The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini

Demirev, Atanas V.; Khanal, Anamika; Sedai, Bhishma R.; Lim, Seung Kwan; Na, MinKyun; Nam, Doo Hyun

doi:10.1007/s00253-010-2587-2

Cited by 10 publications

(15 citation statements)

References 32 publications

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“…First, the acyl-CoA synthetase homologue LstC might catalyze the ATP-dependent activation of octanoic acid to produce an acyl-CoA thioester via an acyl-adenylate intermediate (46). The octanoyl-CoA ester is then carboxylated to give hexylmalonyl-CoA, probably by an acyl-CoA carboxylase (ACCase) complex, the inactivation of which caused a decrease of lipstatin production by ϳ80% in S. toxytricini (30). An unknown route may be present to complement the ACCase pathway for the generation of hexylmalonyl-CoA, however.…”

Section: Discussionmentioning

confidence: 99%

“…S. toxytricini strains were grown on International Streptomyces Project Medium 4 (ISP4; BD Biosciences, San Jose, CA, USA) for sporulation and conjugation. Fermentation seed medium (10 g of soya bean flour, 5 g of Bacto soytone, 5 ml of glycerol, 10 ml of soya oil, and 2 ml of Triton X-100 [pH 6.5] per liter) and fermentation medium (30 g of soya bean flour, 14 ml of glycerol, 1 g of Bacto soytone, 1 ml of Triton X-100, and 60 ml of soya oil [pH 7.0] per liter) (30) were used for shake flask fermentation of S. toxytricini and S. lividans strains.…”

Section: Methodsmentioning

confidence: 99%

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Operon for Biosynthesis of Lipstatin, the Beta-Lactone Inhibitor of Human Pancreatic Lipase

Bai

Zhang

Lin

et al. 2014

Appl Environ Microbiol

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c Lipstatin, isolated from Streptomyces toxytricini as a potent and selective inhibitor of human pancreatic lipase, is a precursor for tetrahydrolipstatin (also known as orlistat, Xenical, and Alli), the only FDA-approved antiobesity medication for long-term use. Lipstatin features a 2-hexyl-3,5-dihydroxy-7,10-hexadecadienoic-␤-lactone structure with an N-formyl-L-leucine group attached as an ester to the 5-hydroxy group. It has been suggested that the ␣-branched 3,5-dihydroxy fatty acid ␤-lactone moiety of lipstatin in S. toxytricini is derived from Claisen condensation between two fatty acid substrates, which are derived from incomplete oxidative degradation of linoleic acid based on feeding experiments. In this study, we identified a six-gene operon (lst) that was essential for the biosynthesis of lipstatin by large-deletion, complementation, and single-gene knockout experiments. lstA, lstB, and lstC, which encode two ␤-ketoacyl-acyl carrier protein synthase III homologues and an acyl coenzyme A (acyl-CoA) synthetase homologue, were indicated to be responsible for the generation of the ␣-branched 3,5-dihydroxy fatty acid backbone. Subsequently, the nonribosomal peptide synthetase (NRPS) gene lstE and the putative formyltransferase gene lstF were involved in decoration of the ␣-branched 3,5-dihydroxy fatty acid chain with an N-formylated leucine residue. Finally, the 3␤-hydroxysteroid dehydrogenase-homologous gene lstD might be responsible for the reduction of the ␤-keto group of the biosynthetic intermediate, thereby facilitating the formation of the unique ␤-lactone ring.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Operon for Biosynthesis of Lipstatin, the Beta-Lactone Inhibitor of Human Pancreatic Lipase

Bai

Zhang

Lin

et al. 2014

Appl Environ Microbiol

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“…Linoleic acid contains the same number of double bonds as lipstatin chain and it is the basic building unit. Further experiments with feeding putative intermediates in the process of fermentation indicated that linoleic acid, caprylic acid and N-formyl-L-leucine or preferably L-leucine are effective precursors of lipstatin biosynthesis (Schuhr et al, 2002;Eisenreich et al, 2003;Demirev et al, 2010).…”

Section: Full Papermentioning

confidence: 99%

“…In S. toxytricini, the acetylCoA carboxylase gene cluster is the pccB gene locus containing accA3, pccB, and pccE, which has been demonstrated to be involved in the secondary metabolism. Disruption of the pccB and pccE genes caused a lipstatin production reduction as much as 80%, indicating their critical role in lipstatin biosynthesis (Demirev et al, 2009(Demirev et al, , 2010. The pccB gene encodes a β subunit of ACCase functioning as carboxyltransferase in the activation of ACCase along with biotinylation mediated by Bp1 protein, which needs divalent cations as a cofactor (Demirev et al, 2011).…”

Section: Full Papermentioning

confidence: 99%

“…4). As for the biosynthetic pathway, the carbon skeleton of the lipstatin molecule is biosynthesized via Claisen condensation of two fatty acid precursors, 8-carbon atoms, and 14-carbon atoms fatty acids (Demirev et al, 2010). Lipstatin contains a β-lactone ring; one of the side chains contains two isolated double bonds and a hydroxyl group esterified to N-formyl-L-leucine.…”

Section: Effects Of Precursors On Lipstatin Productionmentioning

confidence: 99%

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Enhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium

Zhu

Wang

et al. 2014

J. Gen. Appl. Microbiol.

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This paper is concerned with optimization of fermentation conditions for lipstatin production with. Under these conditions, a maximum lipstatin of 4.208 g/ml was achieved in verification experiments in 500 ml shake flasks. Key words: divalent cations; L-leucine; lipstatin; Streptomyces toxytricini IntroductionLipstatin is an irreversible inhibitor of pancreatic lipases produced by Streptomyces toxytricini Weibel et al., 1987). Lipstatin is of considerable importance as the key intermediate for the preparation of tetrahydrolipstatin (THL, Orlistat), which is a drug designed for the prophylaxis and treatment of diseases associated with obesity. Lipstatin contains a β-lactone structure that probably accounts for the irreversible lipase inhibition by covalent modification of the serine residue of its catalytic triad (Luthra et al., 2013b).The wide clinical application of orlistat has promoted the commercial production of lipstatin (Zohrabian, 2100). Hence, investigations into the improvement of the productions of lipstatin are of commercial importance. In an attempt to improve lipstatin production, improvement of lipstatinproducing strains and fermentation processes has been carried out during the past decade.S. toxytricini strains with an increased lipstatin fermentation unit have been achieved through modification and improvement by utilizing physical and chemical mutagenesis. A high lipstatin-producing strain SIPI-HJ-80 was obtained by ultraviolet ray and microwave treatment, which showed an increased productivity by 2.5 times over the original strain SIPI-UM-5 (Huang et al., 2006). More recently, a N-methyl-N -nitro-N-nitrosoguanidine (NTG) mutated strain of S. toxytricini (derived from strain ATCC 19813) was reported to have a lipstatin production of 2.88 mg/g at 264 h of fermentation time in a shake flask (Luthra et al., 2013a).With the strain S. toxytricini ATCC 19813, 8-fold enhancement (from 0.097 g/L to 0.885 g/L) in lipstatin production in a shake flask was obtained through medium optimization. It is also indicated that soya oil, soya lecithin and soya bean flour had significant effects on lipstatin production (Luthra et al., 2012). An increased lipstatin titer of 1.980 g/L was observed with selected S. toxytricini strain NRRL 15443 (Kumar et al., 2012). Further, a maximum lipstatin production of 3.290 g/L was achieved via a full factorial medium Full PaperEnhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium (Received February 28, 2014; Accepted April 3, 2014) None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.

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Development and Optimization of a High-Throughput Screening Assay for Rapid Evaluation of Lipstatin Production by Streptomyces Strains

et al. 2017

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Pancreatic lipase inhibitors, such as tetrahydrolipstatin (orlistat), are used in anti-obesity treatments. Orlistat is the only anti-obesity drug approved by the European Medicines Agency (EMA). The drug is synthesized by saturation of lipstatin, a β-lactone compound, isolated from Streptomyces toxytricini and S. virginiae. To identify producers of novel pancreatic lipase inhibitors or microbial strains with improved lipstatin production and higher chemical purity remains still a priority. In this study, a high-throughput screening method to identify Streptomyces strains producing potent pancreatic lipase inhibitors was established. The assay was optimized and validated using S. toxytricini NRRL 15443 and its mutants. Strains grew in 24-well titer plates. Lipstatin levels were assessed directly in culture medium at the end of cultivation by monitoring lipolytic activity in the presence of a chromogenic substrate, 1,2-Di-O-lauryl-rac-glycero-3-glutaric acid 6-methylresorufin ester (DGGR). The lipase activity decreased in response to lipstatin production, and this was demonstrated by accumulation of red-purple methylresorufin, a product of DGGR digestion. The sensitivity of the assay was achieved by adding a lipase of high lipolytic activity and sensitivity to lipstatin to the reaction mixture. In the assay, the fungal lipase from Mucor javanicus was used as an alternative to the human pancreatic lipase. Many fungal lipases preserve high lipolytic activity in extreme conditions and are not colipase dependent. The assay proved to be reliable in differentiation of strains with high and low lipstatin productivity.

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The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini

Cited by 10 publications

References 32 publications

Operon for Biosynthesis of Lipstatin, the Beta-Lactone Inhibitor of Human Pancreatic Lipase

Operon for Biosynthesis of Lipstatin, the Beta-Lactone Inhibitor of Human Pancreatic Lipase

Enhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium

Development and Optimization of a High-Throughput Screening Assay for Rapid Evaluation of Lipstatin Production by Streptomyces Strains

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