Myeloid-Specific Deficiency of Long-Chain Acyl CoA Synthetase 4 Reduces Inflammation by Remodeling Phospholipids and Reducing Production of Arachidonic Acid-Derived Proinflammatory Lipid Mediators.

In response to infection or tissue damage, resident peritoneal macrophages (rpMACs) produce inflammatory lipid mediators from the polyunsaturated fatty acid (PUFA), arachidonic acid (AA). Long-chain acyl-CoA synthetase 4 (ACSL4) catalyzes the covalent addition of a CoA moiety to fatty acids, with a strong preference for AA and other PUFAs containing three or more double bonds. PUFA-CoA can be incorporated into phospholipids, which is the source of PUFA for lipid mediator synthesis. In this study, we demonstrated that deficiency of Acsl4 in mouse rpMACs resulted in a significant reduction of AA incorporated into all phospholipid classes and a reciprocal increase in incorporation of oleic acid and linoleic acid. After stimulation with opsonized zymosan (opZym), a diverse array of AA-derived lipid mediators, including leukotrienes, PGs, hydroxyeicosatetraenoic acids, and lipoxins, were produced and were significantly reduced in Acsl4-deficient rpMACs. The Acsl4-deficient rpMACs stimulated with opZym also demonstrated an acute reduction in mRNA expression of the inflammatory cytokines, Il6, Ccl2, Nos2, and Ccl5 When Acsl4-deficient rpMACs were incubated in vitro with the TLR4 agonist, LPS, the levels of leukotriene B4 and PGE2 were also significantly decreased. In LPS-induced peritonitis, mice with myeloid-specific Acsl4 deficiency had a significant reduction in leukotriene B4 and PGE2 levels in peritoneal exudates, which was coupled with reduced infiltration of neutrophils in the peritoneal cavity as compared with wild-type mice. Our data demonstrate that chronic deficiency of Acsl4 in rpMACs reduces the incorporation of AA into phospholipids, which reduces lipid mediator synthesis and inflammation.

Biotransformation and recovery of the isoflavones genistein and daidzein from industrial antibiotic fermentations.

The objective of this study was to follow the metabolic fate of isoflavone glucosides from the soybean meal in a model industrial fermentation to determine if commercially useful isoflavones could be harvested as coproducts from the spent broth at the end of the fermentation. The isoflavone aglycones, genistein, and daidzein together make up 0.1-0.2 % of the soybean meal by weight but serve no known function in the manufacturing process. After feeding genistein to washed cells of the erythromycin-producing organism, Saccharopolyspora erythraea, the first biotransformation product (Gbp1) was determined by X-ray crystallography to be genistein-7-O-α-rhamnoside (rhamnosylgenistein). Subsequent feeding of rhamnosylgenistein to growing cells of Saccharopolyspora erythraea led to the production of a second biotransformation product, Gbp2. Chromatographic evidence suggested that Gbp2 accumulated in the spent broth of the erythromycin fermentation. When the spent broth was hydrolyzed with acid or industrial enzyme preparations, the isoflavone biotransformation products were returned back to their parental forms, genistein and daidzein, which were then recovered as coproducts. Desirable features of this method are that it does not require modification of the erythromycin manufacturing process or genetic engineering of the producing organism to be put into practice. A preliminary investigation of five additional antibiotic fermentations of industrial importance also found isoflavone coproduct potential.

An erythromycin process improvement using the diethyl methylmalonate-responsive (Dmr) phenotype of the Saccharopolyspora erythraea mutB strain.

The Saccharopolyspora erythraea mutB knockout strain, FL2281, having a block in the methylmalonyl-CoA mutase reaction, was found to carry a diethyl methylmalonate-responsive (Dmr) phenotype in an oil-based fermentation medium. The Dmr phenotype confers the ability to increase erythromycin A (erythromycin) production from 250-300% when the oil-based medium is supplemented with 15 mM levels of this solvent. Lower concentrations of the solvent stimulated proportionately less erythromycin production, while higher concentrations had no additional benefit. Although the mutB strain is phenotypically a low-level erythromycin producer, diethyl methylmalonate supplementation allowed it to produce up to 30% more erythromycin than the wild-type (control) strain-a strain that does not show the Dmr phenotype. The Dmr phenotype represents a new class of strain improvement phenotype. A theory to explain the biochemical mechanism for the Dmr phenotype is proposed. Other phenotypes found to be associated with the mutB knockout were a growth defect and hyper-pigmentation, both of which were restored to normal by exposure to diethyl methylmalonate. Furthermore, mutB fermentations did not significantly metabolize soybean oil in the presence of diethyl methylmalonate. Finally, a novel method is proposed for the isolation of additional mutants with the Dmr phenotype.

The good and bad of adipose tissue macrophage exosomes in obesity.

Adipose tissue macrophages regulate adipose tissue inflammation and systemic insulin-glucose homeostasis. In a recent study by Ying et al. (2021), M2 polarized bone marrow-derived macrophages secreted exosomes containing miR-690 that, when administered to obese mice, improved glucose-insulin homeostasis. miR-690 reduced expression of Nadk, which decreased inflammation and improved insulin signaling.

Muscle strength is increased in mice that are colonized with microbiota from high-functioning older adults.

Evidence in support of a gut-muscle axis has been reported in rodents, but studies in older adult humans are limited. Accordingly, the primary goals of the present study were to compare gut microbiome composition in older adults that differed in terms of the percentage of whole body lean mass and physical functioning (high-functioning, HF, n = 18; low-functioning, LF, n = 11), and to evaluate the causative role of the gut microbiome on these variables by transferring fecal samples from older adults into germ-free mice. Family-level Prevotellaceae, genus-level Prevotella and Barnesiella, and the bacterial species Barnesiella intestinihominis were higher in HF older adults at the initial study visit, at a 1-month follow-up visit, in HF human fecal donors, and in HF-colonized mice, when compared with their LF counterparts. Grip strength was significantly increased by 6.4% in HF-, when compared with LF-colonized mice. In contrast, despite significant differences for the percentage of whole body lean mass and physical functioning when comparing the human fecal donors, the percentage of whole body lean mass and treadmill endurance capacity were not different when comparing human microbiome-containing mice. In sum, these data suggest a role for gut bacteria on the maintenance of muscle strength, but argue against a role for gut bacteria on the maintenance of the percentage of whole body lean mass or endurance capacity, findings that collectively add to elucidation of the gut-muscle axis in older adults.

Knockout of the erythromycin biosynthetic cluster gene, eryBI, blocks isoflavone glucoside bioconversion during erythromycin fermentations in Aeromicrobium erythreum but not in Saccharopolyspora erythraea.

Isoflavone glucosides are valuable nutraceutical compounds and are present in commercial fermentations, such as the erythromycin fermentation, as constituents of the soy flour in the growth medium. The purpose of this study was to develop a method for recovery of the isoflavone glucosides as value-added coproducts at the end of either Saccharopolyspora erythraea or Aeromicrobium erythreum fermentation. Because the first step in isoflavone metabolism was known to be the conversion of isoflavone glucosides to aglycones by a beta-glucosidase, we chose to knock out the only beta-glucosidase gene known at the start of the study, eryBI, to see what effect this had on metabolism of isoflavone glucosides in each organism. In the unicellular erythromycin producer A. erythreum, knockout of eryBI was sufficient to block the conversion of isoflavone glucosides to aglycones. In S. erythraea, knockout of eryBI had no effect on this reaction, suggesting that other beta-glucosidases are present. Erythromycin production was not significantly affected in either strain as a result of the eryBI knockout. This study showed that isoflavone metabolism could be blocked in A. erythreum by eryBI knockout but that eryBI knockout was not sufficient to block isoflavone metabolism in S. erythraea.

A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction.

OBJECTIVE: Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction. METHODS: We developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings. RESULTS: Ad-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro. CONCLUSION: These studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE).

Controlled release of cytokines using silk-biomaterials for macrophage polarization.

Polarization of macrophages into an inflammatory (M1) or anti-inflammatory (M2) phenotype is important for clearing pathogens and wound repair, however chronic activation of either type of macrophage has been implicated in several diseases. Methods to locally control the polarization of macrophages is of great interest for biomedical implants and tissue engineering. To that end, silk protein was used to form biopolymer films that release either IFN-γ or IL-4 to control the polarization of macrophages. Modulation of the solubility of the silk films through regulation of ß-sheet (crystalline) content enabled a short-term release (4-8 h) of either cytokine, with smaller amounts released out to 24 h. Altering the solubility of the films was accomplished by varying the time that the films were exposed to water vapor. The released IFN-γ or IL-4 induced polarization of THP-1 derived macrophages into the M1 or M2 phenotypes, respectively. The silk biomaterials were able to release enough IFN-γ or IL-4 to repolarize the macrophage from M1 to M2 and vice versa, demonstrating the well-established plasticity of macrophages. High ß-sheet content films that are not soluble and do not release the trapped cytokines were also able to polarize macrophages that adhered to the surface through degradation of the silk protein. Chemically conjugating IFN-γ to silk films through disulfide bonds allowed for longer-term release to 10 days. The release of covalently attached IFN-γ from the films was also able to polarize M1 macrophages in vitro. Thus, the strategy described here offers new approaches to utilizing biomaterials for directing the polarization of macrophages.

Metabolic engineering of antibiotic-producing actinomycetes using in vitro transposon mutagenesis.

A program of mutation and screening, with stepwise reverse engineering or "decoding" of the improved strain, is a way to better understand the genetics and physiology of the strain improvement process. As more is learned about the genetics of strain improvement, it is hoped that more fundamental principles will emerge about the types of mutations and genetic manipulations that reliably lead to higher producing strains. This will accelerate the construction of higher producing strains by metabolic engineering in the future. In this chapter, a detailed tagged mutagenesis approach is described using in vitro transposon mutagenesis which allowed the successful identification of key genes involved in macrolide (erythromycin) antibiotic biosynthesis.

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production.

Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8kb DNA fragment in a plasmid which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1. None of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in a strain containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3' terminus of the mutR coding sequence.

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea.

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102-153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141-236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53-76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.

Physical-genetic map of the erythromycin-producing organism Saccharopolyspora erythraea.

A physical map of the chromosome of the erythromycin-producing actinomycete Saccharopolyspora erythraea NRRL 2338 has been constructed using the restriction enzymes AseI and DraI. The map was constructed by a variety of methods including linking clone analysis, cross-hybridizations using labelled macrorestriction fragments, gene probing, two-dimensional PFGE and restriction enzyme site generation. Analysis of the individual macrorestriction patterns of the 17 AseI-, 6 DraI- and 22 AseI/DraI-digested fragments indicated a chromosome size of about 8 Mb. Linking clones for five contiguous AseI fragments were obtained, covering 32% of the chromosome. The linkage of an additional eight AseI fragments was aided by the finding that the rRNA operons of S. erythraea contain an AseI site within the 16S (rrs) gene. Generation of S. erythraea strains that contain additional DraI sites within selected AseI fragments, followed by PFGE analysis and Southern hybridization to determine specific linkages, facilitated the completion of the AseI map. The entire DraI map was constructed by gene probing and cross-hybridizations. PFGE analysis of agarose-embedded DNA prepared in either the presence or absence of proteinase K suggested that the S. erythraea NRRL 2338 chromosome is linear. A total of 15 genes or gene clusters were mapped to specific AseI and DraI fragments, including the erythromycin-biosynthetic gene cluster and the rRNA operons.

Analysis of an 8.1-kb DNA fragment contiguous with the erythromycin gene cluster of Saccharopolyspora erythraea in the eryCI-flanking region.

An 8.1-kb region of the Saccharopolyspora erythraea genome, significant for its contiguity to the known genes of the erythromycin biosynthetic gene cluster, was mutationally analyzed and its DNA sequence was determined. The region lies immediately adjacent to eryCI. The newly characterized region is notable for a large, 3.0-kb segment, predicted not to be translated, followed by four probable genes: an acetyltransferase gene, a protease inhibitor gene, a methyltransferase gene, and a transposase gene. Because the probable functions of the genes in this region are not required for erythromycin biosynthesis or resistance and because a deletion of a 6.0-kb portion of this region had no effect on erythromycin biosynthesis, this region marks the outside boundary of the erythromycin gene cluster. Therefore, eryCI represents the end of the cluster. These results complete the analysis of the erythromycin gene cluster and eliminate the possibility that additional sought-after pathway-specific structural or regulatory genes might be found within or adjacent to the cluster.

Engineering precursor flow for increased erythromycin production in Aeromicrobium erythreum.

Metabolic engineering technology for industrial microorganisms is under development to create rational, more reliable, and more cost-effective approaches to strain improvement. Strain improvement is a critical component of the drug development process, yet the genetic basis for high production by industrial microorganisms is still a mystery. In this study, a search was begun for genetic modifications critical for high-level antibiotic production. The model system used was erythromycin production studied in the unicellular actinomycete, Aeromicrobium erythreum. A tagged-mutagenesis approach allowed reverse engineering of improved strains, revealing two genes, mutB and cobA, in the primary metabolic branch for methylmalonyl-CoA utilization. Knockouts in these genes created a permanent metabolic switch in the flow of methylmalonyl-CoA, from the primary branch into a secondary metabolic branch, driving erythromycin overproduction. The model provides insights into the regulation and evolution of secondary metabolism.

The erythromycin biosynthetic gene cluster of Aeromicrobium erythreum.

The erythromycin-biosynthetic (ery) gene cluster of Aeromicrobium erythreum was cloned and characterized. The 55.4-kb cluster contains 25 ery genes. Homologues were found for each gene in the previously characterized ery gene cluster from Saccharopolyspora erythraea. In addition, four new predicted ery genes were identified. Two of the new predicted genes, coding for a phosphopantetheinyl transferase (eryP) and a type II thioesterase (eryTII), were internal to the ery cluster. The other two new genes, coding for a thymidine 5'-diphosphate-glucose synthase (eryDI) and a MarR-family transcriptional repressor (ery-ORF25), were found at the two ends of the ery cluster. A knockout in eryDI showed it to be essential for erythromycin biosynthesis. The gene order of the two ery clusters was conserved within a core region of 15 contiguous genes, with the exception of IS1136 which was not found in the A. erythreum cluster. Beyond the core region, gene shuffling had occurred between the two sides of the cluster. The flanking regions of the two ery clusters were not alike in the type of genes found.