Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2421-2427 ◽  
Author(s):  
Eva Radmacher ◽  
Luke J. Alderwick ◽  
Gurdyal S. Besra ◽  
Alistair K. Brown ◽  
Kevin J. C. Gibson ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Corynebacterium Glutamicum ◽  
Fatty Acid Synthase ◽  
Lipid Analysis ◽  
Growth Yield ◽  
Mycolic Acid ◽  
Mycolic Acids ◽  
Fatty Acid Synthases

The lipid-rich Corynebacterianeae, to which Corynebacterium glutamicum and Mycobacterium species belong, produce both fatty acids and mycolic acids. Compared with most other bacteria, C. glutamicum possesses two fatty acid synthases, encoded by fasA (8907 kb; FAS-IA) and fasB (8988 kb; FAS-IB). Here, it was shown by mutational analyses that fasA is essential but fasB is not. However, in a fasA background, the fasB mutation results in a slightly reduced growth yield, l-glutamate production is increased, and comparative lipid analysis suggests that in vivo FAS-IB is active primarily to supply palmitate. Transcript quantifications revealed that the fasB transcript contributes 32 % to both fas transcripts during growth on glucose, affirmative for fasB expression, and that fasB is subordinate to fasA. The fasA transcript is downregulated by 8·3-fold during growth on acetate as compared with glucose. The lipid analyses also demonstrate that cells grown on propionate produce a number of uneven fatty acids (e.g. 15 : 0, 17 : 0, 17 : 1), which are not present in cells grown on glucose or acetate, suggesting that fatty acid synthase in vivo may also use propionyl-CoA as the priming unit in fatty acid synthesis. The fatty acid auxotrophic fasAB double mutant was used to determine the suggested incorporation of fatty acids into mycolic acids. Supplementation of this mutant with uniformly labelled [13C]oleate and analysis of isolated mycolic acids confirmed that mature mycolic acids in the mutant consist exclusively of two fused [13C]oleate molecules. In addition to an altered phospholipid profile, the fasB mutant also exhibits differences in its mycolic acid profile. Taken together, the results show that although FAS-IA is the most relevant fatty acid synthase of C. glutamicum and FAS-IB is supplementary, both synthases are necessary to produce the characteristic lipid environment of this organism.

scholarly journals The Two Carboxylases of Corynebacterium glutamicum Essential for Fatty Acid and Mycolic Acid Synthesis

2007 ◽  
Vol 189 (14) ◽  
pp. 5257-5264 ◽  
Author(s):  
Roland Gande ◽  
Lynn G. Dover ◽  
Karin Krumbach ◽  
Gurdyal S. Besra ◽  
Hermann Sahm ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Corynebacterium Glutamicum ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Α Subunit ◽  
Mycolic Acids

ABSTRACT The suborder Corynebacterianeae comprises bacteria like Mycobacterium tuberculosis and Corynebacterium glutamicum, and these bacteria contain in addition to the linear fatty acids, unique α-branched β-hydroxy fatty acids, called mycolic acids. Whereas acetyl-coenzyme A (CoA) carboxylase activity is required to provide malonyl-CoA for fatty acid synthesis, a new type of carboxylase is apparently additionally present in these bacteria. It activates the α-carbon of a linear fatty acid by carboxylation, thus enabling its decarboxylative condensation with a second fatty acid to afford mycolic acid synthesis. We now show that the acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated α-subunit AccBC, the β-subunit AccD1, and the small peptide AccE of 8.9 kDa, forming an active complex of approximately 812,000 Da. The carboxylase involved in mycolic acid synthesis is made up of the two highly similar β-subunits AccD2 and AccD3 and of AccBC and AccE, the latter two identical to the subunits of the acetyl-CoA carboxylase complex. Since AccD2 and AccD3 orthologues are present in all Corynebacterianeae, these polypeptides are vital for mycolic acid synthesis forming the unique hydrophobic outer layer of these bacteria, and we speculate that the two β-subunits present serve to lend specificity to this unique large multienzyme complex.

scholarly journals A Futile Metabolic Cycle of Fatty Acyl-CoA Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production

2020 ◽  
Author(s):  
Masato Ikeda ◽  
Keisuke Takahashi ◽  
Tatsunori Ohtake ◽  
Ryosuke Imoto ◽  
Haruka Kawakami ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acids ◽  
Corynebacterium Glutamicum ◽  
Candidate Genes ◽  
Outer Layer ◽  
Acid Production ◽  
Fatty Acyl ◽  
Fatty Acid Production ◽  
Mycolic Acids

Fatty acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD) catalyze opposing reactions between acyl-CoAs and free fatty acids. Within the genome of Corynebacterium glutamicum, several candidate genes for each enzyme are present, although their functions remain unknown. Modified expressions of the candidate genes in the fatty acid producer WTΔfasR led to identification of one tes gene (tesA) and two fadD genes (fadD5 and fadD15), which functioned positively and negatively in fatty acid production, respectively. Genetic analysis showed that fadD5 and fadD15 are responsible for utilization of exogenous fatty acids and that tesA plays a role in supplying fatty acids for synthesis of the outer layer components mycolic acids. Enzyme assays and expression analysis revealed that tesA, fadD5, and fadD15 were co-expressed to create a cyclic route between acyl-CoAs and fatty acids. When fadD5 or fadD15 was disrupted in wild-type C. glutamicum, both disruptants excreted fatty acids during growth. Double disruptions of them resulted in a synergistic increase in production. Additional disruption of tesA revealed a canceling effect on production. These results indicate that the FadDs normally shunt the surplus of TesA-generated fatty acids back to acyl-CoAs for lipid biosynthesis and that interception of this shunt provokes cells to overproduce fatty acids. When this strategy was applied to a fatty acid high-producer, the resulting fadDs-disrupted and tesA-amplified strain exhibited a 72% yield increase relative to its parent and produced fatty acids, which consisted mainly of oleic acid, palmitic acid, and stearic acid, on the gram scale per liter from 1% glucose. IMPORTANCE The industrial amino acid producer Corynebacterium glutamicum has currently evolved into a potential workhorse for fatty acid production. In this organism, we obtained evidence showing the presence of a unique mechanism of lipid homeostasis, namely, a formation of a futile cycle of acyl-CoA hydrolysis and resynthesis mediated by acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD), respectively. The biological role of the coupling of Tes and FadD would be to supply free fatty acids for synthesis of the outer layer components mycolic acids and to recycle their surplusage to acyl-CoAs for membrane lipid synthesis. We further demonstrated that engineering of the cycle in a fatty acid high-producer led to dramatically improved production, which provides a useful engineering strategy for fatty acid production in this industrially important microorganism.

scholarly journals Fatty Acid Synthesis Is Essential for Survival of Cryptococcus neoformans and a Potential Fungicidal Target

2007 ◽  
Vol 51 (10) ◽  
pp. 3537-3545 ◽  
Author(s):  
Methee Chayakulkeeree ◽  
Thomas H. Rude ◽  
Dena L. Toffaletti ◽  
John R. Perfect
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cryptococcus Neoformans ◽  
Fatty Acid Synthase ◽  
Acid Synthesis ◽  
Yeast Cells ◽  
Synthase Inhibitor ◽  
The Brain

ABSTRACT Fatty acid synthase in the yeast Cryptococcus neoformans is composed of two subunits encoded by FAS1 and FAS2 genes. We inserted a copper-regulated promoter (P CTR4-2 ) to regulate FAS1 and FAS2 expression in Cryptococcus neoformans (strains P CTR4-2 /FAS1 and P CTR4-2 /FAS2, respectively). Both mutants showed growth rates similar to those of the wild type in a low-copper medium in which FAS1 and FAS2 were expressed, but even in the presence of exogenous fatty acids, strains were suppressed in growth under high-copper conditions. The treatment of C. neoformans with fluconazole was shown to have an increased inhibitory activity and even became fungicidal when either FAS1 or FAS2 expression was suppressed. Furthermore, a subinhibitory dose of fluconazole showed anticryptococcal activity in vitro in the presence of cerulenin, a fatty acid synthase inhibitor. In a murine model of pulmonary cryptococcosis, a tissue census of yeast cells in P CTR4-2 /FAS2 strain at day 7 of infection was significantly lower than that in mice treated with tetrathiomolybdate, a copper chelator (P < 0.05), and a yeast census of P CTR4-2 /FAS1 strain at day 14 of infection in the brain was lower in the presence of more copper. In fact, no positive cultures from the brain were detected in mice (with or without tetrathiomolybdate treatment) infected with the P CTR4-2 /FAS2 strain, which implies that this mutant did not reach the brain in mice. We conclude that both FAS1 and FAS2 in C. neoformans are essential for in vitro and in vivo growth in conditions with and without exogenous fatty acids and that FAS1 and FAS2 can potentially be fungicidal targets for C. neoformans with a potential for synergistic behavior with azoles.

scholarly journals Purification and Biochemical Characterization of theMycobacterium tuberculosisβ-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

2001 ◽  
Vol 276 (50) ◽  
pp. 47029-47037 ◽  
Author(s):  
Merrill L. Schaeffer ◽  
Gautam Agnihotri ◽  
Craig Volker ◽  
Howard Kallender ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Mycolic Acids ◽  
E Coli ◽  
Long Chain

Mycolic acids are vital components of theMycobacterium tuberculosiscell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performsde novofatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of theM. tuberculosisFASII system. KasA and KasB were expressed inE. coliand purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use ofM. tuberculosisAcpM, suggesting that structural differences between AcpM andE. coliACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

scholarly journals Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis.

1996 ◽  
Vol 40 (12) ◽  
pp. 2813-2819 ◽  
Author(s):  
R A Slayden ◽  
R E Lee ◽  
J W Armour ◽  
A M Cooper ◽  
I M Orme ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Type I ◽  
Dependent Manner ◽  
Fas Ii

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.

scholarly journals A tissue-specific increase in lipogenesis in rat brown adipose tissue in hypothyroidism

1988 ◽  
Vol 251 (2) ◽  
pp. 553-557 ◽  
Author(s):  
H S Baht ◽  
E D Saggerson
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Maximum Activity ◽  
Brown Adipocyte ◽  
Glycolytic Flux ◽  
Brown Adipocytes ◽  
White Adipocytes ◽  
Brown Adipose

1. Rats were made hypothyroid by feeding them with propylthiouracil together with a low-iodine diet for 4 weeks. 2. [U-14C]Glucose conversion into fatty acids was substantially enhanced in brown adipocytes isolated from hypothyroid rats. Incorporation of 3H2O into fatty acids in vivo was enhanced in hypothyroidism in interscapular brown fat, but not in epididymal white fat or in liver. Hypothyroidism increased the activities of fatty acid synthase and ATP citrate lyase in brown, but not in white, adipocytes. 3. Glycolytic flux in brown adipocytes, quantified by [3-3H]glucose detritiation, was increased by hypothyroidism. This change was accompanied by increased maximum activity of phosphofructokinase. In white adipocytes a large increase in phosphofructokinase maximum activity was observed in hypothyroidism, but this change was accompanied by only small increases in the rate of glucose detritiation by incubated cells. It is suggested that in the brown adipocyte the overall conversion of glucose into fatty acids is enhanced in thyroid deficiency, but that this change is muted in the white adipocyte, possibly because of limitation of glucose transport. 4. Fatty acid synthesis in brown adipocytes from hypothyroid animals was considerably less sensitive to inhibition by exogenous fatty acids than is the process in cells from euthyroid animals. Consequently, the effect of hypothyroidism to enhance lipogenesis is amplified in the presence of physiological concentrations of fatty acid.

scholarly journals A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

Open Biology ◽  
2017 ◽  
Vol 7 (2) ◽  
pp. 160277 ◽  
Author(s):  
Matías Cabruja ◽  
Sonia Mondino ◽  
Yi Ting Tsai ◽  
Julia Lara ◽  
Hugo Gramajo ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipid Metabolism ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acid Biosynthesis ◽  
Mycolic Acids ◽  
Storage Lipids ◽  
Conditional Mutant

Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo , we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C 12 to C 18 acyl-CoAs, but not of long-chain acyl-CoAs (C 19 to C 24 ). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.

scholarly journals Dietary Coleus Amboinicus Herb Decreases Ruminal Methanogenesis and Biohydrogenation, and Improves Meat Quality and Fatty Acid Composition in Longissimus Thoracis of Lambs

2021 ◽  
Author(s):  
Yulianri Rizki Yanza ◽  
Malgorzata Szumacher-Strabel ◽  
Dorota Lechniak ◽  
Sylwester Ślusarczyk ◽  
Pawel Kolodziejski ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Meat Quality ◽  
Methane Production ◽  
Fatty Acid Synthase ◽  
Unsaturated Fatty Acids ◽  
Biologically Active ◽  
Coleus Amboinicus

Abstract Background: This study aimed to investigate the effect of biologically active compounds (BAC) of Coleus amboinicus Lour. (CAL) herb fed to growing lambs on ruminal methane production, ruminal biohydrogenation of unsaturated fatty acids and meat characteristics. An in vitro trial (Experiment 1) comprising of control and three experimental diets (CAL constituting 10%, 15%, and 20% of the total diet) was conducted to determine an effective dose for in vivo experiments. After the in vitro trial, two in vivo experiments were conducted on six growing, rumen-cannulated lambs (Experiment 2) and 16 growing lambs (Experiment 3), which were assigned into the control (CON) and one experimental diet (20% of CAL). Several parameters were examined in vitro (pH, ammonia and VFA concentrations, protozoa, methanogens and select bacteria populations) and in vivo (methane production, digestibility, ruminal microorganism populations, meat quality, fatty acids profiles in rumen fluid and meat, transcript expression of 5 genes in meat). Results: The CAL lowered in vitro methane production by 51%. In the in vivo experiments, lambs fed CAL decreased methane production by 20% compared with the CON animals (Experiment 3), which corresponded to the reduced total methanogens counts in all experiments up to 28%, notably Methanobacteriales. In Experiment 3, CAL increased or tended to increase the numbers of Ruminococcus albus, Megasphaeraelsdenii, Butyrivibrioproteoclasticus, and Butyrivibriofibrisolvens. Dietary CAL suppressed the Holotricha population, but increased or tended to increase Entodiniomorpha population in Experiments 2 and 3. An increase in the polyunsaturated fatty acid (PUFA) proportion in the rumen of lambs was noted in response to the CAL diet, which was mainly attributable to the increase in C18:3 cis-9 cis-12 cis-15 (LNA) proportion. The CAL reduced the mRNA expressions of four investigated genes in meat (fatty acid synthase, stearoyl-CoA desaturase, lipoprotein lipase, and fatty acid desaturase 1). Conclusions:Summarizing, polyphenols of CAL (20% in diet) origin can mitigate ruminal methane production by inhibiting the methanogens communities. Supplementation of CAL also provides favorable conditions in the rumen by modulating ruminal bacteria involved in fermentation and biohydrogenation of fatty acids. CAL elevated the LNA concentration, which led to improved meat quality through increased deposition of n-3 PUFA.

scholarly journals Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C20 Fatty Acids to C60-to-C90 Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

2008 ◽  
Vol 74 (16) ◽  
pp. 5078-5085 ◽  
Author(s):  
Aner Gurvitz ◽  
J. Kalervo Hiltunen ◽  
Alexander J. Kastaniotis
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipoic Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acid Synthesis ◽  
Acid Biosynthesis ◽  
Mycolic Acids

ABSTRACT We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C20 fatty acids to form C60-to-C90 mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Δ cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Δ cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C4 to C8) than was previously thought (>C12). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.

scholarly journals Dietary Coleus amboinicus Lour. decreases ruminal methanogenesis and biohydrogenation, and improves meat quality and fatty acid composition in longissimus thoracis muscle of lambs

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Yulianri Rizki Yanza ◽  
Malgorzata Szumacher-Strabel ◽  
Dorota Lechniak ◽  
Sylwester Ślusarczyk ◽  
Pawel Kolodziejski ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Meat Quality ◽  
Methane Production ◽  
Fatty Acid Synthase ◽  
Biologically Active ◽  
In Vivo Experiments ◽  
Coleus Amboinicus

Abstract Background Methane production and fatty acids (FA) biohydrogenation in the rumen are two main constraints in ruminant production causing environmental burden and reducing food product quality. Rumen functions can be modulated by the biologically active compounds (BACs) of plant origins as shown in several studies e.g. reduction in methane emission, modulation of FA composition with positive impact on the ruminant products. Coleus amboinicus Lour. (CAL) contains high concentration of polyphenols that may potentially reduce methane production and modulate ruminal biohydrogenation of unsaturated FA. This study aimed to investigate the effect of BAC of Coleus amboinicus Lour. (CAL) fed to growing lambs on ruminal methane production, biohydrogenation of unsaturated FA and meat characteristics. In this study, the in vitro experiment aiming at determining the most effective CAL dose for in vivo experiments was followed by two in vivo experiments in rumen-cannulated rams and growing lambs. Experiment 1 (RUSITEC) comprised of control and three experimental diets differing in CAL content (10%, 15%, and 20% of the total diet). The two in vivo experiments were conducted on six growing, rumen-cannulated lambs (Exp. 2) and 16 growing lambs (Exp. 3). Animals were assigned into the control (CON) and experimental (20% of CAL) groups. Several parameters were examined in vitro (pH, ammonia and VFA concentrations, protozoa, methanogens and select bacteria populations) and in vivo (methane production, digestibility, ruminal microorganism populations, meat quality, fatty acids profiles in rumen fluid and meat, transcript expression of 5 genes in meat). Results CAL lowered in vitro methane production by 51%. In the in vivo Exp. 3, CAL decreased methane production by 20% compared with the CON group, which corresponded to reduction of total methanogen counts by up to 28% in all experiments, notably Methanobacteriales. In Exp. 3, CAL increased or tended to increase populations of some rumen bacteria (Ruminococcus albus, Megasphaera elsdenii, Butyrivibrio proteoclasticus, and Butyrivibrio fibrisolvens). Dietary CAL suppressed the Holotricha population, but increased or tended to increase Entodiniomorpha population in vivo. An increase in the polyunsaturated fatty acid (PUFA) proportion in the rumen of lambs was noted in response to the CAL diet, which was mainly attributable to the increase in C18:3 cis-9 cis-12 cis-15 (LNA) proportion. CAL reduced the mRNA expression of four out of five genes investigated in meat (fatty acid synthase, stearoyl-CoA desaturase, lipoprotein lipase, and fatty acid desaturase 1). Conclusions Summarizing, polyphenols of CAL origin (20% in diet) mitigated ruminal methane production by inhibiting the methanogen communities. CAL supplementation also improved ruminal environment by modulating ruminal bacteria involved in fermentation and biohydrogenation of FA. Besides, CAL elevated the LNA concentration, which improved meat quality through increased deposition of n-3 PUFA.

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