scholarly journals Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield

2020 ◽  
Author(s):  
Yongkun Lv ◽  
Yang Gu ◽  
Jingliang Xu ◽  
Jingwen Zhou ◽  
Peng Xu
Keyword(s):  
Scale Up ◽  
Genetic Circuits ◽  
Inducible Promoters ◽  
Simple Strategy ◽  
Selective Growth Advantage ◽  
Intermediary Metabolite ◽  
Engineered Yeast ◽  
Negative Autoregulation ◽  
Metabolic Heterogeneity ◽  
Strain Stability

AbstractMetabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitioned malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing hybrid promoters to control leucine synthesis, this flavonoid addiction phenotype confers a selective growth advantage to the naringenin-producing cell. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield.

scholarly journals Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield

2020 ◽  
Vol 61 ◽  
pp. 79-88 ◽  
Author(s):  
Yongkun Lv ◽  
Yang Gu ◽  
Jingliang Xu ◽  
Jingwen Zhou ◽  
Peng Xu
Keyword(s):  
Negative Autoregulation ◽  
Strain Stability

scholarly journals Intelligent microbial cell factory with genetic pH shooting (GPS) for cell self-responsive base/acid regulation

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Chenyi Li ◽  
Xiaopeng Gao ◽  
Xiao Peng ◽  
Jinlin Li ◽  
Wenxin Bai ◽  
...  
Keyword(s):  
Ph Regulation ◽  
Self Regulation ◽  
Scale Up ◽  
Microbial Cell ◽  
Production Costs ◽  
Cell Factory ◽  
Ph Sensing ◽  
Genetic Circuits ◽  
Microbial Cell Factory ◽  
Cell Factories

Abstract Background In industrial fermentation, pH fluctuation resulted from microbial metabolism influences the strain performance and the final production. The common way to control pH is adding acid or alkali after probe detection, which is not a fine-tuned method and often leads to increased costs and complex downstream processing. Here, we constructed an intelligent pH-sensing and controlling genetic circuits called “Genetic pH Shooting (GPS)” to realize microbial self-regulation of pH. Results In order to achieve the self-regulation of pH, GPS circuits consisting of pH-sensing promoters and acid-/alkali-producing genes were designed and constructed. Designed pH-sensing promoters in the GPS can respond to high or low pHs and generate acidic or alkaline substances, achieving endogenously self-responsive pH adjustments. Base shooting circuit (BSC) and acid shooting circuit (ASC) were constructed and enabled better cell growth under alkaline or acidic conditions, respectively. Furthermore, the genetic circuits including GPS, BSC and ASC were applied to lycopene production with a higher yield without an artificial pH regulation compared with the control under pH values ranging from 5.0 to 9.0. In scale-up fermentations, the lycopene titer in the engineered strain harboring GPS was increased by 137.3% and ammonia usage decreased by 35.6%. Conclusions The pH self-regulation achieved through the GPS circuits is helpful to construct intelligent microbial cell factories and reduce the production costs, which would be much useful in industrial applications.

scholarly journals Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budget

2020 ◽  
Vol 48 (17) ◽  
pp. 9995-10012
Author(s):  
James A Davey ◽  
Corey J Wilson
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Resource Partitioning ◽  
Inducible Promoter ◽  
Living System ◽  
Coupled Systems ◽  
Genetic Circuits ◽  
Inducible Promoters ◽  
Metabolic Burden ◽  
Resource Budget

Abstract Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden – as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength.

scholarly journals Automated Design and Implementation of a NOR Gate in Pseudomonas Putida

2021 ◽  
Author(s):  
Huseyin Tas ◽  
Lewis Grozinger ◽  
Angel Goñi-Moreno ◽  
Victor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Design Tool ◽  
Genetic Circuits ◽  
Automated Generation ◽  
Inducible Promoters ◽  
E Coli ◽  
Transcriptional Regulatory ◽  
Cross Platform ◽  
Nor Gate

ABSTRACT Boolean NOR gates have been widely implemented in Escherichia coli as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial hosts/chassis is generally hampered by frequent changes in the parameters of the INPUT/OUTPUT response functions brought about by new genetic and biochemical contexts. Here, we have used the circuit design tool CELLO for assembling a NOR gate in the soil bacterium and metabolic engineering platform Pseudomonas putida with components tailored for E. coli. To this end, we capitalized on the functional parameters of 20 genetic inverters for each host and the resulting compatibility between NOT pairs. Moreover, we added to the gate library 3 inducible promoters that are specific to P. putida, thus expanding cross-platform assembly options. While the number of potential connectable inverters decreased drastically when moving the library from E. coli to P. putida, the CELLO software was still able to find an effective NOR gate in the new chassis. Automated generation of the corresponding DNA sequence and in vivo experimental verification accredited that some genetic modules initially optimized for E. coli can indeed be reused to deliver NOR logic in P. putida as well. Furthermore, the results highlight the value of creating host-specific collections of well-characterized regulatory inverters for quick assembly of genetic circuits to meet complex specifications.

scholarly journals Superaerophobic hydrogels for enhanced electrochemical and photoelectrochemical hydrogen production

2020 ◽  
Vol 6 (15) ◽  
pp. eaaz3944
Author(s):  
Dasom Jeon ◽  
Jinwoo Park ◽  
Changhwan Shin ◽  
Hyunwoo Kim ◽  
Ji-Wook Jang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Scale Up ◽  
High Catalytic Activity ◽  
Gas Evolution ◽  
Proof Of Concept ◽  
Practical Application ◽  
M13 Virus ◽  
Simple Strategy ◽  
Hydrogen Bubbles ◽  
Photoelectrochemical Hydrogen Production

The efficient removal of gas bubbles in (photo)electrochemical gas evolution reactions is an important but underexplored issue. Conventionally, researchers have attempted to impart bubble-repellent properties (so-called superaerophobicity) to electrodes by controlling their microstructures. However, conventional approaches have limitations, as they are material specific, difficult to scale up, possibly detrimental to the electrodes’ catalytic activity and stability, and incompatible with photoelectrochemical applications. To address these issues, we report a simple strategy for the realization of superaerophobic (photo)electrodes via the deposition of hydrogels on a desired electrode surface. For a proof-of-concept demonstration, we deposited a transparent hydrogel assembled from M13 virus onto (photo)electrodes for a hydrogen evolution reaction. The hydrogel overlayer facilitated the elimination of hydrogen bubbles and substantially improved the (photo)electrodes’ performances by maintaining high catalytic activity and minimizing the concentration overpotential. This study can contribute to the practical application of various types of (photo)electrochemical gas evolution reactions.

NiAl precipitation in Fe-12w/o Cr-5w/o Ni-4w/o Al

1983 ◽  
Vol 41 ◽  
pp. 202-203
Author(s):  
L.E. Murr ◽  
J.S. Dunning ◽  
S. Shankar
Keyword(s):  
Solid Solution ◽  
Stainless Steel ◽  
Stainless Steels ◽  
Alloy Composition ◽  
Chromium Content ◽  
Scale Up ◽  
Optical Metallography ◽  
Property Evaluation ◽  
310 Stainless Steel ◽  
Microstructural Studies

Aluminum additions to conventional 18Cr-8Ni austenitic stainless steel compositions impart excellent resistance to high sulfur environments. However, problems are typically encountered with aluminum additions above about 1% due to embrittlement caused by aluminum in solid solution and the precipitation of NiAl. Consequently, little use has been made of aluminum alloy additions to stainless steels for use in sulfur or H2S environments in the chemical industry, energy conversion or generation, and mineral processing, for example.A research program at the Albany Research Center has concentrated on the development of a wrought alloy composition with as low a chromium content as possible, with the idea of developing a low-chromium substitute for 310 stainless steel (25Cr-20Ni) which is often used in high-sulfur environments. On the basis of workability and microstructural studies involving optical metallography on 100g button ingots soaked at 700°C and air-cooled, a low-alloy composition Fe-12Cr-5Ni-4Al (in wt %) was selected for scale up and property evaluation.

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