Metabolic engineering and synthetic biology for isoprenoid production in Escherichia coli and Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Govinda R. Navale ◽  
Mahesh S. Dharne ◽  
Sandip S. Shinde
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Synthetic Biology

Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

2019 ◽  
Author(s):  
Thomas Siemon ◽  
Zhangqian Wang ◽  
Guangkai Bian ◽  
Tobias Seitz ◽  
Ziling Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Chemical Synthesis ◽  
Building Block ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Economical Method ◽  
Englerin A ◽  
Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

2019 ◽  
Author(s):  
Thomas Siemon ◽  
Zhangqian Wang ◽  
Guangkai Bian ◽  
Tobias Seitz ◽  
Ziling Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Chemical Synthesis ◽  
Building Block ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Economical Method ◽  
Englerin A ◽  
Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

Metabolic engineering and synthetic biology approaches driving isoprenoid production in Escherichia coli

2017 ◽  
Vol 241 ◽  
pp. 430-438 ◽  
Author(s):  
Chonglong Wang ◽  
Bakht Zada ◽  
Gongyuan Wei ◽  
Seon-Won Kim
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Synthetic Biology

scholarly journals Metabolite Sequestration Enables Rapid Recovery from Fatty Acid Depletion in Escherichia coli

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Christopher J. Hartline ◽  
Ahmad A. Mannan ◽  
Di Liu ◽  
Fuzhong Zhang ◽  
Diego A. Oyarzún
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Metabolic Engineering ◽  
Synthetic Biology ◽  
Transcriptional Regulator ◽  
Nutrient Depletion ◽  
Fast Recovery ◽  
Metabolic Adaptations ◽  
Rapid Release ◽  
Metabolic Recovery

ABSTRACT Microbes adapt their metabolism to take advantage of nutrients in their environment. Such adaptations control specific metabolic pathways to match energetic demands with nutrient availability. Upon depletion of nutrients, rapid pathway recovery is key to release cellular resources required for survival under the new nutritional conditions. Yet, little is known about the regulatory strategies that microbes employ to accelerate pathway recovery in response to nutrient depletion. Using the fatty acid catabolic pathway in Escherichia coli, here, we show that fast recovery can be achieved by rapid release of a transcriptional regulator from a metabolite-sequestered complex. With a combination of mathematical modeling and experiments, we show that recovery dynamics depend critically on the rate of metabolite consumption and the exposure time to nutrients. We constructed strains with rewired transcriptional regulatory architectures that highlight the metabolic benefits of negative autoregulation over constitutive and positive autoregulation. Our results have wide-ranging implications for our understanding of metabolic adaptations, as well as for guiding the design of gene circuitry for synthetic biology and metabolic engineering. IMPORTANCE Rapid metabolic recovery during nutrient shift is critical to microbial survival, cell fitness, and competition among microbiota, yet little is known about the regulatory mechanisms of rapid metabolic recovery. This work demonstrates a previously unknown mechanism where rapid release of a transcriptional regulator from a metabolite-sequestered complex enables fast recovery to nutrient depletion. The work identified key regulatory architectures and parameters that control the speed of recovery, with wide-ranging implications for the understanding of metabolic adaptations as well as synthetic biology and metabolic engineering.

scholarly journals Rapid conversion of replicating and integrating Saccharomyces cerevisiae plasmid vectors via Cre recombinase

2020 ◽  
Author(s):  
Daniel P. Nickerson ◽  
Monique A. Quinn ◽  
Joshua M. Milnes
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Cre Recombinase ◽  
Shuttle Vectors ◽  
Genetics Research ◽  
Complete Set ◽  
Rapid Conversion

ABSTRACTPlasmid shuttle vectors capable of replication in both Saccharomyces cerevisiae and Escherichia coli and optimized for controlled modification in vitro and in vivo are a key resource supporting yeast as a premier system for genetics research and synthetic biology. We have engineered a series of yeast shuttle vectors optimized for efficient insertion, removal and substitution of plasmid yeast replication loci, allowing generation of a complete set of integrating, low copy and high copy plasmids via predictable operations as an alternative to traditional subcloning. We demonstrate the utility of this system through modification of replication loci via Cre recombinase, both in vitro and in vivo, and restriction endonuclease treatments.

scholarly journals The Model System Saccharomyces cerevisiae Versus Emerging Non-Model Yeasts for the Production of Biofuels

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 299
Author(s):  
Maria Priscila Lacerda ◽  
Eun Joong Oh ◽  
Carrie Eckert
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Synthetic Biology ◽  
Biofuel Cell ◽  
Efficient Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
Fatty Acid Production ◽  
Inhibitor Tolerance ◽  
Cell Factories ◽  
Recent Advances

Microorganisms are effective platforms for the production of a variety of chemicals including biofuels, commodity chemicals, polymers and other natural products. However, deep cellular understanding is required for improvement of current biofuel cell factories to truly transform the Bioeconomy. Modifications in microbial metabolic pathways and increased resistance to various types of stress caused by the production of these chemicals are crucial in the generation of robust and efficient production hosts. Recent advances in systems and synthetic biology provide new tools for metabolic engineering to design strategies and construct optimal biocatalysts for the sustainable production of desired chemicals, especially in the case of ethanol and fatty acid production. Yeast is an efficient producer of bioethanol and most of the available synthetic biology tools have been developed for the industrial yeast Saccharomyces cerevisiae. Non-conventional yeast systems have several advantageous characteristics that are not easily engineered such as ethanol tolerance, low pH tolerance, thermotolerance, inhibitor tolerance, genetic diversity and so forth. Currently, synthetic biology is still in its initial steps for studies in non-conventional yeasts such as Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris. Therefore, the development and application of advanced synthetic engineering tools must also focus on these underexploited, non-conventional yeast species. Herein, we review the basic synthetic biology tools that can be applied to the standard S. cerevisiae model strain, as well as those that have been developed for non-conventional yeasts. In addition, we will discuss the recent advances employed to develop non-conventional yeast strains that are efficient for the production of a variety of chemicals through the use of metabolic engineering and synthetic biology.

scholarly journals An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Blake J. Rasor ◽  
Xiunan Yi ◽  
Hunter Brown ◽  
Hal S. Alper ◽  
Michael C. Jewett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Synthetic Biology ◽  
Biosynthetic Pathways ◽  
Cell Extracts ◽  
Systematic Optimization ◽  
Host Metabolism ◽  
Host Strains

AbstractCell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing.

scholarly journals Rapid conversion of replicating and integrating Saccharomyces cerevisiae plasmid vectors via Cre recombinase

2021 ◽  
Author(s):  
Daniel P Nickerson ◽  
Monique A Quinn ◽  
Joshua M Milnes
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Cre Recombinase ◽  
Shuttle Vectors ◽  
Genetics Research ◽  
Complete Set ◽  
Rapid Conversion

Abstract Plasmid shuttle vectors capable of replication in both Saccharomyces cerevisiae and Escherichia coli and optimized for controlled modification in vitro and in vivo are a key resource supporting yeast as a premier system for genetics research and synthetic biology. We have engineered a series of yeast shuttle vectors optimized for efficient insertion, removal and substitution of plasmid yeast replication loci, allowing generation of a complete set of integrating, low copy and high copy plasmids via predictable operations as an alternative to traditional subcloning. We demonstrate the utility of this system through modification of replication loci via Cre recombinase, both in vitro and in vivo, and restriction endonuclease treatments.

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