Parameters That Enhance the Bacterial Expression of Active Plant Polyphenol Oxidases

<div><p>Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert diphenolic compounds into their corresponding quinones. Although there is significant interest in these enzymes because of their role in food deterioration, the lack of a suitable expression system for the production of soluble and active plant PPOs has prevented detailed investigations of their structure and activity. Recently we developed a bacterial expression system that was sufficient for the production of PPO isoenzymes from dandelion (<i>Taraxacum officinale</i>). The system comprised the <i>Escherichia coli</i> Rosetta 2 (DE3) [pLysSRARE2] strain combined with the pET-22b(+)-vector cultivated in auto-induction medium at a constant low temperature (26°C). Here we describe important parameters that enhance the production of active PPOs using dandelion PPO-2 for proof of concept. Low-temperature cultivation was essential for optimal yields, and the provision of CuCl₂ in the growth medium was necessary to produce an active enzyme. By increasing the copper concentration in the production medium to 0.2 mM, the yield in terms of PPO activity per mol purified protein was improved 2.7-fold achieving a v_max of 0.48±0.1 µkat per mg purified PPO-2 for 4-methylcatechol used as a substrate. This is likely to reflect the replacement of an inactive apo-form of the enzyme with a correctly-folded, copper-containing counterpart. We demonstrated the transferability of the method by successfully expressing a PPO from tomato (<i>Solanum lycopersicum</i>) showing that our optimized system is suitable for the analysis of further plant PPOs. Our new system therefore provides greater opportunities for the future of research into this economically-important class of enzymes.</p></div

Influence of copper supplementation on enzymatic parameters.

<p>Production cultures were grown with different concentrations of supplemented CuCl₂ in AIM. <b>A SDS-PAGE of purified proteins.</b> ToPPO-2 was purified via affinity-chromatography using Strep-Tactin. Equal protein amounts were loaded on an acrylamide gel and analyzed by SDS-PAGE and Coomassie-staining. <b>B Maximal activities (v_max)</b> and <b>C Michaelis constants (K_m).</b> Enzyme kinetics were performed on 4-methylcatechol as substrate and enzymatic parameters were determined by non-linear regression to the Michaelis-Menten equation. <i>* P<0.01 in Student's t-test (n = 4); K_m-values were not significantly different (P = 0.955). </i><b>D Correlation between the lower band appearing on SDS-PAGE and v_max.</b> Data given are from three independent experiments. Analysis of the relative density of protein bands was performed using ImageJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077291#pone.0077291-Rasband1" target="_blank">[32]</a> calculating the lower band in relation to the respective upper one. Poor separation of double bands at higher copper concentrations (1 – 2 mM) precluded their accurate quantification so that only data from the lower concentrations (0 – 0.5 mM) are included. <i>Statistics for correlation was performed using SigmaPlot11 and Pearson Product Moment Correlation, giving a significant positive correlation (corr. coefficient = 0.676, p<0.001, n = 23) between the tested parameters.</i></p

Optimal temperature for ToPPO-2 production.

<p>Production cultures were grown under different temperature settings. <b>A SDS-PAGE of crude protein extracts.</b> Samples were taken after 48 h and analyzed by SDS-PAGE (equal total protein amount per well). To visualize protein composition a Coomassie-staining was performed. <b>B SDS-PAGE of purified proteins.</b> ToPPO-2 was purified via affinity-chromatography using Strep-Tactin. Equal protein amounts were loaded on an acrylamide gel and analyzed by SDS-PAGE and Coomassie staining. <b>C Protein yields and specific PPO-activity after purification.</b> The specific PPO-activity (v₀) was analyzed per mg of purified PPO. The average value from all samples is given as a dotted line. Yields of purified protein per volume of bacterial culture were also calculated. <i>c: continuous growth in given temperature; 0.3/0.9: growth at 37°C until OD_{600 nm} of 0.3/0.9 was reached, then grown at 26°C; control: empty vector control grown at 26°C.</i></p

Expression system for dandelion PPOs. A Scheme for the PPO-2 expression plasmid.

<p>Location of important genes, regulatory elements and sites for restriction enzymes are given. The StrepII-tag (WSHPQFEK) and the Enterokinase recognition site (Ek-site: DDDDK) were introduced via PCR. <i>ORF: open reading frame</i>. <b>B Procedure of heterologous PPO-expression.</b> Conditions like media, supplements and temperatures are given and the course of action is schematized. <i>AIM: autoinduction medium; LB: lysogeny broth</i>.</p

Structure and catalysis of PPOs and related enzymes. A Type-3 copper center.

<p>Conserved histidines of the two copper centers (CuA and CuB) are labeled (H_A1-H_A3, H_B1-H_B3) and colored in green. Copper is colored blue and the oxygen molecules of the bound peroxide in red (<i>modified from </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077291#pone.0077291-Decker2" target="_blank">[31]</a>). <b>Phenoloxidase activities.</b> Tyrosinases oxidase monophenolic compounds via their monophenolase activity to diphenols and further to the respective quinones. PPOs mostly just catalyze the conversion of diphenols to quinones (diphenolase activity).</p

Optimal time for harvest of recombinant ToPPO-2. A SDS-PAGE of crude protein extracts.

<p>Samples were taken at different sampling times and analyzed by SDS-PAGE (same total protein amount per well). To visualize protein composition a Coomassie-staining was performed. <b>B Western blot analysis.</b> Proteins were blotted on a nitrocellulose membrane. Strep-Tactin-HRP conjugate was used to specifically detect the StrepII-tag of the recombinant PPO. <b>C Bacterial growth and PPO-activity over sampling time.</b> Samples were checked for their OD at 600 nm. Additionally crude protein extracts were analyzed for PPO-activity per mg of total protein. <i>The presented data show one experiment representative for three replicates</i>.</p

Copper-saturation of recombinant ToPPO-2.

<p>The copper content in aliquots containing defined amounts of purified ToPPO was determined using TXRF. Subsequently the molar ratio of Cu to purified PPO was calculated. <i>* P<0.01 in Student's t-test (n = 4).</i></p

Transferability of the improved expression system to further eukaryotic PPOs.

<p>Further eukaryotic PPOs (s. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077291#pone-0077291-t001" target="_blank">Table 1</a>) were cloned and heterologously expressed in <i>E. coli</i> Rosetta 2 (DE3) [pLysSRARE2]. Production cultures were grown with different concentrations (0 mM, 0.02 mM, 0.2 mM) of supplemented CuCl₂ in AIM. <b>A SDS-PAGE of purified proteins.</b> Cultures were harvested after 48 h and recombinant PPOs were purified via affinity-chromatography using Strep-Tactin. Equal protein amounts were loaded on an acrylamide gel and analyzed via SDS-PAGE using Coomassie staining. <b>B Specific PPO-activity after purification.</b> Activity (v₀) was analyzed per mg purified protein for 4-methylcatechol as substrate. <i>Purified ToPPO-2 produced with 0.2 mM CuCl₂ supplementation served as positive control (+) and buffer as negative control (-).</i></p

A bottom-up approach towards a bacterial consortium for the biotechnological conversion of chitin to L-lysine

Chitin is an abundant waste product from shrimp and mushroom industries and as such, an appropriate secondary feedstock for biotechnological processes. However, chitin is a crystalline substrate embedded in complex biological matrices, and, therefore, difficult to utilize, requiring an equally complex chitinolytic machinery. Following a bottom-up approach, we here describe the step-wise development of a mutualistic, non-competitive consortium in which a lysine-auxotrophic Escherichia coli substrate converter cleaves the chitin monomer N-acetylglucosamine (GlcNAc) into glucosamine (GlcN) and acetate, but uses only acetate while leaving GlcN for growth of the lysine-secreting Corynebacterium glutamicum producer strain. We first engineered the substrate converter strain for growth on acetate but not GlcN, and the producer strain for growth on GlcN but not acetate. Growth of the two strains in co-culture in the presence of a mixture of GlcN and acetate was stabilized through lysine cross-feeding. Addition of recombinant chitinase to cleave chitin into GlcNAc(2), chitin deacetylase to convert GlcNAc(2) into GlcN(2) and acetate, and glucosaminidase to cleave GlcN(2) into GlcN supported growth of the two strains in co-culture in the presence of colloidal chitin as sole carbon source. Substrate converter strains secreting a chitinase or a beta-1,4-glucosaminidase degraded chitin to GlcNAc(2) or GlcN(2) to GlcN, respectively, but required glucose for growth. In contrast, by cleaving GlcNAc into GlcN and acetate, a chitin deacetylase-expressing substrate converter enabled growth of the producer strain in co-culture with GlcNAc as sole carbon source, providing proof-of-principle for a fully integrated co-culture for the biotechnological utilization of chitin

Exploring the role of alpha-1,3-glucan synthases on fungal cell wall integrity in Aspergillus niger (Vortrag)

Alpha-glucan synthases (ags) play a key role in the synthesis of alpha-1,3-glucan, a crucial component of the fungal cell wall that is (i) contributing to its structural integrity and is (ii) involved in cross-linking of different polymers, so that it influences the composition of both: the outer shell and inner membrane of fungal cells. In the filamentous fungi Aspergillus niger five ags genes are annotated, of which agsA and agsE were shown to be the most highly expressed genes during different stages of development. In addition, an agsE deletion mutant caused smaller micro-colonies as well as a shift in the secretome composition of A. niger, without affecting biomass production. Concurrently, intracellular cross-linking between chitin and beta-glucan is primarily mediated by the seven-membered cell wall-related transglycosylase gene family (crh). Although an impact on cell wall integrity has been expected when deleting the entire crh gene cluster in A. niger, significant alterations in cell wall integrity became only evident when the crh gene cluster deletion was combined with the reduction of alpha-glucan and galactomannan by deleting respective agsE or ugmA. With this background, we aimed to further explore the impact of ags gene deletions – both individually and as an entire family – on fungal cell wall integrity of A. niger. Using targeted CRISPR/Cas9 technology, we engineered various ags-deficient strains, including the deletion of the entire gene family (ΔagsA-E) in a mutant strain lacking all chitin-glucan cross-linking enzymes (Δcrh, TLF39). Subsequent morphological and biochemical characterization of these mutants pinpoint the importance of agsE for maintaining cell wall stability and suggesting its potential influence on protein production and/or secretion. These findings therefor provide not only new insights into fungal biology but also potential targets for biotechnological applications