Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains

Rathnasingh, Chelladurai; Raj, Subramanian Mohan; You-Jin, Lee; Catherine, Christy; Ashok, Somasundar; Park, Sunghoon

doi:10.1016/j.jbiotec.2011.06.008

Cited by 146 publications

(93 citation statements)

References 15 publications

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“…Mcr shunts malonyl-CoA away from fatty acid biosynthesis by catalyzing the conversion of malonyl-CoA, first into malonate semialdehyde, and then into 3HP, at the expense of two NADPH. This route from glucose to 3HP has been published, with the authors achieving titers of 60 mg/L with expression of mcr alone (23). Titers were increased to 180 mg/L with overexpression of the ACC complex and pntAB, increasing availability of malonylCoA and NADPH, respectively.…”

Section: Resultsmentioning

confidence: 98%

Genetically encoded sensors enable real-time observation of metabolite production

Rogers

Church

2016

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

167

114

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Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules.biotechnology | directed evolution | biosensor | metabolic engineering | synthetic biology B iological production of valuable products such as pharmaceuticals or renewable chemicals holds the potential to transform the global economy. However, the rate at which bioengineers are able to engineer new living catalysts is hampered by an exceedingly slow design-build-test cycle. We describe a method to accelerate the design-build-test cycle for metabolic engineering by enabling the observation of product formation within microbes as it occurs.Biological production of a desired product is accomplished by guiding a low-cost starting material such as glucose through a series of intracellular enzymatic reactions, ultimately yielding a molecule of economic interest. The choice of culture conditions, the creation of enzyme variants, and the tuning of endogenous cellular metabolism create a vast universe of potential designs. Because of the complexity of biology, appropriate genetic designs and culture conditions are not known a priori. Even sophisticated modeling paradigms can result in very large design spaces (1, 2). Evaluating genetic designs or modulating process parameters to achieve a desired outcome is therefore a major bottleneck in the bioengineering design-build-test cycle.Current methods...

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Section: Resultsmentioning

confidence: 98%

Genetically encoded sensors enable real-time observation of metabolite production

Rogers

Church

2016

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

167

114

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“…1). Previously, the low intracellular malonyl-CoA concentration restricted the efficient production of flavonoids in E. coli (37,38). Therefore, it was necessary to engineer the bacteria for enhanced malonyl-CoA availability so as to achieve a metabolic balance between the requirements of malonyl-CoA for cell growth and product synthesis.…”

Section: Discussionmentioning

confidence: 99%

Efficient Synthesis of Eriodictyol from l -Tyrosine in Escherichia coli

Zhu

et al. 2014

Appl Environ Microbiol

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The health benefits of flavonoids for humans are increasingly attracting attention. Because the extraction of high-purity flavonoids from plants presents a major obstacle, interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoid with anti-inflammatory and antioxidant activities. Its efficient synthesis has been hampered by two factors: the poor expression of cytochrome P450 and the low intracellular malonyl coenzyme A (malonyl-CoA) concentration in Escherichia coli. To address these issues, a truncated plant P450 flavonoid, flavonoid 3=-hydroxylase (tF3=H), was functionally expressed as a fusion protein with a truncated P450 reductase (tCPR) in E. coli. This allowed the engineered E. coli to produce eriodictyol from L-tyrosine by simultaneously coexpressing the fusion protein with tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance the availability of malonyl-CoA so as to achieve a new metabolic balance and rebalance the relative expression of genes to enhance eriodictyol accumulation. This approach made the production of eriodictyol 203% higher than that in the control strain. By using these strategies, the production of eriodictyol from L-tyrosine reached 107 mg/liter. The present work offers an approach to the efficient synthesis of other hydroxylated flavonoids from L-tyrosine or even glucose in E. coli.

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“…Our group (Jan et al, unpublished) and other researchers have combined overexpression or deletion of these genes with other host strain modifications to improve NADPH-dependent pathway performance where NADPH availability is measured in the presence of a NADPH utilizing reaction or "sink". In E. coli engineered for xylitol [53] or platform chemical 3-hydroxypropionic acid production [54], PntAB increased production.…”

Section: Cofactor Considerations In Metabolic Engineeringmentioning

confidence: 99%

Cofactor engineering for advancing chemical biotechnology

Wang

San

Bennett

2013

Current Opinion in Biotechnology

138

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Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.

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Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains

Cited by 146 publications

References 15 publications

Genetically encoded sensors enable real-time observation of metabolite production

Genetically encoded sensors enable real-time observation of metabolite production

Efficient Synthesis of Eriodictyol from l -Tyrosine in Escherichia coli

Cofactor engineering for advancing chemical biotechnology

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