Development of a Transcription Factor-Based Lactam Biosensor

Zhang, Jingwei; Barajas, Jesus F.; Burdu, Mehmet; Ruegg, Thomas L.; Dias, Bryton; Keasling, Jay D.

doi:10.1021/acssynbio.6b00136

Cited by 57 publications

(58 citation statements)

References 56 publications

(70 reference statements)

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“…[15][16][17] Additionally, several bioactive compounds are valerolactam derivatives. [18][19][20][21][22] Only recently, the biotechnological production of valerolactam (1) 23 and a lactam biosensor 24 have been introduced. So far, the molecular structure of 1 has only been described in respective co-crystals [25][26][27][28][29][30][31] and metal complexes.…”

Section: Introductionmentioning

confidence: 99%

Missing Link in the Homologous Series of Lactams: The X-ray Structure of Valerolactam

Weck

Nauha

Gruber

2018

Crystal Growth & Design

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Lactams are an important class of heterocyclic compounds and are widely used for industrial and pharmaceutical purposes. Even decades after initial lactam syntheses, research on their physical and chemical properties is still rewarding. It delivers valuable information on the reactivity of lactams and their conformational behavior. For the small- and medium-sized parent lactams, the X-ray structures have been well-known, except for the “missing link”, the six-membered valerolactam. An X-ray structure of valerolactam is described here for the first time, stimulating a comparative discussion of the homologous lactam series. The experimental solid state conformation of valerolactam differs significantly from the calculated and energy-minimized ones reported in the literature. The amide bond length in valerolactam is more or less equal to that in other lactams. A comparison with the structure of cyclohexene revealed striking similarities arising from the partial C–N double bond in valerolactam caused by amide resonance.

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Section: Introductionmentioning

confidence: 99%

Missing Link in the Homologous Series of Lactams: The X-ray Structure of Valerolactam

Weck

Nauha

Gruber

2018

Crystal Growth & Design

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“…In previous published work the mathematical basis for determining linear range or limits of detection has often been obscure and non-systematic 1,6,22 . Furthermore, a linear range may not be able to adequately capture the performance characteristics of a biosensor over the range of ligand concentrations where the sensor can still resolve differences.…”

Section: Development Of Two Glutarate Biosensor Vectors Smentioning

confidence: 99%

Glutarate metabolism in Pseudomonas putida is regulated by two distinct glutarate sensing transcription factors

Thompson

Cruz-Morales

Krishna

et al. 2019

Preprint

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A significant bottleneck in synthetic biology involves screening the large genetically encoded libraries enabled by the latest technologies for desirable phenotypes such as chemical production. However, transcription factor based biosensors can be leveraged to screen thousands of genetic designs for optimal chemical production in engineered microbes. In this study we characterize two glutarate sensing transcription factors (CsiR and GcdR) from Pseudomonas putida. The genomic contexts of csiR homologs were analyzed and DNA binding sites were bioinformatically predicted. Both CsiR and GcdR were purified and shown to bind upstream of their coding sequencing in vitro. CsiR was shown to dissociate from DNA in vitro when exogenous glutarate was added confirming it acts as a genetic repressor. Both transcription factors and cognate promoters were then cloned into broad host range vectors to create two glutarate biosensors. Their respective sensing performance features were characterized, and more sensitive derivatives of the GcdR biosensor were created by manipulating the expression of the transcription factor. Sensor vectors were then reintroduced into P. putida and were evaluated for their ability to respond to glutarate as well as various lysine metabolites. Additionally, we developed a novel mathematical approach to describe the usable range of detection for genetically encoded biosensors which may be broadly useful to future efforts to better characterize biosensor performance.

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“…The need has been partially addressed by biorefineries, in which metabolically engineered microorganisms convert inexpensive and renewable biomass into materials of high value and usefulness [1][2][3]. Among numerous chemicals being broadly employed in the industry, lactam is an important set of compounds for the manufacture of nylon [4][5][6]. Notably, ε-caprolactam (caprolactam), featuring an annual production of~4,000,000 tons worldwide, is extensively used in the synthesis of nylon-6 [4].…”

Section: Introductionmentioning

confidence: 99%

“…Among numerous chemicals being broadly employed in the industry, lactam is an important set of compounds for the manufacture of nylon [4][5][6]. Notably, ε-caprolactam (caprolactam), featuring an annual production of~4,000,000 tons worldwide, is extensively used in the synthesis of nylon-6 [4]. Butyrolactam is the proposed monomer of nylon-4, the most hydrophilic material in the current nylon family, and is among the C4 (four-carbon) "Top Value-Added Chemical from Biomass" reported by the US Department of Energy [7].…”

Section: Introductionmentioning

confidence: 99%

“…This extremely high scalability is desired for the development of high-throughput parallel biosensing strategies where a large number of sensors are employed. Notably, Zhang and coworkers have successfully developed a lactam-responsive metabolic pathway in Escherichia coli (E. coli) [4]. The engineered bacteria system can specifically detect major lactam species at 10 mM.…”

Section: Introductionmentioning

confidence: 99%

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Hydrogel Microparticles Functionalized with Engineered Escherichia coli as Living Lactam Biosensors

Zhang

et al. 2019

Sensors

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Recently there has been an increasing need for synthesizing valued chemicals through biorefineries. Lactams are an essential family of commodity chemicals widely used in the nylon industry with annual production of millions of tons. The bio-production of lactams can substantially benefit from high-throughput lactam sensing strategies for lactam producer screening. We present here a robust and living lactam biosensor that is directly compatible with high-throughput analytical means. The biosensor is a hydrogel microparticle encapsulating living microcolonies of engineered lactam-responsive Escherichia coli. The microparticles feature facile and ultra-high throughput manufacturing of up to 10,000,000 per hour through droplet microfluidics. We show that the biosensors can specifically detect major lactam species in a dose-dependent manner, which can be quantified using flow cytometry. The biosensor could potentially be used for high-throughput metabolic engineering of lactam biosynthesis.

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Development of a Transcription Factor-Based Lactam Biosensor

Cited by 57 publications

References 56 publications

Missing Link in the Homologous Series of Lactams: The X-ray Structure of Valerolactam

Missing Link in the Homologous Series of Lactams: The X-ray Structure of Valerolactam

Glutarate metabolism in Pseudomonas putida is regulated by two distinct glutarate sensing transcription factors

Hydrogel Microparticles Functionalized with Engineered Escherichia coli as Living Lactam Biosensors

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