Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Koma, Daisuke; Yamanaka, Hayato; Moriyoshi, Kunihiko; Ohmoto, Takashi; Sakai, Kiyofumi

doi:10.1128/aem.01148-12

Cited by 141 publications

(163 citation statements)

References 45 publications

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“…Although the activity of Ahr has recently been hinted at (18), we unequivocally demonstrate that this enzyme catalyzes the reduction of aliphatic aldehydes to alcohols with a substrate profile ranging, at least, from C 4 to C 16 and a selective preference for NADPH. Based on the experimental verification of its catalytic activity in this study, as well as others (18,23), a replacement of the current gene name, yjgB, with ahr (aldehyde reductase) is proposed to denote the catalytic function of this gene product.…”

Section: Discussionmentioning

confidence: 87%

Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities

Akhtar

Turner

Jones

2012

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

321

349

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Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C 6 –C 18 ) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C 8 –C 16 ) or fatty alkanes (C 7 –C 15 ) was reconstituted in vitro. This concept was applied in vivo , in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L −1 was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C 8 –C 18 ). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities.

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Section: Discussionmentioning

confidence: 87%

Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities

Akhtar

Turner

Jones

2012

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

321

349

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“…We also demonstrated that several aromatic homoamino acid analogs, including L-homotyrosine, could be produced using this system, even though the range of substrate specificity is moderately limited. We propose that various fine chemical materials, such as keto acids, hydroxy acids, and alco-hols, derived from aromatic homoamino acids can be produced by combining the L-Hph-producing strain with various enzymes that have been developed for industrial production of useful chiral building blocks (37). Additionally, detailed structure-based comparison between enzymes for producing L-Hph and other homoamino acids will allow us to generate novel homoamino acidproducing systems through protein engineering in the future.…”

Section: Discussionmentioning

confidence: 99%

Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli

Koketsu

Mitsuhashi

Tabata

2013

Appl Environ Microbiol

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L-Homophenylalanine (L-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While the chemoenzymatic route of synthesis is fully developed, we investigated microbial production of L-Hph to explore the possibility of a more efficient and sustainable approach to L-Hph production. We hypothesized that L-Hph is synthesized from L-Phe via a mechanism homologous to 3-methyl-2-oxobutanoic acid conversion to 4-methyl-2-oxopentanoic acid during leucine biosynthesis. Based on bioinformatics analysis, we found three putative homophenylalanine biosynthesis genes, hphA (Npun_F2464), hphB (Npun_F2457), and hphCD (Npun_F2458), in the cyanobacterium Nostoc punctiforme PCC73102, located around the gene cluster responsible for anabaenopeptin biosynthesis. We constructed Escherichia coli strains harboring hphABCD-expressing plasmids and achieved the fermentative production of L-Hph from L-Phe. To our knowledge, this is the first identification of the genes responsible for homophenylalanine synthesis in any organism. Furthermore, to improve the low conversion efficiency of the initial strain, we optimized the expression of hphA, hphB, and hphCD, which increased the yield to ϳ630 mg/liter. The L-Hph biosynthesis and L-Leu biosynthesis genes from E. coli were also compared. This analysis revealed that HphB has comparatively relaxed substrate specificity and can perform the function of LeuB, but HphA and HphCD show tight substrate specificity and cannot complement the LeuA and LeuC/LeuD functions, and vice versa. Finally, the range of substrate tolerance of the L-Hph-producing strain was examined, which showed that m-fluorophenylalanine, o-fluorophenylalanine, and L-tyrosine were accepted as substrates and that the corresponding homoamino acids were generated.

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“…The fatty aldehyde content however was small compared with total pool size for alkanes and FAs. Fatty aldehydes may be converted to fatty alcohols in E. coli cells by endogenous aldehyde reductases (32,33). We therefore also examined the fatty alcohol content of these cells.…”

Section: Conversion Of Fa To Alkanes Is Limited By the In Vivo Use Ofmentioning

confidence: 99%

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli

Howard

Middelhaufe

Moore

et al. 2013

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

163

142

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Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n-and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (C n ) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane C n and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.branched fatty acid biosynthesis | lux genes | metabolic engineering | synthetic biology

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Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Cited by 141 publications

References 45 publications

Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities

Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities

Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli

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