Probing cell proliferation: Considerations for dye selection.

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

Facile fabrication of temperature/pH dual sensitive hydrogels based on cellulose and polysuccinimide through aqueous amino-succinimide reaction.

A temperature/pH dual sensitive hydrogel with a semi-interpenetrating network (semi-IPN) structure was synthesized through an aqueous amino-succinimide reaction between water-soluble polysuccinimide and polyethyleneimine in the presence of thermosensitive cellulose derivatives. Single-factor experiments were carried out to optimize the preparation conditions of the semi-IPN hydrogel. The swelling behavior and cytotoxicity assay of the hydrogel were tested. Finally, taking 5- fluorouracil (5-Fu) as a model drug, the release performance of the 5-Fu-loaded hydrogel was investigated. The results indicated that the swelling ratio (SR) first decreased and then increased when the pH of the solutions ascended from 2 to 10. The SR decreased with the increase in temperature. In addition, the swelling behavior of the hydrogel was reversible and reproducible under different pH values and temperatures. The prepared hydrogels had good cytocompatibility. The release behavior of 5-Fu was most consistent with the Korsmeyer-Peppas model and followed the case II diffusion. The acidic environment was beneficial for the release of 5-Fu. The preparation process of the semi-IPN hydrogel is simple and the reaction can proceed quickly in water. The strategy introduced here has great potential for application in the preparation of drug carriers.

In situ Raman spectral observation of succinimide intermediates in amyloid fibrillation kinetics.

Succinimide intermediates play the crucial role in the nucleation process for protein amyloid fibril formation, as they can usually induce a non-native conformation in a fraction of soluble proteins to render amyloidogenicity and neurotoxicity. Thus, in situ detection of succinimide intermediates during amyloid fibrillation kinetics is of considerable importance, albeit challenging, because these succinimides are generally unstable in physiological conditions. Here, we found an in situ Raman spectral fingerprint to trace the succinimide intermediates in amyloid fibril formation, wherein the carbonyl symmetric stretching of cyclic imide in the succinimide derivative is located at ca. 1790 cm-1. Using its intensity as an indicator of succinimide intermediates, we have in situ detected and unravelled the role of succinimide intermediates during the oligomer formation from the Bz-Asp-Gly-NH2 dipeptide or the amyloid fibrillation kinetics of lysozyme with thermal/acid treatment.

Discovery and Control of Succinimide Formation and Accumulation at Aspartic Acid Residues in The Complementarity-Determining Region of a Therapeutic Monoclonal Antibody.

PURPOSE: Succinimide formation and isomerization alter the chemical and physical properties of aspartic acid residues in a protein. Modification of aspartic acid residues within complementarity-determining regions (CDRs) of therapeutic monoclonal antibodies (mAbs) can be particularly detrimental to the efficacy of the molecule. The goal of this study was to characterize the site of succinimide accumulation in the CDR of a therapeutic mAb and understand its effects on potency. Furthermore, we aimed to mitigate succinimide accumulation through changes in formulation. METHODS: Accumulation of succinimide was identified through intact and reduced LC-MS mass measurements. A low pH peptide mapping method was used for relative quantitation and localization of succinimide formation in the CDR. Statistical modeling was used to correlate levels of succinimide with basic variants and potency measurements. RESULTS: Succinimide accumulation in Formulation A was accelerated when stored at elevated temperatures. A strong correlation between succinimide accumulation in the CDR, an increase in basic charge variants, and a decrease in potency was observed. Statistical modeling suggest that a combination of ion exchange chromatography and potency measurements can be used to predict succinimide levels in a given sample. Reformulation of the mAb to Formulation B mitigates succinimide accumulation even after extended storage at elevated temperatures. CONCLUSION: Succinimide formation in the CDR of a therapeutic mAb can have a strong negative impact on potency of the molecule. We demonstrate that thorough characterization of the molecule by LC-MS, ion exchange chromatography, and potency measurements can facilitate changes in formulation that mitigate succinimide formation and the corresponding detrimental changes in potency.

Restoring the biological activity of crizanlizumab at physiological conditions through a pH-dependent aspartic acid isomerization reaction.

In this study, we report the isomerization of an aspartic acid residue in the complementarity-determining region (CDR) of crizanlizumab as a major degradation pathway. The succinimide intermediate and iso-aspartic acid degradation products were successfully isolated by ion exchange chromatography for characterization. The isomerization site was identified at a DG motif in the CDR by peptide mapping. The biological characterization of the isolated variants showed that the succinimide variant exhibited a loss in target binding and biological activity compared to the aspartic acid and iso-aspartic acid variants of the molecule. The influence of pH on this isomerization reaction was investigated using capillary zone electrophoresis. Below pH 6.3, the succinimide formation was predominant, whereas at pH values above 6.3, iso-aspartic acid was formed and the initial amounts of succinimide dropped to levels even lower than those observed in the starting material. Importantly, while the succinimide accumulated at long-term storage conditions of 2 to 8°C at pH values below 6.3, a complete hydrolysis of succinimide was observed at physiological conditions (pH 7.4, 37°C), resulting in full recovery of the biological activity. In this study, we demonstrate that the critical quality attribute succinimide with reduced potency has little or no impact on the efficacy of crizanlizumab due to the full recovery of the biological activity within a few hours under physiological conditions.

Fully automated 18F-fluorination of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) for indirect labelling of nanobodies.

N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), a widely used labeling agent to introduce the 4-[18F]fluorobenzoyl-prosthetic group, is normally obtained in three consecutive steps from [18F]fluoride ion. Here, we describe an efficient one-step labeling procedure of [18F]SFB starting from a tin precursor. This method circumvents volatile radioactive side-products and simplifies automatization. [18F]SFB was obtained after HPLC purification in a yield of 42 + 4% and a radiochemical purity (RCP) > 99% (n = 6). In addition, we investigate the automation of the coupling of [18F]SFB to a nanobody (cAbBcII10, targeting ß-lactamase enzyme) and purification by size exclusion chromatography (PD-10 desalting column) to remove unconjugated reagent. Production and use of [18F]SFB were implemented on a radiosynthesis unit (Neptis®). The fully automated radiosynthesis process including purification and formulation required 160 min of synthesis time. [18F]SFB-labeled nanobody was obtained in a yield of 21 + 2% (activity yield 12 + 1% non-decay corrected) and a radiochemical purity (RCP) of > 95% (n = 3). This approach simplifies [18F]SFB synthesis to one-step, enhances the yield in comparison to the previous report and enables the production of radiolabeled nanobody on the same synthesis module.

An Automatic Immunoaffinity Pretreatment of Deoxynivalenol Coupled with UPLC-UV Analysis.

An immunoaffinity magnetic beads (IMBs) based automatic pretreatment method was developed for the quantitative analysis of deoxynivalenol (DON) by ultra-performance liquid chromatography and ultraviolet detector (UPLC-UV). First, N-hydroxysuccinimide-terminated magnetic beads (NHS-MBs) with good magnetic responsivity and dispersibility were synthesized and characterized by optical microscopy, scanning electron microscopy (SEM), and laser diffraction-based particle size analyzer. Then, the amino groups of anti-DON monoclonal antibody (mAb) and the NHS groups of NHS-MBs were linked by covalent bonds to prepare IMB, without any activation reagent. The essential factors affecting the binding and elution of DON were meticulously tuned. Under optimal conditions, DON could be extracted from a real sample and eluted from IMB by water, enabling environmentally friendly and green analysis. Hence, there was no need for dilution or evaporation prior to UPLC-UV analysis. DON in 20 samples could be purified and concentrated within 30 min by the mycotoxin automated purification instrument (MAPI), allowing for automated, green, high-throughput and simple clean-up. Recoveries at four distinct spiking levels in corn and wheat ranged from 92.0% to 109.5% with good relative standard deviations (RSD, 2.1-7.0%). Comparing the test results of IAC and IMB in commercial samples demonstrated the reliability and superiority of IMB for quantitatively analyzing massive samples.

PIMT-Mediated Labeling of l-Isoaspartic Acid with Tris Facilitates Identification of Isomerization Sites in Long-Lived Proteins.

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate formed during reaction of l-isoAsp-containing peptides with PIMT and S-adenosyl methionine (SAM) is reactive with Tris base and results in a Tris-modified aspartic acid residue with a mass shift of +103 Da. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that Tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.

PEGylation and antioxidant effects of a human glutathione peroxidase 1 mutant.

Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, Mw = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo, and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo. Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.

Rapidly Self-Deactivating and Injectable Succinyl Ester-Based Bioadhesives for Postoperative Antiadhesion.

Postoperative adhesion not only causes severe complications for patients but also increases their economic burden. Injectable bioadhesives with adhesiveness to tissues can cover irregular wounds and stay stable in situ, which is a promising barrier for antiadhesion. However, the potential tissue adhesion caused by bioadhesives' indiscriminate adhesiveness between normal and wounded tissue is still a problem. Herein, by using poly(ethylene glycol) succinimidyl succinate (PEG-SS) and gelatin, a succinyl ester-based bioadhesive (SEgel) was fabricated with self-deactivating properties for postoperative antiadhesion. Because N-hydroxysuccinimide esters (NHS-esters) were used as the adhesive group, the bioadhesives' side in contact with the tissue built covalent anchors quickly to maintain the stability, but the superficial layer facing outward withstood fast hydrolysis and then lost its adhesion within minutes, avoiding the indiscriminate adhesiveness. In addition, because of the specific degradation behavior of succinyl ester, the SEgel with proper in vivo retention was achieved without the worry of causing foreign body reactions and unexpected tissue adhesion. Both the cecum-sidewall adhesion and hepatic adhesion models showed that the SEgel markedly reduced the severity of tissue adhesion. These results, together with the ease of the preparation process and well-proven biocompatibility of raw materials, revealed that the SEgel might be a promising solution for postoperative antiadhesion.

Near-Infrared Fluorescence Imaging of Carotid Plaques in an Atherosclerotic Murine Model.

Successful imaging of atherosclerosis, one of the leading global causes of death, is crucial for diagnosis and intervention. Near-infrared fluorescence (NIRF) imaging has been widely adopted along with multimodal/hybrid imaging systems for plaque detection. We evaluate two macrophage-targeting fluorescent tracers for NIRF imaging (TLR4-ZW800-1C and Feraheme-Alexa Fluor 750) in an atherosclerotic murine cohort, where the left carotid artery (LCA) is ligated to cause stenosis, and the right carotid artery (RCA) is used as a control. Imaging performed on dissected tissues revealed that both tracers had high uptake in the diseased vessel compared to the control, which was readily visible even at short exposure times. In addition, ZW800-1C's renal clearance ability and Feraheme's FDA approval puts these two tracers in line with other NIRF tracers such as ICG. Continued investigation with these tracers using intravascular NIRF imaging and larger animal models is warranted for clinical translation.

In vivo biocompatibility evaluation of in situ-forming polyethylene glycol-collagen hydrogels in corneal defects.

The available treatment options include corneal transplantation for significant corneal defects and opacity. However, shortage of donor corneas and safety issues in performing corneal transplantation are the main limitations. Accordingly, we adopted the injectable in situ-forming hydrogels of collagen type I crosslinked via multifunctional polyethylene glycol (PEG)-N-hydroxysuccinimide (NHS) for treatment and evaluated in vivo biocompatibility. The New Zealand White rabbits (N = 20) were randomly grouped into the keratectomy-only and keratectomy with PEG-collagen hydrogel-treated groups. Samples were processed for immunohistochemical evaluation. In both clinical and histologic observations, epithelial cells were able to migrate and form multilayers over the PEG-collagen hydrogels at the site of the corneal stromal defect. There was no evidence of inflammatory or immunological reactions or increased IOP for PEG-collagen hydrogel-treated corneas during the four weeks of observation. Immunohistochemistry revealed the presence of α-smooth muscle actin (α-SMA) in the superior corneal stroma of the keratectomy-only group (indicative of fibrotic healing), whereas low stromal α-SMA expression was detected in the keratectomy with PEG-collagen hydrogel-treated group. Taken together, we suggest that PEG-collagen may be used as a safe and effective alternative in treating corneal defect in clinical setting.

A rapid lateral flow immunoassay strip for detection of SARS-CoV-2 antigen using latex microspheres.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious and concealed virus that causes pneumonia, severe acute respiratory syndrome, and even death. Although the epidemic has been controlled since the development of vaccines and quarantine measures, many people are still infected, particularly in third-world countries. Several methods have been developed for detection of SARS-CoV-2, but owing to its price and efficiency, the immune strip could be a better method for the third-world countries. METHODS: In this study, two antibodies were linked to latex microspheres, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, as the bridge to decrease the cost further and improve the detection performance. The specificity of the lateral flow immunoassay strip (LFIA) was tested by several common viruses and respiratory bacterial infections. Besides, the reproducibility and stability of the LFIAs were tested on the same batch of test strips. Under optimal conditions, the sensitivity of LFIA was determined by testing different dilutions of the positive specimens. RESULTS: The proposed LFIAs were highly specific, and the limit of detection was as low as 25 ng/mL for SARS-CoV-2 antigens. The clinical applicability was evaluated with 659 samples (230 positive and 429 negative samples) by using both LFIA and rRT-PCR. Youden's index (J) was used to assess the performance of these diagnostic tests. The sensitivity and specificity were 98.22% and 97.93%, respectively, and J is 0.9615. The sensitivity and specificity were 98.22% and 97.93%, respectively, and J is 0.9615. In addition, the consistency of our proposed LFIA was analyzed using Cohen's kappa coefficient (κ = 0.9620). CONCLUSION: We found disease stage, age, gender, and clinical manifestations have only a slight influence on the diagnosis. Therefore, the lateral flow immunoassay SARS-CoV-2 antigen test strip is suitable for point-of-care detection and provides a great application for SARS-CoV-2 epidemic control in the third-world countries.

Synthesis and kinetic characterization of hyperbolic inhibitors of human cathepsins K and S based on a succinimide scaffold.

Cathepsins K and S are closely related papain-like cysteine peptidases and potential therapeutic targets for metabolic and inflammatory diseases such as osteoporosis and arthritis. Here we describe the reduction of a previously characterized succinimide (2,5-dioxopyrrolidine)-containing hyperbolic inhibitor of cathepsin K (methyl (RS)-N-[1-(4-methoxyphenyl)-2,5-dioxopyrrolidin-3-yl]glycinate), to obtain a better and more selective compound (compound 4a - methyl (2,5-dioxopyrrolidin-3-yl)glycinate), which acted as a hyperbolic mixed inhibitor/activator similar to already known allosteric effectors of cathepsin K. We then investigated the potential of the succinimide scaffold as inhibitors of cathepsins K and/or S and synthesized a library of such compounds by 1,4-addition of α-amino acid esters and related compounds to N-substituted maleimides. From the generated library, we identified the first small molecule hyperbolic inhibitors of cathepsin S (methyl ((R)-2,5-dioxopyrrolidin-3-yl)-l-threoninate (compound R-4c) and 3-{[(1S,2R,3'S)-2-hydroxycyclohexyl]amino}pyrrolidine-2,5-dione (compound (1S,2R,3'S-10)). The former acted via a similar mechanism to compound 4a, while the latter was a hyperbolic specific inhibitor of cathepsin S. Given the versatility of the scaffold, the identified compounds will be used as the basis for the development of high-affinity hyperbolic inhibitors of the individual peptidases and to explore the potential of hyperbolic inhibitors for the inhibition of cysteine cathepsins in in vitro models.

Chemoselective Installation of Diverse Succinimides on Fused Benzimidazoles via Rhodium-Catalyzed C-H Activation/Annulation: Chemosensor for Heavy Metals.

A novel Rh-catalyzed cascade C-H activation/annulation of 2-arylbenzimidazoles with maleimides is reported. Rapid chemoselective access to two structurally distinct succinimide-bearing benzoimidazoisoquinolinones is achieved, depending on the acidic and basic conditions. This atom- and step-economic strategy features a wide substrate scope, excellent functional group tolerance, and site-specific functionalization. Application of the methodology yields a novel benzimidazole-based probe as a fluorescent chemosensor for the nanomolar detection of Hg2+, Cu2+, and Fe3+ ions.

Label-retention expansion microscopy.

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

High-Affinity Points of Interaction on Antibody Allow Synthesis of Stable and Highly Functional Antibody-Gold Nanoparticle Conjugates.

Many emerging nanobiotechnologies rely on the proper function of proteins immobilized on gold nanoparticles. Often, the surface chemistry of the AuNP is engineered to control the orientation, surface coverage, and structure of the adsorbed protein to maximize conjugate function. Here, we chemically modified antibody to investigate the effect of protein surface chemistries on adsorption to AuNPs. A monoclonal anti-horseradish peroxidase IgG antibody (anti-HRP) was reacted with N-succinimidyl acrylate (NSA) or reduced dithiobissuccinimidyl propionate (DSP) to modify lysine residues. Zeta potential measurements confirmed that both chemical modifications reduced the localized regions of positive charge on the protein surface, while the DSP modification incorporated additional free thiols. Dynamic light scattering confirmed that native and chemically modified antibodies adsorbed onto AuNPs to form bioconjugates; however, adsorption kinetics revealed that the NSA-modified antibody required significantly more time to allow for the formation of a hard corona. Moreover, conjugates formed with the NSA-modified antibody lost antigen-binding function, whereas unmodified and DSP-modified antibodies adsorbed onto AuNPs to form functional conjugates. These results indicate that high-affinity functional groups are required to prevent protein unfolding and loss of function when adsorbed on the AuNP surface. The reduced protein charge and high-affinity thiol groups on the DSP-modified antibody enabled pH-dependent control of protein orientation and the formation of highly active conjugates at solution pHs (<7.5) that are inaccessible with unmodified antibody due to conjugate aggregation. This study establishes parameters for protein modification to facilitate the formation of highly functional and stable protein-AuNP conjugates.

Design, synthesis and biological evaluation of double fatty chain-modified glucagon-like peptide-1 conjugates.

Twelve double fatty chains and Aib8-Arg34-GLP-1 (7-37) were designed and obtained by microwave-assisted solid-phase synthesis. Then, twelve conjugates of Aib8-Arg34-GLP-1 (7-37) were synthesized in 1% triethylamine aqueous solution. Conjugates 2, 3, 6, 7, 10 and 11 showed better GLP-1 receptor activation potency than semaglutide. However, conjugates 2, 6 and 10 showed slightly worse glucose-lowering effects in vivo than semaglutide but better effects than conjugates 3, 7 and 11. The CD spectra of conjugates 2, 6 and 10 indicated that they had the same secondary structure as liraglutide and semaglutide. The receptor affinity results for conjugates 2, 6 and 10 measured by SPR (surface plasmon resonance) showed that conjugate 2 had higher receptor affinity than conjugates 6 and 10. In addition, albumin binding assays indicated that double fatty acid chains had obvious synergistic effects compared with single fatty acid chains. In conclusion, the structure-activity relationship of different side chains was summarized and one candidate, conjugate 2, was screened.

Ex vivo evaluation of a multilayered sealant patch for watertight dural closure: cranial and spinal models.

Cerebrospinal fluid leakage is a frequent complication after cranial and spinal surgery. To prevent this complication and seal the dura watertight, we developed Liqoseal, a dural sealant patch comprising a watertight polyesterurethane layer and an adhesive layer consisting of poly(DL-lactide-co-ε-caprolactone) copolymer and multiarmed N-hydroxylsuccinimide functionalized polyethylene glycol. We compared acute burst pressure and resistance to physiological conditions for 72 h of Liqoseal, Adherus, Duraseal, Tachosil, and Tisseel using computer-assisted models and fresh porcine dura. The mean acute burst pressure of Liqoseal in the cranial model (145 ± 39 mmHg) was higher than that of Adherus (87 ± 47 mmHg), Duraseal (51 ± 42 mmHg) and Tachosil (71 ± 16 mmHg). Under physiological conditions, cranial model resistance test results showed that 2 of 3 Liqoseal sealants maintained dural attachment during 72 hours as opposed to 3 of 3 for Adherus and Duraseal and 0 of 3 for Tachosil. The mean burst pressure of Liqoseal in the spinal model (233 ± 81 mmHg) was higher than that of Tachosil (123 ± 63 mmHg) and Tisseel (23 ± 16 mmHg). Under physiological conditions, spinal model resistance test results showed that 2 of 3 Liqoseal sealants maintained dural attachment for 72 hours as opposed to 3 of 3 for Adherus and 0 of 3 for Duraseal and Tachosil. This novel study showed that Liqoseal is capable of achieving a strong watertight seal over a dural defect in ex vivo models.

Internal Illumination to Overcome the Cell Density Limitation in the Scale-up of Whole-Cell Photobiocatalysis.

Cyanobacteria have the capacity to use photosynthesis to fuel their metabolism, which makes them highly promising production systems for the sustainable production of chemicals. Yet, their dependency on visible light limits the cell-density, which is a challenge for the scale-up. Here, it was shown with the example of a light-dependent biotransformation that internal illumination in a bubble column reactor equipped with wireless light emitters (WLEs) could overcome this limitation. Cells of the cyanobacterium Synechocystis sp. PCC 6803 expressing the gene of the ene-reductase YqjM were used for the reduction of 2-methylmaleimide to (R)-2-methylsuccinimide with high optical purity (>99 % ee). Compared to external source of light, illumination by floating wireless light emitters allowed a more than two-fold rate increase. Under optimized conditions, product formation rates up to 3.7 mm h-1 and specific activities of up to 65.5 U gDCW -1 were obtained, allowing the reduction of 40 mm 2-methylmaleimide with 650 mg isolated enantiopure product (73 % yield). The results demonstrate the principle of internal illumination as a means to overcome the intrinsic cell density limitation of cyanobacterial biotransformations, obtaining high reaction rates in a scalable photobioreactor.