the MIXED procedure of SAS with water treatment and time (when appropriate) in the model. During the 28-d grazing period, ADG was greater ( < 0.05) for GLY than for CON or FRU. During the 2-d fasting period, BW shrink was lower ( < 0.05) for GLY compared with CON or FRU. Hot carcass weight was greater ( < 0.05) for GLY than for FRU. The interaction for glycogen content × postmortem time was significant ( = 0.003) in LM and semitendinosus (ST) muscles. Glycogen content in the LM was greater ( < 0.05) for GLY at 2 and 3 h and for FRU at 1 h postmortem compared with CON. Glycogen content in ST did not differ between treatments ( > 0.05). Liver glycogen content was over 14-fold greater ( < 0.05) for GLY compared with FRU or CON. Liver free glucose was greater ( < 0.05) for GLY than for CON, whereas liver lipid content was higher ( < 0.05) for CON than for GLY. Supplementation with GLY increased ( < 0.05) odd-chain fatty acids in LM, subcutaneous fat (SQ), and the liver. Stearic acid (C18:0) concentrations were reduced in LM ( = 0.064) and subcutaneous adipose tissue (SQ; = 0.018), whereas oleic acid (C18:1 -9) concentration tended to be increased ( = 0.066) in SQ with FRU and GLY. Linolenic acid (C18:3 -3) was reduced ( = 0.031) and all long-chain -3 fatty acid (eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid) concentrations were increased ( < 0.05) with FRU and GLY compared with CON. Glycerol supplementation upregulated ( < 0.05) stearoyl-CoA desaturate () and fatty acid synthase () mRNA by over 40-fold in the SQ and 5-fold in the liver. Glycerol supplementation also upregulated ( < 0.05) glucose transporters and glycogen branching enzyme in the liver. Overall, glycerol supplementation improved growth, reduced BW shrink during fasting, increased glycogen content in muscle and the liver, and stimulated de novo lipogenesis.