scholarly journals Optogenetic Amplification Circuits for Light-Induced Metabolic Control

2020 ◽  
Author(s):  
Evan M. Zhao ◽  
Makoto A. Lalwani ◽  
Jhong-Min Chen ◽  
Paulina Orillac ◽  
Jared E. Toettcher ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Control ◽  
Transcriptional Response ◽  
Attractive Alternative ◽  
Chemical Production ◽  
Chemical Inducers ◽  
High Tunability ◽  
Three Phase ◽  
Fold Induction ◽  
Cell Densities

AbstractDynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 21.8-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light doses as low as ~1%, and strong homogeneous light-induction in bioreactors of at least 5L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

scholarly journals Engineering and Evolution of Methanol Assimilation in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Monica I. Espinosa ◽  
Ricardo A. Gonzalez-Garcia ◽  
Kaspar Valgepea ◽  
Manuel Plan ◽  
Colin Scott ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Extract ◽  
Glyoxylate Cycle ◽  
Carbon Assimilation ◽  
Tca Cycle ◽  
Ethanol Metabolism ◽  
Attractive Alternative ◽  
Chemical Production ◽  
Adaptive Laboratory Evolution ◽  
Methanol Yeast

AbstractMicrobial fermentation for chemical production is becoming more broadly adopted as an alternative to petrochemical refining. Fermentation typically relies on sugar as a feedstock, however, one-carbon compounds like methanol are an attractive alternative as they can be derived from organic waste and natural gas. This study focused on engineering methanol assimilation in the yeast Saccharomyces cerevisiae. Three methanol assimilation pathways were engineered and tested: a synthetic xylulose monophosphate (XuMP), a ‘hybrid’ methanol dehydrogenase-XuMP, and a bacterial ribulose monophosphate (RuMP) pathway, with the latter identified as the most effective at assimilating methanol. Additionally, 13C-methanol tracer analysis uncovered a native capacity for methanol assimilation in S. cerevisiae, which was optimized using Adaptive Laboratory Evolution. Three independent lineages selected in liquid methanol-yeast extract medium evolved premature stop codons in YGR067C, which encodes an uncharacterised protein that has a predicted DNA-binding domain with homology to the ADR1 transcriptional regulator. Adr1p regulates genes involved in ethanol metabolism and peroxisomal proliferation, suggesting YGR067C has a related function. When one of the evolved YGR067C mutations was reverse engineered into the parental CEN.PK113-5D strain, there were up to 5-fold increases in 13C-labelling of intracellular metabolites from 13C-labelled methanol when 0.1 % yeast extract was a co-substrate, and a 44 % increase in final biomass. Transcriptomics and proteomics revealed that the reconstructed YGR067C mutation results in down-regulation of genes in the TCA cycle, glyoxylate cycle, and gluconeogenesis, which would normally be up-regulated during growth on a non-fermentable carbon source. Combining the synthetic RuMP and XuMP pathways with the reconstructed Ygr067cp truncation led to further improvements in growth. These results identify a latent methylotrophic metabolism in S. cerevisiae and pave the way for further development of native and synthetic one-carbon assimilation pathways in this model eukaryote.

Optimized Modulation and Dynamic Control of a Three-Phase Dual Active Bridge Converter With Variable Duty Cycles

2019 ◽  
Vol 34 (3) ◽  
pp. 2856-2873 ◽  
Author(s):  
Jun Huang ◽  
Zhuoqiang Li ◽  
Ling Shi ◽  
Yue Wang ◽  
Jinda Zhu
Keyword(s):  
Dynamic Control ◽  
Dual Active Bridge ◽  
Duty Cycles ◽  
Three Phase

Metabolic engineering of Escherichia coli: A sustainable industrial platform for bio-based chemical production

2013 ◽  
Vol 31 (8) ◽  
pp. 1200-1223 ◽  
Author(s):  
Xianzhong Chen ◽  
Li Zhou ◽  
Kangming Tian ◽  
Ashwani Kumar ◽  
Suren Singh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Chemical Production

scholarly journals Transcriptional co-operativity between distant retinoic acid response elements in regulation of Cyp26A1 inducibility

2005 ◽  
Vol 392 (1) ◽  
pp. 241-248 ◽  
Author(s):  
Olivier Loudig ◽  
Glenn A. Maclean ◽  
Naomi L. Dore ◽  
Luong Luu ◽  
Martin Petkovich
Keyword(s):  
Retinoic Acid ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Critical Role ◽  
Transcriptional Response ◽  
Luciferase Reporter ◽  
Maximal Response ◽  
Fold Induction

Cyp26A1 encodes an RA (retinoic acid)-catabolizing CYP (cytochrome P450) protein that plays a critical role in regulating RA distribution in vivo. Cyp26A1 expression is inducible by RA, and the locus has previously been shown to contain a RARE (RA response element), R1, within the minimal promoter [Loudig, Babichuk, White, Abu-Abed, Mueller and Petkovich (2000) Mol. Endocrinol. 14, 1483–1497]. In the present study, we report the identification of a second functional RARE (R2) located 2.0 kb upstream of the Cyp26A1 transcriptional start site. Constructs containing murine sequences encompassing both R1 and R2 showed that these elements work together to generate higher transcriptional activity upon treatment with RA than those containing R1 alone. Inclusion of R2 also dramatically enhanced the sensitivity of reporter constructs to RA, as even treatment with 10−8 M RA resulted in a 5-fold induction of reporter activity. Mutational analysis identified R2 as the functional element responsible for the increased RA inducibility of promoter constructs. The element was shown to bind RARγ (RA receptor γ)/RXRα (retinoid X receptor α) heterodimers in vitro, and inclusion of nuclear receptors in transfections boosted the transcriptional response. A construct containing both R1 and R2 was used to generate a stable luciferase reporter cell line that can be used as a tool to identify factors regulating Cyp26A1 expression. The analysis of R1 and R2 has led to the proposal that the two elements work synergistically to provide a maximal response to RA and that R2 is an upstream enhancer.

scholarly journals Dynamic control of pathway expression with riboregulated switchable feedback promoters

2019 ◽  
Author(s):  
Cameron J. Glasscock ◽  
John T. Lazar ◽  
Bradley W. Biggs ◽  
Jack H. Arnold ◽  
Min Kyoung Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Engineering ◽  
Stress Response ◽  
Dynamic Control ◽  
Transcriptional Networks ◽  
Cytochrome P450 Enzyme ◽  
Control Of Gene Expression ◽  
System Productivity ◽  
P450 Enzyme ◽  
Pathway Regulation

AbstractDynamic pathway regulation has emerged as a promising strategy in metabolic engineering for improved system productivity and yield, and continues to grow in sophistication. Bacterial stress-response promoters allow dynamic gene regulation using the host’s natural transcriptional networks, but lack the flexibility to control the expression timing and overall magnitude of pathway genes. Here, we report a strategy that uses RNA transcriptional regulators to introduce another layer of control over the output of natural stress-response promoters. This new class of gene expression cassette, called a riboregulated switchable feedback promoter (rSFP), can be modularly activated using a variety of mechanisms, from manual induction to quorum sensing. We develop and apply rSFPs to regulate a toxic cytochrome P450 enzyme in the context of a Taxol precursor biosynthesis pathway and show this leads to 2.4x fold higher titers than from the best reported strain. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, protein and biologic production, and many other applications.

scholarly journals Automation assisted anaerobic phenotyping for metabolic engineering

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Kaushik Raj ◽  
Naveen Venayak ◽  
Patrick Diep ◽  
Sai Akhil Golla ◽  
Alexander F. Yakunin ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
High Throughput ◽  
Large Scale ◽  
Plastic Waste ◽  
Small Scale ◽  
Chemical Production ◽  
Liquid Handling ◽  
Strain Design ◽  
Wide Range ◽  
Scale Down

Abstract Background Microorganisms can be metabolically engineered to produce a wide range of commercially important chemicals. Advancements in computational strategies for strain design and synthetic biological techniques to construct the designed strains have facilitated the generation of large libraries of potential candidates for chemical production. Consequently, there is a need for high-throughput laboratory scale techniques to characterize and screen these candidates to select strains for further investigation in large scale fermentation processes. Several small-scale fermentation techniques, in conjunction with laboratory automation have enhanced the throughput of enzyme and strain phenotyping experiments. However, such high throughput experimentation typically entails large operational costs and generate massive amounts of laboratory plastic waste. Results In this work, we develop an eco-friendly automation workflow that effectively calibrates and decontaminates fixed-tip liquid handling systems to reduce tip waste. We also investigate inexpensive methods to establish anaerobic conditions in microplates for high-throughput anaerobic phenotyping. To validate our phenotyping platform, we perform two case studies—an anaerobic enzyme screen, and a microbial phenotypic screen. We used our automation platform to investigate conditions under which several strains of E. coli exhibit the same phenotypes in 0.5 L bioreactors and in our scaled-down fermentation platform. We also propose the use of dimensionality reduction through t-distributed stochastic neighbours embedding (t-SNE) in conjunction with our phenotyping platform to effectively cluster similarly performing strains at the bioreactor scale. Conclusions Fixed-tip liquid handling systems can significantly reduce the amount of plastic waste generated in biological laboratories and our decontamination and calibration protocols could facilitate the widespread adoption of such systems. Further, the use of t-SNE in conjunction with our automation platform could serve as an effective scale-down model for bioreactor fermentations. Finally, by integrating an in-house data-analysis pipeline, we were able to accelerate the ‘test’ phase of the design-build-test-learn cycle of metabolic engineering.

Modular Metabolic Engineering for Biobased Chemical Production

2019 ◽  
Vol 37 (2) ◽  
pp. 152-166 ◽  
Author(s):  
Hongyuan Lu ◽  
Juan C. Villada ◽  
Patrick K.H. Lee
Keyword(s):  
Metabolic Engineering ◽  
Chemical Production
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