8 research outputs found
A Two-Layer Gene Circuit for Decoupling Cell Growth from Metabolite Production
SummaryWe present a synthetic gene circuit for decoupling cell growth from metabolite production through autonomous regulation of enzymatic pathways by integrated modules that sense nutrient and substrate. The two-layer circuit allows Escherichia coli to selectively utilize target substrates in a mixed pool; channel metabolic resources to growth by delaying enzymatic conversion until nutrient depletion; and activate, terminate, and re-activate conversion upon substrate availability. We developed two versions of controller, both of which have glucose nutrient sensors but differ in their substrate-sensing modules. One controller is specific for hydroxycinnamic acid and the other for oleic acid. Our hydroxycinnamic acid controller lowered metabolic stress 2-fold and increased the growth rate 2-fold and productivity 5-fold, whereas our oleic acid controller lowered metabolic stress 2-fold and increased the growth rate 1.3-fold and productivity 2.4-fold. These results demonstrate the potential for engineering strategies that decouple growth and production to make bio-based production more economical and sustainable
Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid short- and branched-chain alkyl esters biodiesel
Engineering synthetic promoters as dynamic controllers in saccharomyces cerevisiae
Metabolic engineering of yeast is an attractive way to produce advanced biofuels. However, engineering of yeast by introducing heterologous proteins and pathways can reduce growth rates and impact productivity. Towards optimizing yeast strains, sensor-regulators can assist the optimal usage of cellular resources, where protein expression can be regulated by the concentration of important metabolites. In this thesis, synthetic promoters were engineered and heterologous transcriptional repressors were expressed in order to create dynamic controllers in yeast. First, fatty acids/fatty acyl-CoA sensor-regulators were made as they are key intermediates in the production of fatty acid derived biofuels, which are suitable for direct use in current transportation infrastructure. This enables fatty acid dependant control of fatty acid derivative producing proteins. Second, AND-gate dynamic controllers that combine inducible promoter function, which enables cells to quickly accumulate biomass before triggering the production of biofuel producing proteins, and fatty acid sensing-regulation were constructed. Third, xylose sensor-regulators were created, where xylose is a major sugar component in the affordable lignocellulose biomass carbon source. This allows regulation of xylose utilizing proteins based on the amount of xylose sugars detected. The function and performance of these synthetic promoters to dynamically control yEGFP reporter protein were demonstrated.DOCTOR OF PHILOSOPHY (SCBE
Development and characterization of AND-gate dynamic controllers with a modular synthetic GAL1 core promoter in Saccharomyces cerevisiae
Expression of heterologous proteins in metabolic engineering endeavors can be detrimental to host cells due to increased usage of cellular resources. Dynamic controls, where protein expression can be triggered on-demand, are effective for the engineering and optimization of bio-catalysts towards robust cell growth and enhanced biochemical productivity. Here, we describe the development and characterization of AND-gate dynamic controllers in Saccharomyces cerevisiae which combine two dynamic control strategies, inducible promoters and sensing-regulation. These dynamic controllers were constructed based on synthetic hybrid promoters. Promoter enhancer sequences were fused to a synthetic GAL1 core promoter containing DNA binding sites for the binding of a repressor that reduced DNA affinity upon interaction with key intermediates in a biochemical pathway. As fatty acids are key intermediates for production of fatty alcohols, fatty acid esters, alkenes, and alkanes, which are advanced biofuels, we used the fatty acid responsive FadR repressor and its operator sequence to demonstrate the functionality of the dynamic controllers. We established that the synthetic GAL1 core promoter can be used as a modular promoter part for constructing synthetic hybrid promoters and conferring fatty acid inducibility. We further showed the performance of the AND-gate dynamic controllers, where two inputs (fatty acid and copper presence/phosphate starvation) were required to switch the AND-gate ON. This work provides a convenient platform for constructing AND-gate dynamic controllers, that is, promoters that combine inducible functionality with regulation of protein expression levels upon detection of key intermediates towards the engineering and optimization of bio-catalytic yeast cells
Bacterial FadR and synthetic promoters function as modular fatty acid sensor regulators in Saccharomyces cerevisiae
Fatty acid derivatives have ideal properties for use as drop-in biofuels. An effective strategy in engineering microbial cells to maximize productivity and yield involves dynamic control of protein production in response to concentrations of key intermediates. In Saccharomyces cerevisiae, the activities of the native transcription factors responsive to fatty acids are repressed in the presence of a glucose carbon source. In order to develop a modular fatty acid regulation system in S. cerevisiae, we constructed fatty acid/fatty acyl-CoA biosensors in S. cerevisiae using bacterial FadR transcriptional repressors and yeast synthetic promoters containing DNA-binding operators. We demonstrated the functionality of FadR repressors in S. cerevisiae, and tuned the sensing system by varying the promoter strength upstream to the FadR-coding sequence by varying the number of operator sites in the synthetic promoter and by using FadR from two bacterial sources (Escherichia coli and Vibrio cholerae) with different ligand sensitivities. We envision that our fatty acid/fatty acyl-CoA biosensors can be used for regulation of protein expression based on the availability of fatty acid intermediates, which will assist in balancing of cellular metabolism during fatty acid derivatives production in yeast
Engineering a riboswitch-based genetic platform for the self-directed evolution of acid-tolerant phenotypes
Cells are exposed to shifts in environmental pH, which direct their metabolism and behavior. Here the authors design pH-sensing riboswitches to create a gene expression program, digitalize the system to respond to a narrow pH range and apply it to evolve host cells with improved tolerance to a variety of organic acids
Synthetic biology toolkits and applications in Saccharomyces cerevisiae
10.1016/j.biotechadv.2018.07.005Biotechnology advances3671870-188