Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

Yang, Joseph; Seo, Sang Woo; Jang, Se Bok; Shin, So-I; Lim, Chae Hyun; Roh, Tae-Young; Jung, Gyoo Yeol

doi:10.1038/ncomms2404

Cited by 155 publications

(127 citation statements)

References 45 publications

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“…Biosensors that report on the concentration of a chemical within each individual cell can alleviate this screening bottleneck (10). Such sensor reporters transduce the binding of a target small molecule by a sensory protein or RNA into a gene expression readout (7).…”

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confidence: 99%

Evolution-guided optimization of biosynthetic pathways

Raman

Rogers

Taylor

et al. 2014

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

262

247

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Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers. We convert the intracellular presence of the target chemical into a fitness advantage for the cell by using a sensor domain responsive to the chemical to control a reporter gene necessary for survival under selective conditions. Because artificial selection tends to amplify unproductive cheaters, we devised a negative selection scheme to eliminate cheaters while preserving library diversity. This scheme allows us to perform multiple rounds of evolution (addressing ∼10 9 cells per round) with minimal carryover of cheaters after each round. Based on candidate genes identified by flux balance analysis, we used targeted genome-wide mutagenesis to vary the expression of pathway genes involved in the production of naringenin and glucaric acid. Through up to four rounds of evolution, we increased production of naringenin and glucaric acid by 36-and 22-fold, respectively. Naringenin production (61 mg/L) from glucose was more than double the previous highest titer reported. Wholegenome sequencing of evolved strains revealed additional untargeted mutations that likely benefit production, suggesting new routes for optimization.evolution | metabolic engineering | synthetic biology | sensors | biosynthetic pathways

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mentioning

confidence: 99%

Evolution-guided optimization of biosynthetic pathways

Raman

Rogers

Taylor

et al. 2014

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

262

247

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“…Engineers have begun developing strategies for multiplexed evaluation of metabolite production phenotypes to enable a fully multiplexed design-build-test cycle (1,(4)(5)(6)(7). In cases of genetic engineering, such a design cycle would more closely resemble biological evolution, rather than the design approaches inspired by electrical engineering that currently dominate the fields of synthetic biology and metabolic engineering.…”

mentioning

confidence: 99%

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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“…Structural analysis of the Llysine binding aptamer domain of this RNA revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing procedure to regulate the enzyme activity upon interaction with Llysine (Muranaka et al 2009;Yang et al 2013). A genetic RNA device comprising the L-lysine riboswitch could be used to monitor the alteration of phenotypes caused by changes in the intracellular concentration of L-lysine and to facilitate the strain improvement (Yang et al 2013). However, this device has not been used in screening the evolved aspartate kinase.…”

Section: Introductionmentioning

confidence: 98%

“…As a majority of target products are not associated with a conspicuous phenotype of the host, the desired property cannot be readily obtained without a general screening tool with the ability of detecting the target products and capacity for high-throughput screening (Dietrich et al 2010;Yang et al 2013). Recent studies demonstrated that natural RNA molecules, such as riboswitch, can recognize small organic compounds, harness allosteric changes, and control the expression of adjacent genes at the levels of transcription, translation, and RNA degradation (Mironov et al 2002;Nahvi et al 2002;Winkler et al 2002).…”

Section: Introductionmentioning

confidence: 99%

Evolution of a chimeric aspartate kinase for L-lysine production using a synthetic RNA device

Wang

Gao

et al. 2015

Appl Microbiol Biotechnol

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Aspartate kinase (AK) is a key enzyme involved in catalyzing the first step of the aspartate-derived amino acid biosynthesis, including L-lysine and L-threonine, which is regulated by the end-metabolites through feedback inhibition. In order to accumulate the end-metabolites in the host, the feedback inhibition of AK has to be released. In this study, a chimeric aspartate kinase, which is composed of the N-terminal catalytic region from Bacillus subtilis AKII and the C-terminal region from Thermus thermophilus, was evolved through random mutagenesis and then screened using a high-throughput synthetic RNA device which comprises of an L-lysine-sensing riboswitch and a selection module. Of three evolved aspartate kinases, the best mutant BT3 showed 160 % increased in vitro activity compared to the wild-type enzyme. Recombinant Escherichia coli harboring BT3 produced 674 mg/L L-lysine in batch cultivation, similar to that produced by the strain harboring the typical commercial widely used feedback resistant aspartate kinase AKC (fbr) from E. coli. The results suggested that this strategy can be extended for screening of other key enzymes involved in lysine biosynthesis pathways.

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Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

Cited by 155 publications

References 45 publications

Evolution-guided optimization of biosynthetic pathways

Evolution-guided optimization of biosynthetic pathways

Genetically encoded sensors enable real-time observation of metabolite production

Evolution of a chimeric aspartate kinase for L-lysine production using a synthetic RNA device

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