Metabolic engineering and synthetic biology endeavors benefit from promoters that perform consistently(or robustly)with respect to cellular growth phase(exponential and stationary)and fermentation scale(microtiter pla...Metabolic engineering and synthetic biology endeavors benefit from promoters that perform consistently(or robustly)with respect to cellular growth phase(exponential and stationary)and fermentation scale(microtiter plates,tubes,flasks,and bioreactors).However,nearly all endogenous promoters(especially in Saccharomyces cerevisiae)do not perform in this manner.In this work,a hybrid promoter engineering strategy is leveraged to create novel synthetic promoters with robustness across these conditions.Using a multi-dimensional RNA-seq dataset,promoters with specific phase dependencies were identified.Fragments enriched with functional transcription factors were identified using MEME suite.These motif-containing fragments could impart activity dependence in the opposing condition.Specifically,we obtain two new promoters with high and consistent expression across both phases by increasing the exponential phase activity of the starting stationary-phase scaffold by 38 and 23-fold respectively.Further,we show that these promoters function consistently across various laboratory growth scales over time in a microtiter plate and in flasks.Overall,this work presents and validates a new strategy for engineering promoters in S.cerevisiae with high levels of expression that are robust to cellular growth phase and the scale of the culture.展开更多
Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner.However,most industrial applications have focused on only a small subset of elements from the periodic table,centered a...Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner.However,most industrial applications have focused on only a small subset of elements from the periodic table,centered around carbon biochemistry.This review aims to illustrate the expanse of chemical elements that can currently(and potentially)be integrated into useful products using cellular systems.Specifically,we describe recent advances in expanding the cellular scope to include the halogens,selenium and the metalloids,and a variety of metal incorporations.These examples range from small molecules,heteroatom-linked uncommon elements,and natural products to biomining and nanotechnology applications.Collectively,this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology.展开更多
Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity.As a result,these systems are not easily portable or reusable for on-demand protei...Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity.As a result,these systems are not easily portable or reusable for on-demand protein production applications.In this study,we employ Bioproduced Proteins On Demand(Bio-POD),a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable,on-demand production and secretion of medium-and high-molecular weight proteins(in this case,SEAP,α-amylase,and anti-HER2).The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration.The hydrogel platform described here is the first hydrogel immobilization using a P.pastoris system to produce recombinant proteins of this breadth.These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production.展开更多
Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair,genetic recombination,and gene expression.Here,we present CRISPR-PIN,a CRISPR/dCas9-based tool that allows control...Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair,genetic recombination,and gene expression.Here,we present CRISPR-PIN,a CRISPR/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae.This approach utilizes a cohesindockerin interaction between dCas9 and a perinuclear protein.In doing so,we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery.We demonstrate the utility of this approach for two applications:the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci.In both cases,we obtain results on par with prior reports using traditional,more cumbersome genetic systems.Thus,CRISPR-PIN offers the opportunity for future studies of chromosome biology and gene localization.展开更多
Organisms can be engineered to produce a wide variety of compounds by either enhancing endogenous metabolic pathways,or by introducing exogenous pathways that are either borrowed from other organism,or de novo-designe...Organisms can be engineered to produce a wide variety of compounds by either enhancing endogenous metabolic pathways,or by introducing exogenous pathways that are either borrowed from other organism,or de novo-designed pathways unknown to nature.While overexpression of bottleneck enzymes and deletion of competing pathways remain at the core of metabolic pathway engineering,there are many other key elements that need to be considered to successfully develop strains for the production of valuable products.展开更多
基金We acknowledge support from the Air Force Office of Scientific Research under Award No.FA9550-14-1-0089.Sequencing was conducted at the Genomic Sequencing and Analysis Facility(RRID no.SCR_021713)flow cytometry as conducted at the Microscopy and Imaging Facility(RRID no.SCR_021756)at the UT Austin Center for Biomedical Research Support.
文摘Metabolic engineering and synthetic biology endeavors benefit from promoters that perform consistently(or robustly)with respect to cellular growth phase(exponential and stationary)and fermentation scale(microtiter plates,tubes,flasks,and bioreactors).However,nearly all endogenous promoters(especially in Saccharomyces cerevisiae)do not perform in this manner.In this work,a hybrid promoter engineering strategy is leveraged to create novel synthetic promoters with robustness across these conditions.Using a multi-dimensional RNA-seq dataset,promoters with specific phase dependencies were identified.Fragments enriched with functional transcription factors were identified using MEME suite.These motif-containing fragments could impart activity dependence in the opposing condition.Specifically,we obtain two new promoters with high and consistent expression across both phases by increasing the exponential phase activity of the starting stationary-phase scaffold by 38 and 23-fold respectively.Further,we show that these promoters function consistently across various laboratory growth scales over time in a microtiter plate and in flasks.Overall,this work presents and validates a new strategy for engineering promoters in S.cerevisiae with high levels of expression that are robust to cellular growth phase and the scale of the culture.
基金We acknowledge support from The Camille and Henry Dreyfus Teacher Scholar Program,The Welch Foundation under Grant F-1753,and the Air Force Office of Scientific Research under Award No.FA9550-14-1-0089.
文摘Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner.However,most industrial applications have focused on only a small subset of elements from the periodic table,centered around carbon biochemistry.This review aims to illustrate the expanse of chemical elements that can currently(and potentially)be integrated into useful products using cellular systems.Specifically,we describe recent advances in expanding the cellular scope to include the halogens,selenium and the metalloids,and a variety of metal incorporations.These examples range from small molecules,heteroatom-linked uncommon elements,and natural products to biomining and nanotechnology applications.Collectively,this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology.
基金This work was supported by the Camille and Henry Dreyfus Foundation(H.A.).A.N.acknowledges both UW CoMotion and Royalty Research Fund for supporting this work.We thank Dr.Hung
文摘Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity.As a result,these systems are not easily portable or reusable for on-demand protein production applications.In this study,we employ Bioproduced Proteins On Demand(Bio-POD),a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable,on-demand production and secretion of medium-and high-molecular weight proteins(in this case,SEAP,α-amylase,and anti-HER2).The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration.The hydrogel platform described here is the first hydrogel immobilization using a P.pastoris system to produce recombinant proteins of this breadth.These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production.
基金This work was supported by the Camille Dreyfus Teacher-ScholarAward.
文摘Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair,genetic recombination,and gene expression.Here,we present CRISPR-PIN,a CRISPR/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae.This approach utilizes a cohesindockerin interaction between dCas9 and a perinuclear protein.In doing so,we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery.We demonstrate the utility of this approach for two applications:the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci.In both cases,we obtain results on par with prior reports using traditional,more cumbersome genetic systems.Thus,CRISPR-PIN offers the opportunity for future studies of chromosome biology and gene localization.
文摘Organisms can be engineered to produce a wide variety of compounds by either enhancing endogenous metabolic pathways,or by introducing exogenous pathways that are either borrowed from other organism,or de novo-designed pathways unknown to nature.While overexpression of bottleneck enzymes and deletion of competing pathways remain at the core of metabolic pathway engineering,there are many other key elements that need to be considered to successfully develop strains for the production of valuable products.
