Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

A brief review of extrusion‐based tissue scaffold bio‐printing

Extrusion‐based bio‐printing has great potential as a technique for manipulating biomaterials and living cells to create three‐dimensional (3D) scaffolds for damaged tissue repair and function restoration. Over the last two decades, advances in both engineering techniques and life sciences have evolved extrusion‐based bio‐printing from a simple technique to one able to create diverse tissue scaffolds from a wide range of biomaterials and cell types. However, the complexities associated with synthesis of materials for bio‐printing and manipulation of multiple materials and cells in bio‐printing pose many challenges for scaffold fabrication. This paper presents an overview of extrusion‐based bio‐printing for scaffold fabrication, focusing on the prior‐printing considerations (such as scaffold design and materials/cell synthesis), working principles, comparison to other techniques, and to‐date achievements. This paper also briefly reviews the recent development of strategies with regard to hydrogel synthesis, multi‐materials/cells manipulation, and process‐induced cell damage in extrusion‐based bio‐printing. The key issue and challenges for extrusion‐based bio‐printing are also identified and discussed along with recommendations for future, aimed at developing novel biomaterials and bio‐printing systems, creating patterned vascular networks within scaffolds, and preserving the cell viability and functions in scaffold bio‐printing. The address of these challenges will significantly enhance the capability of extrusion‐based bio‐printing.     

Prediction of Rate‐Limiting Reactions for Growth‐Associated Production Using a Constraint‐Based Approach

Identification of a rate‐limiting step in pathways is a key challenge in metabolic engineering. Although the prediction of rate‐limiting steps using a kinetic model is a powerful approach, there are several technical hurdles for developing a kinetic model. In this study, an in silico screening algorithm of key enzyme for metabolic engineering is developed to identify the possible rate‐limiting reactions for the growth‐coupled target production using a stoichiometric model without any experimental data and kinetic parameters. In this method, for each reaction, an upper‐bound flux constraint is imposed and the target production is predicted by linear programming. When the constraint decreases the target production at the optimal growth state, the reaction is thought to be a possible rate‐limiting step. For validation, this method is applied to the production of succinate or 1,4‐butanediol (1,4‐BDO) in Escherichia coli, in which the experimental engineering for eliminating rate‐limiting steps has been previously reported. In succinate production from glycerol, nine reactions including phosphoenolpyruvate carboxylase are predicted as the rate‐limiting steps. In 1,4‐BDO production from glucose, eight reactions including pyruvate dehydrogenase are predicted as the rate‐limiting steps. These predictions include experimentally identified rate‐limiting steps, which would contribute to metabolic engineering as a practical tool for screening candidates of rate‐limiting reactions.     

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l‐Homoserine to 4‐Hydroxy‐2‐ketobutyrate in a Glycerol‐Independent Pathway of 1,3‐Propanediol Production from Glucose

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4‐hydroxy‐2‐ketobutyrate, a key reaction in producing 1,3‐propanediol (1,3‐PDO) from glucose in a novel glycerol‐independent metabolic pathway. To this end, a computation‐based rational approach is used to change the substrate specificity of SerC from l ‐phosphoserine to l ‐homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerC_R42W/R77W, is successfully improved by 4.2‐fold in comparison to the wild type when l ‐homoserine is used as the substrate, while its activity toward the natural substrate l ‐phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3‐PDO production, the “homoserine to 1,3‐PDO” pathway is constructed in E. coli by coexpression of SerC_R42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L^?1 1,3‐PDO in fed‐batch fermentation, which is 13‐fold higher than the wild‐type strain and represents an important step forward to realize the promise of the glycerol‐independent synthetic pathway for 1,3‐PDO production from glucose.     

Surface‐directed assembly of cell‐laden microgels

Cell‐laden microscale hydrogels (microgels) can be used as tissue building blocks and assembled to create 3D tissue constructs with well‐defined microarchitecture. In this article, we present a bottom‐up approach to achieve microgel assembly on a patterned surface. Driven by surface tension, the hydrophilic microgels can be assembled into well‐defined shapes on a glass surface patterned with hydrophobic and hydrophilic regions. We found that the cuboidic microgels (～100–200 µm in width) could self‐assemble into defined shapes with high fidelity to the surface patterns. The microgel assembly process was improved by increasing the hydrophilicity of the microgels and reducing the surface tension of the surrounding solution. The assembled microgels were stabilized by a secondary crosslinking step. Assembled microgels containing cells stained with different dyes were fabricated to demonstrate the application of this approach for engineering microscale tissue constructs containing multiple cell types. This bottom‐up approach enables rapid fabrication of cell‐laden microgel assemblies with pre‐defined geometrical and biological features, which is easily scalable and can be potentially used in microscale tissue engineering applications. Biotechnol. Bioeng. 2010; 105: 655–662. © 2009 Wiley Periodicals, Inc.     

miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins

In recent years, the number of complex but clinically effective biologicals such as multi‐specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult‐to‐express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next‐generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult‐to‐express proteins. In a previous study we identified miR‐143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR‐143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR‐143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen‐activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR‐143‐3p in CHO cells. Finally, siRNA‐mediated knock‐down of MAPK7 could be demonstrated to phenocopy pro‐productive effects of miR‐143. In summary, our data suggest that miR‐143 might represent a novel genetic element to enhance production of difficult‐to‐express proteins in CHO cells which may be partly mediated by down‐regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046–1058, 2017     

EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker‐free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone‐MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113‐7D) and a diploid industrial strain (Ethanol Red) for production of 3‐hydroxypropionic acid, where we tested three different acetyl‐CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone‐MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.     

Molecular Engineered Hole‐Extraction Materials to Enable Dopant‐Free,Efficient p‐i‐n Perovskite Solar Cells

Two hole‐extraction materials (HEMs), TPP‐OMeTAD and TPP‐SMeTAD, have been developed to facilitate the fabrication of efficient p‐i‐n perovskite solar cells (PVSCs). By replacing the oxygen atom on HEM with sulfur (from TPP‐OMeTAD to TPP‐SMeTAD), it effectively lowers the highest occupied molecular orbital of the molecule and provides stronger Pb? S interaction with perovskites, leading to efficient charge extraction and surface traps passivation. The TPP‐SMeTAD‐based PVSCs exhibit both improved photovoltaic performance and reduced hysteresis in p‐i‐n PVSCs over those based on TPP‐OMeTAD. This work not only provides new insights on creating perovskite‐HEM heterojunction but also helps in designing new HEM to enable efficient organic–inorganic hybrid PVSCs.     

Interface Engineering for Highly Efficient and Stable Planar p‐i‐n Perovskite Solar Cells

Organic‐inorganic halide perovskite materials have become a shining star in the photovoltaic field due to their unique properties, such as high absorption coefficient, optimal bandgap, and high defect tolerance, which also lead to the breathtaking increase in power conversion efficiency from 3.8% to over 22% in just seven years. Although the highest efficiency was obtained from the TiO₂ mesoporous structure, there are increasing studies focusing on the planar structure device due to its processibility for large‐scale production. In particular, the planar p‐i‐n structure has attracted increasing attention on account of its tremendous advantages in, among other things, eliminating hysteresis alongside a competitive certified efficiency of over 20%. Crucial for the device performance enhancement has been the interface engineering for the past few years, especially for such planar p‐i‐n devices. The interface engineering aims to optimize device properties, such as charge transfer, defect passivation, band alignment, etc. Herein, recent progress on the interface engineering of planar p‐i‐n structure devices is reviewed. This review is mainly focused on the interface design between each layer in p‐i‐n structure devices, as well as grain boundaries, which are the interfaces between polycrystalline perovskite domains. Promising research directions are also suggested for further improvements.     

Two‐Photon polymerization for microfabrication of three‐dimensional scaffolds for tissue engineering application

In natural tissues cells are embedded in a three‐dimensional fibrous network of biopolymers like collagen, hyaluronic acid etc. This extracellular matrix (ECM) influences the cell fate, the differentiation status, metabolic processes and provides structural integrity. For a three‐dimensional or physiological cell cultivation that are required in biomedical applications (e.g. tissue engineering, BioMEMS) scaffolds are needed. These scaffolds mimic the ECM according to their biocompatibility which comprises aspects of surface compatibility and importantly for tissue engineering applications aspects of structural compatibility. We have evaluated scaffold design parameters for the three‐dimensional cultivation of chondrocytes for the tissue engineering of artificial cartilage. Two‐photon polymerization is a powerful technique for fabrication of polymeric three‐dimensional micro‐ and submicro‐structures. The photoinitiation system for two‐photon polymerization is excited by simultaneous absorption of two photons leading to chemical polymerization reactions. Due to a tight confinement of the excitation volume around the focal point, this method can produce micrometer sized objects maintaining a high spatial resolution down to 100 nm. Two‐photon processes require very high photon densities which are provided by pulsed femtosecond lasers. The potential of this approach for microfabrication of scaffolds for tissue engineering is demonstrated by investigation of the cell response to microstructures with complex three‐dimensional geometry and feature sizes in the range of few micrometers.     

The N‐glycan on Asn54 affects the atypical N‐glycan composition of plant‐produced interleukin‐22, but does not influence its activity

Human interleukin‐22 (IL‐22) is a member of the IL‐10 cytokine family that has recently been shown to have major therapeutic potential. IL‐22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL‐22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL‐22 in Nicotiana benthamiana plants and investigated the role of N‐glycosylation on protein folding and biological activity. Expression levels of IL‐22 were up to 5.4 μg/mg TSP, and N‐glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human‐like N‐glycans on IL‐22 by co‐expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6‐fucoylation did not improve the biological activity of IL‐22. The combination of site‐directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N‐glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N‐glycan on Asn54 contributes to the atypical N‐glycan composition of plant‐produced IL‐22 and influences the N‐glycan composition of N‐glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N‐glycosylation on folding and activity of recombinant glycoproteins, such as IL‐22.     

Surface Engineering Strategies of Layered LiCoO2 Cathode Material to Realize High‐Energy and High‐Voltage Li‐Ion Cells

Battery industries and research groups are further investigating LiCoO₂ to unravel the capacity at high‐voltages (>4.3 vs Li). The research trends are towards the surface modification of the LiCoO₂ and stabilize it structurally and chemically. In this report, the recent progress in the surface‐coating materials i.e., single‐element, binary, and ternary hybrid‐materials etc. and their coating methods are illustrated. Further, the importance of evaluating the surface‐coated LiCoO₂ in the Li‐ion full‐cell is highlighted with our recent results. Mg,P‐coated LiCoO₂ full‐cells exhibit excellent thermal stability, high‐temperature cycle and room‐temperature rate capabilities with high energy‐density of ≈1.4 W h cc^?1 at 10 C and 4.35 V. Besides, pouch‐type full‐cells with high‐loading (18 mg cm^?2) electrodes of layered‐Li(Ni,Mn)O₂ ‐coated LiCoO₂ not only deliver prolonged cycle‐life at room and elevated‐temperatures but also high energy‐density of ≈2 W h cc^?1 after 100 cycles at 25 °C and 4.47 V (vs natural graphite). The post‐mortem analyses and experimental results suggest enhanced electrochemical performances are attributed to the mechanistic behaviour of hybrid surface‐coating layers that can mitigate undesirable side reactions and micro‐crack formations on the surface of LiCoO₂ at the adverse conditions. Hence, the surface‐engineering of electrode materials could be a viable path to achieve the high‐energy Li‐ion cells for future applications.     

RNA‐Schalter

Riboswitches Synthetic Biology intends to develop novel biological systems such as artificial metabolic pathways. With these, microorganisms are employed to make useful substances such as drugs, biofuels or other chemicals from cheap and renewable resources. Besides genes, regulators are essential parts of genetic circuits. Riboswitches represent a new class of such regulators. They can be developed with customized features using ligand binding RNAs.     

Improvements to the Overpotential of All‐Solid‐State Lithium‐Ion Batteries during the Past Ten Years

After the research that shows that Li₁₀GeP₂S₁₂ (LGPS)‐type sulfide solid electrolytes can reach the high ionic conductivity at the room temperature, sulfide solid electrolytes have been intensively developed with regard to ionic conductivity and mechanical properties. As a result, an increasing volume of research has been conducted to employ all‐solid‐state lithium batteries in electric automobiles within the next five years. To achieve this goal, it is important to review the research over the past decade, and understand the requirements for future research necessary to realize the practical applications of all‐solid‐state lithium batteries. To date, research on all‐solid‐state lithium batteries has focused on achieving overpotential properties similar to those of conventional liquid‐lithium‐ion batteries by increasing the ionic conductivity of the solid electrolytes. However, the increase in the ionic conductivity should be accompanied by improvements of the electronic conductivity within the electrode to enable practical applications. This essay provides a critical overview of the recent progress and future research directions of the all‐solid‐state lithium batteries for practical applications.     

Investigating the role of native propionyl‐CoA and methylmalonyl‐CoA metabolism on heterologous polyketide production in Escherichia coli

6‐Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over‐expression of a native prpE gene (encoding a propionyl‐CoA synthetase) and heterologous pcc genes (encoding a propionyl‐CoA carboxylase) to supply the needed propionyl‐CoA and (2S)‐methylmalonyl‐CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl‐CoA mutase), YgfG (a methylmalonyl‐CoA decarboxylase), and YgfH (a propionyl‐CoA:succinate CoA transferase), also involved in propionyl‐CoA and methylmalonyl‐CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over‐expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over‐expression of sbm did not influence 6dEB production; ygfG over‐expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L. Biotechnol. Bioeng. 2010; 105: 567–573. © 2009 Wiley Periodicals, Inc.     

Green‐Solvent‐Processed All‐Polymer Solar Cells Containing a Perylene Diimide‐Based Acceptor with an Efficiency over 6.5%

To realize high power conversion efficiencies (PCEs) in green‐solvent‐processed all‐polymer solar cells (All‐PSCs), a long alkyl chain modified perylene diimide (PDI)‐based polymer acceptor PPDIODT with superior solubility in nonhalogenated solvents is synthesized. A properly matched PBDT‐TS1 is selected as the polymer donor due to the red‐shifted light absorption and low‐lying energy level in order to achieve the complementary absorption spectrum and matched energy level between polymer donor and polymer acceptor. By utilizing anisole as the processing solvent, an optimal efficiency of 5.43% is realized in PBDT‐TS1/PPDIODT‐based All‐PSC with conventional configuration, which is comparable with that of All‐PSCs processed by the widely used binary solvent. Due to the utilization of an inverted device configuration, the PCE is further increased to over 6.5% efficiency. Notably, the best‐performing PCE of 6.58% is the highest value for All‐PSCs employing PDI‐based polymer acceptors and green‐solvent‐processed All‐PSCs. The excellent photovoltaic performance is mainly attributed to a favorable vertical phase distribution, a higher exciton dissociation efficiency (P_diss) in the blend film, and a higher electrode carrier collection efficiency. Overall, the combination of rational molecular designing, material selection, and device engineering will motivate the efficiency breakthrough in green‐solvent‐processed All‐PSCs.