AraC Regulatory Protein Mutants with Altered Effector Specificity

The AraC regulatory protein of the Escherichia coli ara operon has been engineered to activate transcription in response to d-arabinose and not in response to its native effector l-arabinose. Two different AraC mutant libraries, each with four randomized binding pocket residues, were subjected to FACS-mediated dual screening using a GFP reporter. Both libraries yielded mutants with the desired switch in effector specificity, and one mutant we describe maintains tight repression in the absence of effector. The presence of 100 mM l-arabinose does not influence the response of the reported mutants to d-arabinose, and the mutants are not induced by other sugars tested (d-xylose, d-fucose, d-lyxose). Co-expression of the FucP transporter in E. coli enabled induction by d-arabinose in the 0.1 mM range. Our results demonstrate the power of dual screening for altering AraC inducer specificity and represent steps toward the design of customized in vivo molecular reporters and gene switches for metabolic engineering

Data_Sheet_1_The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli.docx

Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2′3’ - cyclic Guanosine Monophosphate–Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate–Adenosine monophosphate synthase (mcGAS). In E. coli BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with <20 EU/mL (<0.3 EU/μg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes.</p

Subnanometric Roughness Affects the Deposition and Mobile Adhesion of <i>Escherichia coli</i> on Silanized Glass Surfaces

We investigate the deposition and transient adhesion of <i>Escherichia coli</i> on alkyl and fluoroalkyl silanized glass surfaces of different carbon chain lengths. The rate at which bacteria deposit onto these surfaces decreases as the shear stress is increased from 3 to 67 mPa, but trends in the deposition rate across all surfaces cannot be predicted from extended DLVO calculations of the interaction potential. As the surface root-mean-square (rms) roughness increases, the deposition rate increases and the percentage of motile tethered cells decreases. Furthermore, on surfaces of root-mean-square roughness of less than 0.2 nm, bacteria exhibit mobile adhesion, for which surface-associated cells linearly translate distances greater than approximately 1.5 times their average body length along the flow direction. <i>E. coli</i> bacteria with and without flagella exhibit mobile adhesion, indicating that this behavior is not driven by these appendages. Cells that express fimbriae do not exhibit mobile adhesion. These results suggest that even subnanoscale roughness can influence the deposition and transient adhesion of bacteria and imply that strategies to reduce frictional interactions by making cells or surfaces smoother may help to control the initial fouling of surfaces by <i>E. coli</i> bacteria

Screening for Enhanced Triacetic Acid Lactone Production by Recombinant Escherichia coli Expressing a Designed Triacetic Acid Lactone Reporter

Triacetic acid lactone (TAL) is a signature byproduct of polyketide synthases (PKSs) and a valuable synthetic precursor. We have developed an endogenous TAL reporter by engineering the Escherichia coli regulatory protein AraC to activate gene expression in response to TAL. The reporter enabled in vivo directed evolution of Gerbera hybrida 2-pyrone synthase activity in E. coli. Two rounds of mutagenesis and high-throughput screening yielded a variant conferring ∼20-fold increased TAL production. The catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) of the variant toward the substrate malonyl-CoA was improved 19-fold. This study broadens the utility of engineered AraC variants as customized molecular reporters. In addition, the TAL reporter can find applications in other basic PKS activity screens

OptZyme: Computational Enzyme Redesign Using Transition State Analogues

<div><p>OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., k_cat or k_cat/K_M) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using <i>Escherichia coli</i> β-glucuronidase as a benchmark system, we confirm that K_M correlates (R² = 0.960) with the computed interaction energy between the enzyme and the <i>para</i>-nitrophenyl- β, D-glucuronide substrate, k_cat/K_M correlates (R² = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and k_cat correlates (R² = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved K_M, k_cat, and k_cat/K_M for a new substrate, <i>para</i>-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving K_M, k_cat, or k_cat/K_M. Mutants predicted to enhance the activity for <i>para</i>-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely <i>H162S</i>, <i>L361G</i>, <i>W549R</i>, and <i>N550S</i>.</p></div

Proposed catalytic reaction mechanism of GUS from <i>in vacuo</i> QM calculations (Text S1).

<p>In the first step, substrate binds to the active site of GUS. Next, the lone pair on the glycosidic bond attacks the proton of E413 (<i>A</i>). This forms a hypothetical TS (<i>B</i>) with the glycosidic bond partially broken. The glycosidic bond is fully cleaved, releasing <i>para</i>-nitrophenolate and forming a carbocation intermediate (<i>C</i>). The electrons on the anionic E504 then attack the anomeric carbon, resulting in a hypothetical TS (<i>D</i>) where the carbocation and E504 are electrostatically attractive. A covalent intermediate (<i>E</i>) is formed between the carbohydrate moiety of pNP-GLU and E504. Presumably, in the next step, the basic E413 attacks a proton of a water molecule. The resulting hydroxide anion attacks the anomeric carbon to yield the product of the reaction. The two catalytic residues are regenerated for further turnover.</p

Correlation between pNP-GAL IE_TSA and ln(k_cat/K_M).

<p>The correlation found here is significantly lower than the one found for pNP-GLU (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075358#pone-0075358-g007" target="_blank">Figure 7</a>) primarily due to mutant <i>T509S</i>. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075358#pone.0075358.s004" target="_blank">Figure S4</a>.</p

Top 10 mutants identified using OptZyme for optimizing K_M, k_cat/K_M, and k_cat for pNP-GAL.

<p>One-letter amino acid abbreviations for each design position and WT residue. Energy values are in kJ/mol.</p

Top 10 mutants identified using OptZyme for optimizing K_M, k_cat/K_M, and k_cat for pNP-GLU.

<p>One-letter amino acid abbreviations for each design position and WT residue. Energy values are in kJ/mol.</p

Comparison between ground state, hypothetical TS, and TSA for pNP-GLU and pNP-GAL.

<p>Differences between pNP-GLU (<i>A</i>) and pNP-GAL (<i>B</i>) include reversal of the stereospecificity of the C4 carbon and replacement of a carboxylic acid (pNP-GLU) at the C5 carbon with an alcohol (pNP-GAL). The previously-suggested <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075358#pone.0075358-Marsh1" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075358#pone.0075358-Conchie1" target="_blank">[53]</a> TSA for pNP-GLU, 1,5-glucarolactone (<i>D</i>), resembles the proposed TS (<i>C</i>) in terms of charge distribution and stereospecificity of the carbohydrate. In contrast to the proposed TS structure, the TSA lacks the <i>para</i>-nitrophenyl (pNP) moiety and a hydrogen atom from the C1 carbon. In addition, the TSA (<i>D</i>) differs from pNP-GLU (<i>A</i>) by assuming a more flattened sugar ring geometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075358#pone.0075358.s001" target="_blank">Figure S1</a> for dihedral angles) and partial positive charge at the anomeric carbon. The TSA for pNP-GAL, 1,5-galactonolactone (<i>E</i>), is similar to 1,5-glucaronolactone (<i>D</i>). The differences between 1,5-galactonolactone and 1,5-glucaronolactone are identical to the differences between pNP-GAL and pNP-GLU.</p