Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

Vick, Jacob E.; Johnson, Ethan T.; Choudhary, Swati; Bloch, Sarah E.; López‐Gallego, Fernando; Srivastava, Poonam; Tikh, Ilya B.; Wawrzyn, Grayson T.; Schmidt‐Dannert, Claudia

doi:10.1007/s00253-011-3633-4

Cited by 58 publications

(72 citation statements)

References 29 publications

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“…To enable this, the construction of pUCBB-pCT5-ntH6-eGFP (see Fig. S1 and Table S1 in the supplemental material for the sequence and map of the construct) was first completed based on previous BioBrick vector designs (31). E. coli atoB and fadB genes were PCR amplified, digested with BglII and NotI, and ligated by T4 ligase (Invitrogen, Carlsbad, CA) into pUCBB-P CT5 -ntH6-eGFP that was previously digested with BglII and NotI to produce pUCBB-P CT5 -atoB and pUCBB-P CT5 -fadB.…”

Section: Methodsmentioning

confidence: 99%

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Vick

Clomburg

Blankschien

et al. 2015

Appl Environ Microbiol

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We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the ␤-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541-554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a ␤-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ⌬fabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the ␤-oxidation reversal in E. coli. R ecent advances in synthetic biology and enzyme and metabolic engineering (1, 2) are allowing the development of an ever-expanding array of microbial hosts for the production of advanced fuels and industrially relevant chemicals (3, 4). Most efforts are now focused on moving past the historically successful biofuels and industrial products, such as ethanol (5), toward "drop-in" fuels and additives that can be incorporated into existing infrastructure (4). The production of such molecules requires the synthesis of higher-chain-length (C Ն 4) products from 1-, 2-or 3-carbon metabolic intermediates and hence pathways that can mediate the formation of carbon-carbon bonds. A variety of native and engineered pathways have been utilized for this purpose, including the clostridial n-butanol pathway (6), isoprenoid biosynthesis (7,8), the ␣-keto-acids pathway (9, 10), and fatty acid biosynthesis (11,12).Among the aforementioned pathways, the bacterial type II fatty acid biosynthesis system (FAB II) (13) is probably the most widely engineered and has been harnessed for the synthesis of many products, including fatty acids (14), fatty acid methyl esters (15), fatty acid ethyl esters (11, 16), fatty alcohols (11), and alkanes (12). At the core of the FAB II system is an elongation cycle that uses discrete enzymes to catalyze each of its four ste...

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Section: Methodsmentioning

confidence: 99%

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Vick

Clomburg

Blankschien

et al. 2015

Appl Environ Microbiol

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“…All pathway genes were initially cloned into pUCBB, one of our group’s custom set of BioBrick™-compatible vectors, for subsequent assembly of multi-gene plasmids to create the biosynthetic pathway modules described later. [50] Monocistronic “bricks” containing gene expression cassettes allow for “stacking” using the standard BioBrick™ restriction sites [51] that can be reused repeatedly.…”

Section: Resultsmentioning

confidence: 99%

“…To this end, we assembled and tested three modular plasmid systems for the production of RA 7 (Figure 1) using our previously designed, compatible BioBrick™ plasmids for pathway assembly in E. coli . [50] …”

Section: Resultsmentioning

confidence: 99%

Construction of a Chimeric Biosynthetic Pathway for the De Novo Biosynthesis of Rosmarinic Acid in Escherichia coli

Bloch

Schmidt‐Dannert

2014

ChemBioChem

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Hydroxycinnamic acid esters (HCEs) are widely-distributed phenylpropanoid-derived plant natural products. Rosmarinic acid (RA 7), the most well-known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, a microbial production platform for HCEs could provide a sustainable, controlled means of production. Through the overexpression of a six-enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA 7 and the related HCE isorinic acid (IA 8) in E. coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We show HCE biosynthesis using three plant RAS orthologs exhibiting different levels of HCE biosynthesis, but the same ratio of IA 8 to RA 7 produced. This work serves as a proof of concept for a microbial production platform for HCEs using a modular biosynthetic approach to access diverse natural and non-natural HCEs.

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“…[31c, 35] Other BioBrick TM standard compliant vectors featuring additional restriction sites [36] or regulatory elements [37] were also constructed.…”

Section: The Challenges Of Multiple Recombinant Protein Expressionmentioning

confidence: 99%

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

et al. 2015

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Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

Cited by 58 publications

References 29 publications

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Construction of a Chimeric Biosynthetic Pathway for the De Novo Biosynthesis of Rosmarinic Acid in Escherichia coli

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

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