Biosensor-Enabled Directed Evolution to Improve Muconic Acid Production in Saccharomyces cerevisiae.

Muconic acid is a valuable platform chemical with potential applications in the production of polymers such as nylon and polyethylene terephthalate (PET). The conjugate base, muconate, has been previously biosynthesized in the bacterial host Escherichia coli. Likewise, previous significant pathway engineering lead to the first reported instance of rationally engineered production of muconic acid in the yeast Saccharomyces cerevisiae. To further increase muconic acid production in this host, a combined adaptive laboratory evolution (ALE) strategy and rational metabolic engineering is employed. To this end, a biosensor module that responds to the endogenous aromatic amino acid (AAA) as a surrogate for pathway flux is adapted. Following two rounds of ALE coupled with an anti-metabolite feeding strategy, the strains with improved AAA pathway flux is isolated. Next, it is demonstrated that this increased flux can be redirected into the composite muconic acid pathway with a threefold increase in the total titer of the composite pathway compared to our previously engineered strain. Finally, a truncation of the penta-functional ARO1 protein is complemented and overexpress an endogenous aromatic decarboxylase to establish a final strain capable of producing 0.5 g L-1 muconic acid in shake flasks and 2.1 g L-1 in a fed-batch bioreactor with a yield of 12.9 mg muconic acid/g glucose at the rate of 9.0 mg h-1 . This value represents the highest titer of muconic acid reported to date in S. cerevisiae, in addition to the highest reported titer of a shikimate pathway derivative in this host.

Coordinated transcription factor and promoter engineering to establish strong expression elements in Saccharomyces cerevisiae.

Gene expression requires the coordination of trans-acting factors and cis-DNA elements to initiate transcription. Here we present a coordinated approach that combines cis-acting element engineering with mutant trans-acting factors to engineer yeast promoters. Specifically, we first construct a hybrid promoter based on the ARO9 upstream region that exhibits high constitutive and inducible expression with respect to exogenous tryptophan. Next, we perform protein engineering to identify a mutant Aro80p that affords both high constitutive expression while retaining inducible traits. We then use this mutant trans-acting factor to drive expression and generate ultra-strong promoters with transcriptional output roughly 2 fold higher than TDH3 (GPD), one of the strongest promoters to-date. Finally, we used this element to construct a modular expression system capable of staged outputs resulting in a system with nearly 6-fold, 12-fold and 15-fold expression relative to the off-state. This work further highlights the potential of using endogenous transcription factors (including mutant factors) along with hybrid promoters to expand the yeast synthetic biology toolbox.

Advances and current limitations in transcript-level control of gene expression.

Gene expression control is critical to increase production of recombinant proteins, fine-tune metabolic pathways and reliably express synthetic pathways. The importance of transcriptional control seems to be most important in eukaryotic systems. In this review, we highlight recent developments in the field of transcriptional engineering with an emphasis on the opportunities and challenges. We discuss the engineering of 'parts' that influence transcriptional throughput including promoters, terminators, and transcription factors as well as the genetic context of the expression cassette. While great strides have been made in the area, the robustness of these parts has been largely untested. This review highlights the importance of considering robustness in biological systems and the limitations that current synthetic parts possess.

Metabolic engineering of muconic acid production in Saccharomyces cerevisiae.

The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141mg/L muconic acid in a shake-flask culture, a value nearly 24-fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts.

Synthesis and evaluation of quinoxaline derivatives as potential influenza NS1A protein inhibitors.

A library of quinoxaline derivatives were prepared to target non-structural protein 1 of influenza A (NS1A) as a means to develop anti-influenza drug leads. An in vitro fluorescence polarization assay demonstrated that these compounds disrupted the dsRNA-NS1A interaction to varying extents. Changes of substituent at positions 2, 3 and 6 on the quinoxaline ring led to variance in responses. The most active compounds (35 and 44) had IC(50) values in the range of low micromolar concentration without exhibiting significant dsRNA intercalation. Compound 44 was able to inhibit influenza A/Udorn/72 virus growth.

Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

Control of a pneumatic orthosis for upper extremity stroke rehabilitation.

A key challenge in rehabilitation robotics is the development of a lightweight, large force, high degrees-of-freedom device that can assist in functional rehabilitation of the arm. Pneumatic actuators can potentially help meet this challenge because of their high power-to-weight ratio. They are currently not widely used for rehabilitation robotics because they are difficult to control. This paper describes the control development of a pneumatically actuated, upper extremity orthosis for rehabilitation after stroke. To provide the sensing needed for good pneumatic control, position and velocity of the robot are estimated by a unique implementation of a Kalman filter using MEMS accelerometers. To compensate for the nonlinear behavior of the pneumatic servovalves, force control is achieved using a new method for air flow mapping using experimentally measured data in a least-squares regression. To help patients move with an inherently compliant robot, a high level controller that assists only as needed in reaching exercises is developed. This high level controller differs from traditional trajectory-based, position controllers, allowing free voluntary movements toward a target while resisting movements away from the target. When the target cannot be reached voluntarily, the controller slowly builds up force, pushing the arm toward the target. As each target position is reached, the controller builds an internal model of the subject's capability, learning the forces necessary to complete movements. Preliminary testing performed on a non-disabled subject demonstrated the ability of the orthosis to complete reaching movements with graded assistance and to adapt to the effort level of the subject. Thus, the orthosis is a promising tool for upper extremity rehabilitation after stroke.