Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster

Miyahisa, Ikuo; Kaneko, Masakatsu; Funa, Nobutaka; Kawasaki, Hisashi; Kojima, Hiroyuki; Ohnishi, Yasuo; Horinouchi, Sueharu

doi:10.1007/s00253-005-1916-3

Cited by 173 publications

(149 citation statements)

References 25 publications

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“…For expression of the Ars proteins in E. coli, pETDuet-ArsA, pACYC-ArsB, pCDFArsC, pETDuet-ArsD, and pETDuet-ArsAD were constructed. In addition to these plasmids, pRSF-ACC, which carries the genes encoding the two subunits of acetyl-CoA carboxylase of Corynebacterium glutamicum, was used to increase the intracellular pool of malonyl-CoA (20,21). The vector plasmids pETDuet, pACYC, pCDF, and pRSF, which contain different replication origins and different selective markers, can be maintained together in the same E. coli cell.…”

Section: Resultsmentioning

confidence: 99%

Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Miyanaga

Funa

Awakawa

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C 22-C26 fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C 22-C26 fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonylCoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.Azotobacter vinelandii ͉ alkylresorcinol ͉ alkylpyrone ͉ long-chain fatty acid ͉ cyst

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Section: Resultsmentioning

confidence: 99%

Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Miyanaga

Funa

Awakawa

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…As a consequence the host organisms provide precursors from their own primary and secondary metabolism that are converted to the expected secondary product through the expression of foreign genes. Our success in fermentative production of plant-specific polyketides by E. coli carrying an artificially assembled phenylpropanoid pathway was the first example to show that a nearly complete biosynthetic pathway in plants was established in a heterologous microorganism for production of flavanones from the amino acid precursors, phenylalanine and tyrosine [5,6]. Later, Yan et al [7] produced 5-deoxyflavanones in E. coli and Beekwilder et al [8] produced a natural raspberry ketone in E. coli.…”

Section: Introductionmentioning

confidence: 99%

“…This was accomplished by assembling PAL from the yeast Rhodotorula rubra; 4CL (ScCCL) from the actinomycete S. coelicolor A3(2); CHS from the licorice plant Glycyrrhiza echinata; and CHI from the plant Pueraria lobata on a single pET plasmid in E. coli (Fig. 4B) [5,6,30]. To maximize the expression of the four genes, various constructions of the artificial gene clusters were tested: for example, the arrangement of PAL, 4CL, CHS and CHI such that the termination codon of the preceding gene overlapped with the start codon of the following gene.…”

Section: -1 Production Of (2s)-flavanonesmentioning

confidence: 99%

Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms

Horinouchi

2008

J Antibiot

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One of the approaches of combinatorial biosynthesis is combining genes from different organisms and designing a new set of gene clusters to produce bioactive compounds, leading to diversification of both chemical and natural product libraries. This makes efficient use of the potential of the host organisms, especially when microorganisms are used. An Escherichia coli system, in which artificial biosynthetic pathways for production of plant-specific medicinal polyketides, such as flavonoids, stilbenoids, isoflavonoids, and curcuminoids, are assembled, has been designed and expressed. Starting with amino acids tyrosine and phenylalanine as substrates, this system yields naringenin, resveratrol, genistein, and curcumin, for example, all of which are beneficial to human health because of their wide variety of biological activities. Supplementation of unnatural carboxylic acids to the recombinant E. coli cells carrying the artificial pathways by precursor-directed biosynthesis results in production of unnatural compounds. Addition of decorating or modification enzymes to the artificial pathway leads to production of natural and unnatural flavonols, flavones, and methylated resveratrols. This microbial system is promising for construction of larger libraries by employing other polyketide synthases and decorating enzymes of various origins. In addition, the concept of building and expressing artificial biosynthetic pathways for production of non-bacterial and unnatural compounds in microorganisms should be successfully applied to production of not only plant-specific polyketides but also many other useful compound classes.

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“…Similarly, coexpression of two genes of C. glutamicum ACCase proved to greatly increase the overall production efficiency of flavanones, the direct precursors of the vast majority of flavonoids (Miyahisa et al, 2005). In this study (2S)-naringenin and (2S)-pinocembrin were synthesized in E. coli by employing an artificial biosynthetic cluster of genes that included phenylalanine ammonia lyase from yeast, cinnamate/coumarate:CoA ligase from the actinomycete Streptomyces coelicolor, chalcone synthase from a licorice plant, and chalcone isomerase from a Pueraria plant.…”

Section: Accase In Bacteria and Algae-based Biotech Projectsmentioning

confidence: 99%

“…In this study, coexpression of phlD and four ACCase subunits genes demonstrated the productivity of 3.8 g/L (2.1-fold change to the control strain) at large scale fed-batch fermentation conditions. This was the highest phloroglucinol production efficiency reported to date and proved that single ACCase overexpression is sufficient to greatly improve the production rate of the entire synthesis pathway.Similarly, coexpression of two genes of C. glutamicum ACCase proved to greatly increase the overall production efficiency of flavanones, the direct precursors of the vast majority of flavonoids (Miyahisa et al, 2005). In this study (2S)-naringenin and (2S)-pinocembrin were synthesized in E. coli by employing an artificial biosynthetic cluster of genes that included phenylalanine ammonia lyase from yeast, cinnamate/coumarate:CoA ligase from the actinomycete Streptomyces coelicolor, chalcone synthase from a licorice plant, and chalcone isomerase from a Pueraria plant.…”

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confidence: 99%

OPINIONS Acetyl-coenzyme A carboxylase – an attractive enzyme for biotechnology

Podkowiński¹,

Tworak²

2011

bta

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Acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. This reaction constitutes the first committed step of fatty acid synthesis in most organisms, while its product also serves as a universal precursor for various other high-value compounds. The important regulatory and rate-limiting role of acetyl-CoA carboxylase makes this enzyme a powerful tool in a variety of biotechnological and medical projects. This review presents the current knowledge on structural and functional features of the enzyme and focuses on its divergent applications. Different metabolic engineering attempts that lead to the production of various compounds, such as fatty acids, polyketides or flavonoids, are presented and their advantages and limitations discussed. The importance of studies on acetyl-CoA carboxylase activity for design and development of new herbicides and antibiotics as well as for medical intervention is also highlighted. Acetyl-coenzyme A carboxylase -enzyme architecture, biochemistry and its role in cell physiologyA variety of biotechnological projects targeted to modulate acetyl-coenzyme A carboxylase activity in bacteria, plants and humans have been proposed and experimentally tested. Here, we would like to present a comprehensive review of these strategies together with a survey of the potential of acetyl-CoA carboxylase for biotechnology.Acetyl-coenzyme A carboxylase (ACC, E.C.6.4.1.2), recognized as an essential enzyme for all Eukaryotes and the majority of Bacteria, is responsible for carboxylation of acetyl-CoA that results in malonyl-coenzyme A formation (Fig. 1). Malonyl-CoA is a major building block for compounds such as fatty acids, polyketides and flavonoids -important components for cell growth, as well as for food, pharmaceuticals and energy production.The malonyl-CoA synthesis consists of two half-reactions (Nikolau et al., 2003). The first one, adenosine triphosphate-dependent carboxylation of biotin prosthetic group covalently bound to a module named biotin carboxyl carrier protein (BCCP), is catalyzed by ACCase segment of biotin carboxylase activity (BC) (Fig. 2). This reaction is immediately followed by the second half-re action: the transfer of a carboxyl group from carboxylated biotin to acetyl-CoA, resulting in malonyl-CoA formation. Carboxyl transferase (CT) is a module that catalyzes the second reaction and provides the required specificity in recognition of the carboxyl acceptor (acetyl-CoA). The ACCase model usually presents BCCP, a module with covalently attached biotin, as a beam responsible for biotin dislocation between two active centers -one with BT and the other with CT activity. The three (Fig. 3). The prokaryotic and eukaryotic types of ACCases are evolutionary related and the phylogeny of the modules provides some hints about their cenancestor, a split between Archaea and Bacteria, and arising of Eukaryota (Lombard and Moreira, 2011).Phylogeny is out of scope of this manuscript, but acetyl-coenzyme A carboxylases from various organisms representing v...

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Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster

Cited by 173 publications

References 25 publications

Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms

OPINIONS Acetyl-coenzyme A carboxylase – an attractive enzyme for biotechnology

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