Design of a stable ethanologenic bacterial strain without heterologous plasmids and antibiotic resistance genes for efficient ethanol production from concentrated dairy waste.

Engineering sustainable bioprocesses that convert abundant waste into fuels is pivotal for efficient production of renewable energy. We previously engineered an Escherichia coli strain for optimized bioethanol production from lactose-rich wastewater like concentrated whey permeate (CWP), a dairy effluent obtained from whey valorization processes. Although attractive fermentation performances were reached, significant improvements are required to eliminate recombinant plasmids, antibiotic resistances and inducible promoters, and increase ethanol tolerance. Here, we report a new strain with chromosomally integrated ethanologenic pathway under the control of a constitutive promoter, without recombinant plasmids and resistance genes. The strain showed extreme stability in 1-month subculturing, with CWP fermentation performances similar to the ethanologenic plasmid-bearing strain. We then investigated conditions enabling efficient ethanol production and sugar consumption by changing inoculum size and CWP concentration, revealing toxicity- and nutritional-related bottlenecks. The joint increase of ethanol tolerance, via adaptive evolution, and supplementation of small ammonium sulphate amounts (0.05% w/v) enabled a fermentation boost with 6.6% v/v ethanol titer, 1.2 g/L/h rate, 82.5% yield, and cell viability increased by three orders of magnitude. Our strain has attractive features for industrial settings and represents a relevant improvement in the existing ethanol production biotechnologies.

CRISPR Interference Modules as Low-Burden Logic Inverters in Synthetic Circuits.

CRISPR and CRISPRi systems have revolutionized our biological engineering capabilities by enabling the editing and regulation of virtually any gene, via customization of single guide RNA (sgRNA) sequences. CRISPRi modules can work as programmable logic inverters, in which the dCas9-sgRNA complex represses a target transcriptional unit. They have been successfully used in bacterial synthetic biology to engineer information processing tasks, as an alternative to the traditionally adopted transcriptional regulators. In this work, we investigated and modulated the transfer function of several model systems with specific focus on the cell load caused by the CRISPRi logic inverters. First, an optimal expression cassette for dCas9 was rationally designed to meet the low-burden high-repression trade-off. Then, a circuit collection was studied at varying levels of dCas9 and sgRNAs targeting three different promoters from the popular tet, lac and lux systems, placed at different DNA copy numbers. The CRISPRi NOT gates showed low-burden properties that were exploited to fix a high resource-consuming circuit previously exhibiting a non-functional input-output characteristic, and were also adopted to upgrade a transcriptional regulator-based NOT gate into a 2-input NOR gate. The obtained data demonstrate that CRISPRi-based modules can effectively act as low-burden components in different synthetic circuits for information processing.

Engineering endogenous fermentative routes in ethanologenic Escherichia coli W for bioethanol production from concentrated whey permeate.

Whey permeate (WP) is a lactose-rich waste effluent, generated during cheese manufacturing and further valorization steps, such as protein extraction. The production of ethanol by WP fermentation has been proposed to increase cost-competitiveness of dairy waste processing. In previous work, the Escherichia coli W strain was selected for its efficient growth in dairy waste and it was engineered to convert lactose into ethanol as the main fermentation product from WP and concentrated WP (CWP). To improve its performance, here the lactate dehydrogenase, fumarate reductase and pyruvate formate lyase fermentative routes were disrupted, obtaining new deletion strains. In test tubes, growth and fermentation profiles obtained in standard laboratory media and CWP showed large differences, and were affected by oxygen, medium and ethanologenic gene expression level. Among the tested strains, the one with triple deletion was superior in both high-oxygen and low-oxygen test tube fermentations, in terms of ethanol titer, rate and yield. The improved performance was due to a lower inhibition by medium acidification rather than an improved ethanol flux. The parent and triple deletion strains showed similar performance indexes in pH-controlled bioreactor experiments. However, the deletion strain showed lower base consumption and residual waste, in terms of both dry matter and chemical oxygen demand after distillation. It thus represents a step towards sustainable dairy wastewater valorization for bioenergy production by decreasing process operation costs.

A Synthetic Close-Loop Controller Circuit for the Regulation of an Extracellular Molecule by Engineered Bacteria.

Feedback control is ubiquitous in biological systems. It can also play a crucial role in the design of synthetic circuits implementing novel functions in living systems, to achieve self-regulation of gene expression, noise reduction, rise time decrease, or adaptive pathway control. Despite in vitro, in vivo, and ex vivo implementations have been successfully reported, the design of biological close-loop systems with quantitatively predictable behavior is still a major challenge. In this work, we tested a model-based bottom-up design of a synthetic close-loop controller in engineered Escherichia coli, aimed to automatically regulate the concentration of an extracellular molecule, N-(3-oxohexanoyl)-L-homoserine lactone (HSL), by rewiring the elements of heterologous quorum sensing/quenching networks. The synthetic controller was successfully constructed and experimentally validated. Relying on mathematical model and experimental characterization of individual regulatory parts and enzymes, we evaluated the predictability of the interconnected system behavior in vivo. The culture was able to reach an HSL steady-state level of 72 nM, accurately predicted by the model, and showed superior capabilities in terms of robustness against cell density variation and disturbance rejection, compared with a corresponding open-loop circuit. This engineering-inspired design approach may be adopted for the implementation of other close-loop circuits for different applications and contribute to decreasing trial-and-error steps.

The effect of pulsed electromagnetic field exposure on osteoinduction of human mesenchymal stem cells cultured on nano-TiO2 surfaces.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by interfering with selective calcium-related osteogenic pathways, and greatly enhanced hBM-MSCs osteogenic features such as the expression of early/late osteogenic genes and protein production (e.g., ALP, COL-I, osteocalcin and osteopontin) and ALP activity. Finally, PEMF-treated cells resulted to secrete into conditioned media higher amounts of BMP-2, DCN and COL-I than untreated cell cultures. These findings confirm once more the osteoinductive potential of PEMF, suggesting that its combination with TiO2 nanostructured surface might be a great option in bone tissue engineering applications.

Evaluation of Poly(Lactic-co-glycolic) Acid Alone or in Combination with Hydroxyapatite on Human-Periosteal Cells Bone Differentiation and in Sinus Lift Treatment.

Most recent advances in tissue engineering in the fields of oral surgery and dentistry have aimed to restore hard and soft tissues. Further improvement of these therapies may involve more biological approaches and the use of dental tissue stem cells in combination with inorganic/organic scaffolds. In this study, we analyzed the osteoconductivity of two different inorganic scaffolds based on poly (lactic-co-glycolic) acid alone (PLGA-Fisiograft) or in combination with hydroxyapatite (PLGA/HA-Alos) in comparison with an organic material based on equine collagen (PARASORB Sombrero) both in vitro and in vivo. We developed a simple in vitro model in which periosteum-derived stem cells were grown in contact with chips of these scaffolds to mimic bone mineralization. The viability of cells and material osteoconductivity were evaluated by osteogenic gene expression and histological analyses at different time points. In addition, the capacity of scaffolds to improve bone healing in sinus lift was examined. Our results demonstrated that the osteoconductivity of PLGA/HA-Alos and the efficacy of scaffolds in promoting bone healing in the sinus lift were increased. Thus, new clinical approaches in sinus lift follow-up should be considered to elucidate the clinical potential of these two PLGA-based materials in dentistry.

Re-using biological devices: a model-aided analysis of interconnected transcriptional cascades designed from the bottom-up.

BACKGROUND: The study of simplified, ad-hoc constructed model systems can help to elucidate if quantitatively characterized biological parts can be effectively re-used in composite circuits to yield predictable functions. Synthetic systems designed from the bottom-up can enable the building of complex interconnected devices via rational approach, supported by mathematical modelling. However, such process is affected by different, usually non-modelled, unpredictability sources, like cell burden. METHODS: Here, we analyzed a set of synthetic transcriptional cascades in Escherichia coli. We aimed to test the predictive power of a simple Hill function activation/repression model (no-burden model, NBM) and of a recently proposed model, including Hill functions and the modulation of proteins expression by cell load (burden model, BM). To test the bottom-up approach, the circuit collection was divided into training and test sets, used to learn individual component functions and test the predicted output of interconnected circuits, respectively. RESULTS: Among the constructed configurations, two test set circuits showed unexpected logic behaviour. Both NBM and BM were able to predict the quantitative output of interconnected devices with expected behaviour, but only the BM was also able to predict the output of one circuit with unexpected behaviour. Moreover, considering training and test set data together, the BM captures circuits output with higher accuracy than the NBM, which is unable to capture the experimental output exhibited by some of the circuits even qualitatively. Finally, resource usage parameters, estimated via BM, guided the successful construction of new corrected variants of the two circuits showing unexpected behaviour. CONCLUSIONS: Superior descriptive and predictive capabilities were achieved considering resource limitation modelling, but further efforts are needed to improve the accuracy of models for biological engineering.

In Vitro and In Vivo Studies of Alar-Nasal Cartilage Using Autologous Micro-Grafts: The Use of the Rigenera® Protocol in the Treatment of an Osteochondral Lesion of the Nose.

Cartilage defects represent a serious problem due to the poor regenerative properties of this tissue. Regarding the nose, nasal valve collapse is associated with nasal blockage and persistent airway obstruction associated with a significant drop in the quality of life for patients. In addition to surgical techniques, several cell-based tissue-engineering strategies are studied to improve cartilage support in the nasal wall, that is, to ameliorate wall insufficiency. Nevertheless, there are no congruent data available on the benefit for patients during the follow-up time. In this manuscript, we propose an innovative approach in the treatment of cartilage defects in the nose (nasal valve collapse) based on autologous micro-grafts obtained by mechanical disaggregation of a small portion of cartilage tissue (Rigenera® protocol). In particular, we first analyzed in vitro murine and human cartilage micro-grafts; secondly, we analyzed the clinical results of a patient with pinched nose deformity treated with autologous micro-grafts of chondrocytes obtained by Rigenera® protocol. The use of autologous micro-graft produced promising results in surgery treatment of cartilage injuries and could be safely and easily administrated to patients with cartilage tissue defects.

Fermentation of lactose to ethanol in cheese whey permeate and concentrated permeate by engineered Escherichia coli.

BACKGROUND: Whey permeate is a lactose-rich effluent remaining after protein extraction from milk-resulting cheese whey, an abundant dairy waste. The lactose to ethanol fermentation can complete whey valorization chain by decreasing dairy waste polluting potential, due to its nutritional load, and producing a biofuel from renewable source at the same time. Wild type and engineered microorganisms have been proposed as fermentation biocatalysts. However, they present different drawbacks (e.g., nutritional supplements requirement, high transcriptional demand of recombinant genes, precise oxygen level, and substrate inhibition) which limit the industrial attractiveness of such conversion process. In this work, we aim to engineer a new bacterial biocatalyst, specific for dairy waste fermentation. RESULTS: We metabolically engineered eight Escherichia coli strains via a new expression plasmid with the pyruvate-to-ethanol conversion genes, and we carried out the selection of the best strain among the candidates, in terms of growth in permeate, lactose consumption and ethanol formation. We finally showed that the selected engineered microbe (W strain) is able to efficiently ferment permeate and concentrated permeate, without nutritional supplements, in pH-controlled bioreactor. In the conditions tested in this work, the selected biocatalyst could complete the fermentation of permeate and concentrated permeate in about 50 and 85 h on average, producing up to 17 and 40 g/l of ethanol, respectively. CONCLUSIONS: To our knowledge, this is the first report showing efficient ethanol production from the lactose contained in whey permeate with engineered E. coli. The selected strain is amenable to further metabolic optimization and represents an advance towards efficient biofuel production from industrial waste stream.

Exploring Wound-Healing Genomic Machinery with a Network-Based Approach.

The molecular mechanisms underlying tissue regeneration and wound healing are still poorly understood despite their importance. In this paper we develop a bioinformatics approach, combining biology and network theory to drive experiments for better understanding the genetic underpinnings of wound healing mechanisms and for selecting potential drug targets. We start by selecting literature-relevant genes in murine wound healing, and inferring from them a Protein-Protein Interaction (PPI) network. Then, we analyze the network to rank wound healing-related genes according to their topological properties. Lastly, we perform a procedure for in-silico simulation of a treatment action in a biological pathway. The findings obtained by applying the developed pipeline, including gene expression analysis, confirms how a network-based bioinformatics method is able to prioritize candidate genes for in vitro analysis, thus speeding up the understanding of molecular mechanisms and supporting the discovery of potential drug targets.

Emerging Perspectives in Scaffold for Tissue Engineering in Oral Surgery.

Bone regeneration is currently one of the most important and challenging tissue engineering approaches in regenerative medicine. Bone regeneration is a promising approach in dentistry and is considered an ideal clinical strategy in treating diseases, injuries, and defects of the maxillofacial region. Advances in tissue engineering have resulted in the development of innovative scaffold designs, complemented by the progress made in cell-based therapies. In vitro bone regeneration can be achieved by the combination of stem cells, scaffolds, and bioactive factors. The biomimetic approach to create an ideal bone substitute provides strategies for developing combined scaffolds composed of adult stem cells with mesenchymal phenotype and different organic biomaterials (such as collagen and hyaluronic acid derivatives) or inorganic biomaterials such as manufactured polymers (polyglycolic acid (PGA), polylactic acid (PLA), and polycaprolactone). This review focuses on different biomaterials currently used in dentistry as scaffolds for bone regeneration in treating bone defects or in surgical techniques, such as sinus lift, horizontal and vertical bone grafts, or socket preservation. Our review would be of particular interest to medical and surgical researchers at the interface of cell biology, materials science, and tissue engineering, as well as industry-related manufacturers and researchers in healthcare, prosthetics, and 3D printing, too.

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

A BioBrick™-Compatible Vector for Allelic Replacement Using the XylE Gene as Selection Marker.

BACKGROUND: Circular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host. Common limitations of existing methods include high false positive rates due to mutations in counter-selection genes, and limited applicability to specific strains or growth media. Finally, solutions compatible with physical standards, such as the BioBrick™, are not currently available, although they proved to be successful in the design of other replicative or integrative plasmids. FINDINGS: We illustrate pBBknock, a novel BioBrick™-compatible vector for allelic replacement in Escherichia coli. It includes a temperature-sensitive replication origin and enables marker-less genome engineering via two homologous recombination events. Chloramphenicol resistance allows positive selection of clones after the first event, whereas a colorimetric assay based on the xylE gene provides a simple way to screen clones in which the second recombination event occurs. Here we successfully use pBBknock to delete the lactate dehydrogenase gene in E. coli W, a popular host used in metabolic engineering. CONCLUSIONS: Compared with other plasmid-based solutions, pBBknock has a broader application range, not being limited to specific strains or media. We expect that pBBknock will represent a versatile solution both for practitioners, also among the iGEM competition teams, and for research laboratories that use BioBrick™-based assembly procedures.

Quantification of the gene silencing performances of rationally-designed synthetic small RNAs.

Small RNAs (sRNAs) are genetic tools for the efficient and specific tuning of target genes expression in bacteria. Inspired by naturally occurring sRNAs, recent works proposed the use of artificial sRNAs in synthetic biology for predictable repression of the desired genes. Their potential was demonstrated in several application fields, such as metabolic engineering and bacterial physiology studies. Guidelines for the rational design of novel sRNAs have been recently proposed. According to these guidelines, in this work synthetic sRNAs were designed, constructed and quantitatively characterized in Escherichia coli. An sRNA targeting the reporter gene RFP was tested by measuring the specific gene silencing when RFP was expressed at different transcription levels, under the control of different promoters, in different strains, and in single-gene or operon architecture. The sRNA level was tuned by using plasmids maintained at different copy numbers. Results demonstrated that RFP silencing worked as expected in an sRNA and mRNA expression-dependent fashion. A mathematical model was used to support sRNA characterization and to estimate an efficiency-related parameter that can be used to compare the performance of the designed sRNA. Gene silencing was also successful when RFP was placed in a two-gene synthetic operon, while the non-target gene (GFP) in the operon was not considerably affected. Finally, silencing was evaluated for another designed sRNA targeting the endogenous lactate dehydrogenase gene. The quantitative study performed in this work elucidated interesting performance-related and context-dependent features of synthetic sRNAs that will strongly support predictable gene silencing in disparate basic or applied research studies.

Multi-Faceted Characterization of a Novel LuxR-Repressible Promoter Library for Escherichia coli.

The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system.

Methods for genetic optimization of biocatalysts for biofuel production from dairy waste through synthetic biology.

Whey is an abundant by-product of cheese production process and it is considered a special waste due to its high nutritional load and hypertrophic potential. Technologies for whey valorization are available. They can convert such waste into high-value products, like whey proteins. However, the remaining liquid (called permeate) is still considered as a polluting waste due to its high lactose concentration. The alcoholic fermentation of lactose into ethanol will simultaneously achieve two important goals: safe disposal of a pollutant waste and green energy production. This methodology paper illustrates the workflow carried out to design and realize an optimized microorganism that can efficiently perform the lactose-to-ethanol conversion, engineered via synthetic biology experimental and computational approaches.

Half-life measurements of chemical inducers for recombinant gene expression.

BACKGROUND: Inducible promoters are widely spread genetic tools for triggering, tuning and optimizing the expression of recombinant genes in engineered biological systems. Most of them are controlled by the addition of a specific exogenous chemical inducer that indirectly regulates the promoter transcription rate in a concentration-dependent fashion. In order to have a robust and predictable degree of control on promoter activity, the degradation rate of such chemicals should be considered in many applications like recombinant protein production. RESULTS: In this work, we use whole-cell biosensors to assess the half-life of three commonly used chemical inducers for recombinant Escherichia coli: Isopropyl ß-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (ATc) and N-(3-oxohexanoyl)-L-homoserine lactone (HSL). A factorial study was conducted to investigate the conditions that significantly contribute to the decay rate of these inducers. Temperature has been found to be the major factor affecting ATc, while medium and pH have been found to highly affect HSL. Finally, no significant degradation was observed for IPTG among the tested conditions. CONCLUSIONS: We have quantified the decay rate of IPTG, ATc and HSL in many conditions, some of which were not previously tested in the literature, and the main effects affecting their degradation were identified via a statistics-based framework. Whole-cell biosensors were successfully used to conduct this study, yielding reproducible measurements via simple multiwell-compatible assays. The knowledge of inducer degradation rate in several contexts has to be considered in the rational design of synthetic biological systems for improving the predictability of induction effects, especially for prolonged experiments.

Low-amplitude high frequency vibration down-regulates myostatin and atrogin-1 expression, two components of the atrophy pathway in muscle cells.

Whole body vibration (WBV) is a very widespread mechanical stimulus used in physical therapy, rehabilitation and fitness centres. It has been demonstrated that vibration induces improvements in muscular strength and performance and increases bone density. We investigated the effects of low-amplitude, high frequency vibration (HFV) at the cellular and tissue levels in muscle. We developed a system to produce vibrations adapted to test several parameters in vitro and in vivo. For in vivo experiments, we used newborn CD1 wild-type mice, for in vitro experiments, we isolated satellite cells from 6-day-old CD1 mice, while for proliferation studies, we used murine cell lines. Animals and cells were treated with high frequency vibration at 30 Hz. We analyzed the effects of mechanical stimulation on muscle hypertrophy/atrophy pathways, fusion enhancement of myoblast cells and modifications in the proliferation rate of cells. Results demonstrated that mechanical vibration strongly down-regulates atrophy genes both in vivo and in vitro. The in vitro experiments indicated that mechanical stimulation promotes fusion of satellite cells treated directly in culture compared to controls. Finally, proliferation experiments indicated that stimulated cells had a decreased growth rate compared to controls. We concluded that vibration treatment at 30 Hz is effective in suppressing the atrophy pathway both in vivo and in vitro and enhances fusion of satellite muscle cells.

Ultrasound stimulus to enhance the bone regeneration capability of gelatin cryogels.

In the present study, gelatin-based cryogels have been seeded with human SAOS-2 osteoblasts. In order to overcome the drawbacks associated with in vitro culture systems, such as limited diffusion and inhomogeneous cell-matrix distribution, this work describes the application of ultrasounds (average power, 149 mW; frequency, 1.5 MHz) to physically enhance the cell culture in vitro. The results indicate that the physical stimulation of cell-seeded gelatin-based cryogels upregulates the bone matrix production.