Biotechnology and Bioengineering

Cover Legend Morphology and phenotypic characteristics of cardiomyocytes on 2D Chitosan-FN film. This figure depicts cardiomyocytes cocultured with 3T3-J2 fibroblasts. (Image courtesy of Dr. Cheul H. Cho.)

The glutamine substrates of transglutaminase (TG) from Streptomyces mobaraensis can be rapidly and efficiently screened using mRNA display. Results demonstrate the screened substrates are short, specific and highly reactive for TG from Streptomyces mobaraensis. This method shows the significant potential to investigate the subsite specificities of unknown TGs and to screen their specific substrates.

A three-component P450pyrTM monooxygenase was successfully co-expressed with a GDH in E. coli as an efficient whole-cell biocatalyst for highly regio- and stereoselective hydroxylation of alicyclic substrates at non-activated carbon atoms. Enhanced productivity resulted via intracellular recycling of NAD(P)H, providing simple and useful syntheses of several alicyclic alcohols in high ee that are valuable pharmaceutical intermediates and difficult to prepare by other methods. E. coli (P450pyrTM-GDH) represents the most productive system known thus far for enhancing P450-catalyzed hydroxylations via cofactor recycling.

Peroxidase (HRP) was immobilized on dimyristoylphosphatidylcholine (DMPC) bilayers supported on Au (111) by dithiotreitol (DTT) self-assembled monolayers. The presence of white spots on immuno-gold labeled samples in the atomic force microscopy (AFM) images confirms the immobilization of HRP on lipid bilayers. Square-wave voltammetry (SWV) experiments were performed to investigate the performance of the HRP–DMPC bilayer system as a nanostructured electrochemical biosensor. The present results indicate that the system can catalyze the redox reaction of dopamine efficiently.

Cytochrome P450 monooxygenases are multi-functional enzymes with widespread applications in chemoenzymatic synthesis of complex chemicals as well as in studies of metabolism and xenobiotics. Lee and coworkers performed a visible light-driven photoenzymatic O-dealkylation reaction by using a cell-free synthesized P450 BM3 monooxygenase variant (BM3m2). A sustainable photoenzymatic reaction of the P450 BM3 was performed through visible light-driven regeneration of NADPH from NADPH. The P450 biocatalysis, coupled with photochemical cofactor regeneration, suggests an excellent strategy for visible a light-driven selective oxidation reaction.

Enhanced biological phosphorus removal (EBPR) from wastewater was obtained with methanol as a sole carbon source using a syntrophic consortium of acetogens and PAO. Methanol-degrading acetogens survive under conventional anaerobic/aerobic EPBR configuration, which favors PAO coexistence and avoids methanogenic activity. By contrast, a propionate-fed PAO-enriched sludge didn't survive with methanol as a sole carbon source due to the lack of acetogenic VFA production from methanol.

Binding characteristics of a bacterial expansin (BsEXLX1) to microcrystalline cellulose were analyzed in comparison with those of CtCBD3 that is a Type A carbohydrate-binding module. Binding parameters of the Langmuir isotherm of BsEXLX1 to Avicel, binding competition between BsEXLX1 and CtCBD3 for Avicel, and lack of binding activity to cellooligosaccharides revealed that the binding behavior of BsEXLX1 follows that of Type A CBMs. However, the binding capacities of cellulose and xylans for BsEXLX1 were significantly lower than those for CtCBD3.

A heterogeneously expressed synthetic porin OprF dramatically increased the membrane permeability of E. coli, resulting in a remarkable decrease in charge-transfer resistance, enhancement in extracellular electron transfer (EET). The oprF-expression strain can efficiently use riboflavin as the electron shuttle, however, its parental strain cannot. Thus, the oprF-expression strain delivered a much higher current output than its parent strain. The results substantiated the observation that membrane permeability can determine the usage spectrum of electron shuttles and the EET efficiency.

The structural determination of membrane proteins is still a huge challenge in structural biology, as it is often difficult to isolate them in a native form. There is an urgent need to set up efficient refolding processes to circumvent such a problem. This contribution describes how a method, based on the association of a detergent (SDS) and 2-methyl-2,4-pentanediol (MPD) was able to refold a trimeric membrane protein. The efficiency of our technique is demonstrated and the results also confirm its transferability.

This study evaluates the technical feasibility for continuous large scale production of fine chemicals, such as n-octanol and (S)-styrene oxide, with engineered catalytic biofilms. Based on results obtained in a micro-scale membrane reactor for the here described selective alkane hydroxylation and a previously reported asymmetric styrene epoxidation, process parameters for a 1,000 tons/year production scale are presented. The results indicate that this biofilm application can be a sustainable alternative compared to traditional suspended cells processes in terms of product yield and biomass waste production.

A virtual microbial colony, the growth of which is directed by a hyphal density field (red) and nutrient density field (green) was studied. The branching frequency of hyphae within a population of such colonies may be accurately determined through fractal analysis.

A detailed study of the spectral differences and similarities of 26 proteins underlined the usefulness of spectral data for high throughput selective protein quantification. A measure of spectral similarity was defined and shown to correlate with the precision of selective protein quantification in protein mixtures based on their absorption spectra. Knowledge of this correlation can be used to estimate the precision of the method for a certain protein combination a priori.

Ralstonia eutropha cells produce an intracellular biopolymer (bio-based, biodegradable plastic) when grown on plant oils, such as palm oil. In this work, the authors recover and purify polymer from bacterial cells using solvents that are safer and more environmentally friendly than chloroform. In addition, the authors find that they have the ability to separate polymers with different monomer compositions using solvents methyl isobutyl ketone (MIBK, pictured) and butyl acetate.

Eight different promoter/5′UTR combinations were cloned, introduced into Agrobacterium tumefaciens and used for transient expression of the fluorescent reporter protein DsRed in different leaves of Nicotiana tabacum. The resulting expression profiles were compiled to a predictive model using a design of experiments approach.

Aggregation of baby hamster kidney (BHK) cells cultivated in perfusion mode was studied by comparing expression profiles of 84 genes in the extracellular adhesion molecules (ECM) pathway by qRT-PCR. In aggregated cultures, 4 genes (COL1A1, COL4A1 THBS2, and VCAN) were consistently up-regulated while 2 (NCAM1 and THBS1) were consistently down-regulated. Based on the differential gene expression results, two mechanistic models were proposed for cellular aggregation. The first suggested cell aggregation was mediated by secreted thrombospondin, while collagen and fibronectin proteins, which have affinity for one another as well as binding sites for cells in culture, were implicated in the second model.

Anion exchange membrane adsorbers are used in the biopharmaceutical industry for clearance of host cell proteins, DNA and virus particles during the manufacture of monoclonal antibodies. Strong anion exchange ligands, which contain quaternary ammonium groups, are ineffective at high ionic strength. Primary amine based ligands are much less sensitive to ionic strength. However they are sensitive to the presence of other ionic species such as phosphate ions. The performance of primary amine based ligands is much more difficult to predict.

Anion exchange membrane adsorbers are used in the biopharmaceutical industry for clearance of host cell proteins, DNA and viruses. While the effectiveness of primary amine based ligands is not compromised at high ionic strength, the presence of hydrogen phosphate ions leads to reduced capacity. Monoclonal antibody recovery using primary amine based anion-exchange ligands may be lower if significant binding occurs due to secondary interactions. The removal of a specific contaminant is affected by the level of removal of the other contaminants.

Klebsiella pneumoniae showed potential to produce 3-hydroxypropionic acid (3-HP) in a significant amount. Carbon flux to 3-HP was critically controlled by oxygen in the opposite direction at two important nodes of glycerol (down) and 3-hydroxypropanealdehyde (up). Controlled aeration was necessary to produce 3-HP at a high amount. Recombinant strain KpBCΔdhaTΔyqhD overexpressing glycerol dehydratase and aldehyde dehydrogenase produced >28 g/L 3-HP at 5% DO in a bioreactor.

In this study, the authors characterized and compared the passive detachment of monoculture Nitrosomonas europaea and Pseudmonas aeruginosa biofilms grown in hollow fiber membrane bioreactors. A unique set of non-invasive experimental methods was used to quantify the correlation between biofilm physiology (surface morphology and metabolism), fluid shear, and detachment.

Klein, Schneider, and Heinzle describe a system of 8 parallel bioreactors for continuous cultivation with only 10 mL working volume. Non-invasive optical measurement of dissolved oxygen ensures aerobic cultivation conditions and is a strong indicator of steady state. Stirring of the bioreactors improves oxygen transfer and prevents sedimentation of cells. Mass spectrometric off gas analysis permits determination of O₂ uptake and CO₂ production rates and further allows complete carbon balancing. Biological validation has been performed by cultivating fission yeast under various physiological conditions.

A high throughput method for the screening and selection of Aspergillus niger transformants expressing high yields of five recombinant laccase-like multicopper oxidases was used to assess the effect of different mRNA 5′ untranslated regions (5′UTRs) in extracellular protein production. The results obtained show that modifications in the 5′UTR of the widely used A. niger glaA expression system can lead to improvements in protein yields.

Chitosan–alginate capsules were formed and subsequently loaded with an AI-2 synthase “nanofactory” to modulate QS activity in Escherichia coli. We propose that biologically inspired carriers of molecular synthesis systems in native settings can modulate bacterial populations, including those of the human microbiome.

Differences between 2D and 3D cell culture systems has been previously reported. Cells cultured in 3D interact and remodel their microenvironment and can develop into complex structures that resemble the observed in vivo phenotypes. Cells cultured in 2D are not always able to recapitulate them, yet 2D cell culture is considered to be a standard technique for cellular analysis, and is a simpler technique for other applications (i.e., therapeutic compound screening). Bellis and coworkers have employed their new technique to determine some of the cellular signaling processes that might be leading to the observed differences between 2D and 3D cell culture outcomes. Their developed transcription factor (TF) activity array uses bioluminescence imaging (BLI) that can monitor the cellular processes for longer culture periods of time while the cells are alive. The array captured expected changes in TF activity to stimuli, and it also provided dynamic profiles within 2D and 3D that have not been previously characterized. The development of the technology to dynamically track TF activity within cells cultured in both 2D and 3D can provide greater understanding of complex cellular processes, such a tissue development and disease progression.

By applying reduction steps to the culture redox potential (CRP in mV) the authors investigated metabolic and regulatory events responsible for the microaerobic formation of highly pigmented photosynthetic membranes (PM)in R. rubrum. As indicated by the photographs, the purple color of these bacteria reflects the amount of membranes produced. The authors argue that the available electron source, the activity of the terminal oxidases and the direction of the TCA cycle alter the intracellular redox state and thus influence PM production in R. rubrum.

The authors have combined a layer-by-layer polyelectrolyte multilayer deposition with a micro-molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days.

The authors develop a microfluidic biochip for liver renal tissues coculture. Liver HepaRG cells metabolize ifosfamide drugs whereas MDCK renal cells did not. Calcium release is enhanced in renal cells in the ifosfamide coculture demonstrating “systemic-like interaction” between liver and kidney cells.

The surface of devitalized cortical bone is modified with chitosan-heparin polyelectrolyte multilayers. These biomimetic periosteum coatings are characterized spectroscopically, and shown to support the adhesion and proliferation of mesenchymal stem cells, while suppressing both Gram positive (S. aureus) and Gram negative (E. coli) bacteria. These coatings have potential as synthetic periosteum to improve the outcomes of cortical bone allografts.

A three-dimensional in vitro cell culture chamber was designed, built using micro-fabrication techniques and validated. This cell culture system allowed study of the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cells insulin secretion. Sabra and Vermette report the importance of the separation distance between two cell niches for cell culture design and the possibility to enhance beta cells function.

In this study, the authors fabricate bioactive 3-D chitosan nanofiber scaffolds using an electrospinning technique and explore its potential for long-term cardiac function in the 3-D co-culture model. The results suggest that the cardiac co-culture model is a promising system for the maintenance of long-term survival and function of cardiomyocytes. The engineered 3-D cardiac co-culture model will be useful for the design and improvement of engineered tissues for the repair of myocardial infarcts, tissue engineering applications, and drug testing.

Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated cultures limiting robust scale-up of cell production, on-line control and optimization of culture conditions. Baptista, Fluri, and Zandstra demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen-controlled perfusion culture. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable, and intensified stirred suspension culture.

Overexpressing enzymes from a heterologous de novo vanillin biosynthesis pathway in baker's yeast leads to increased vanillin production only when combined with model-guided metabolic engineering. This study illustrates how the production of valuable chemicals can be improved through a synergistic approach, which combines metabolic engineering strategies emerging from both local pathway-based and global network-based analyses.

The authors studied the metabolism of CHO cells before and after a metabolic switch from lactate production to consumption when glucose is depleted. The activity of TCA cycle remained the same despite the switch in carbon source, but the fraction of acetyl-CoA catabolized to CO₂ increased from 3% to 32%. The metabolic model in this study was derived from a mouse genome scale model. Despite the model being underdetermined, the estimated flux ranges were sufficiently constrained to allow meaningful comparative analyses.