Using Synthetic Biological Parts and Microbioreactors to Explore the Protein Expression Characteristics of <i>Escherichia coli</i>

Synthetic biology has developed numerous parts for the precise control of protein expression. However, relatively little is known about the burden these place on a host, or their reliability under varying environmental conditions. To address this, we made use of synthetic transcriptional and translational elements to create a combinatorial library of constructs that modulated expression strength of a green fluorescent protein. Combining this library with a microbioreactor platform, we were able to perform a detailed large-scale assessment of transient expression and growth characteristics of two <i>Escherichia coli</i> strains across several temperatures. This revealed significant differences in the robustness of both strains to differing types of protein expression, and a complex response of transcriptional and translational elements to differing temperatures. This study supports the development of reliable synthetic biological systems capable of working across different hosts and environmental contexts. Plasmids developed during this work have been made publicly available to act as a reference set for future research

Internal standard corrected concentrations (RR = response ratio) of secondary metabolites from roquefortine/meleagrin pathway.

<p>The metabolite concentrations in the culture broth of the Δ<i>roqR</i> (A), Δ<i>roqD</i> (B), Δ<i>roqM</i> (C), Δ<i>roqO</i> (D), Δ<i>roqN</i> (E) and Δ<i>roqT</i> (F) strains was compared to the host strain <i>P. chrysogenum</i> DS54555.</p

Southern blot analysis for deletion of the genes in the roquefortine/meleagrin pathway.

<p>Southern blot hybridization was performed with total DNA extracted from <i>P. chrysogenum</i> DS54555 strains with a deletion of the following genes: <i>roqA</i> (A), <i>roqR</i> (B), <i>roqD</i> (C), <i>roqM</i> (D), <i>roqO</i> (E), <i>roqN</i> (F) and <i>roqT</i> (G). The DNA was digested with the restriction enzymes as indicated in the schemes.</p

Change in production of roquefortine/meleagrin metabolites after precursor addition compared to production in cultures without addition.

<p>Colored cells show mean levels that are significant (P<0.05) different.</p

Organization of the roquefortine/meleagrin biosynthetic gene cluster and transcriptomic analysis.

<p>(A) Roquefortine/meleagrin biosynthetic gene cluster and their orthologs in phylogenetically relative species. Homologous proteins are indicated with the same color. (B) Microarray analysis of the roquefortine biosynthetic genes in <i>P. chrysogenum</i> DS54555 using shake flask culture conditions in the absence (−) or presence (+) phenylacetic acid (PAA). (C) Correlation between the expression level of <i>roqA</i> and the concentration of the product HTD (<b>1)</b> present in the growth media. The concentration of <b>1</b> was determined by HPLC-UV-MS.</p

Födoval av torsk (Gadus morrhua L.) i Skagerrak och Kattegatt under februari 1981 /

<div><p>Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus <i>Penicillium chrysogenum</i> and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.</p></div

Total ion chromatogram for culture broth of <i>P. chrysogenum</i>.

<p>Total ion chromatogram (TIC, black) and normalized extracted ion chromatograms (EIC, colored) of secondary metabolites from the roquefortine/meleagrin pathway in the culture broth of <i>P. chrysogenum</i> DS54555. HTD (<b>1</b>, 8.8 min), DHTD (<b>2</b>, 9.4 min), glandicoline A (<b>5</b>, 16.8 min), roquefortine D (<b>3</b>, 18.3 min), glandicoline B (<b>6</b>, 18.4 min), meleagrin (<b>7</b>, 19.6 min), roquefortine C (<b>4</b>, 21.4 min).</p

Proposed roquefortine/meleagrin biosynthetic pathway.

<p>See text for details.</p

Quantification of produced β-lactam compounds in HpPen3 and HpPen4 cultures.

<p>Strains HpPen3 and HpPen4 were grown in glucose/methanol-limited chemostat cultures in the presence of 1 mM AAA and 1 mM PAA. β-lactam compounds were quantified by IP-LC–ESI-ID-MS/MS in spent medium and cell extracts. PenG could not be detected in HpPen3 cultures, but was clearly detectable in HpPen4 cultures, where it was efficiently excreted into the medium. The PenG precursor IPN was present in both HpPen3 and HpPen4 cells; however, the amount was significantly lower in the PenG producing HpPen4 cells. Concentrations are expressed as µmol/l culture. Samples were taken in triplicate. The bars represent the standard error (SE).</p

Subcellular localization of ACVS in <i>H. polymorpha</i>.

<p>Immunocytochemistry using anti-ACVS antibodies showing the presence of ACVS protein in the cytosol of strain HpPen4. Cells were fixed in 3% glutaraldehyde for 1 h on ice, dehydrated in an ethanol series and embedded in Lowicryl, polymerized by UV light. Post-staining was with 0.5% uranylacetate. M – mitochondrion; P – peroxisome; V – vacuole. The bar represents 0.5 µm.</p