Protein design for pathway engineering

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.     

Biocatalysis in ionic liquids for lignin valorization: Opportunities and recent developments

Lignin holds tremendous potential as a renewable feedstock for upgrading to a number of high-value chemicals and products that are derived from the petroleum industry at present. Since lignin makes up a significant fraction of lignocellulosic biomass, co-utilization of lignin in addition to cellulose and hemicelluloses is vital to the economic viability of cellulosic biorefineries. The recalcitrant nature of lignin, originated from the molecule's compositional and structural heterogeneity, however, poses great challenges toward effective and selective lignin depolymerization and valorization. Ionic liquid (IL) is a powerful solvent that has demonstrated high efficiency in fractionating lignocellulosic biomass into sugar streams and a lignin stream of reduced molecular weight. Compared to thermochemical methods, biological lignin deconstruction takes place at mild temperature and pressure while product selectivity can be potentially improved via the specificity of biocatalysts (lignin degrading enzymes, LDEs). This review focuses on a lignin valorization strategy by harnessing the biomass fractionating capabilities of ILs and the substrate and product selectivity of LDEs. Recent advances in elucidating enzyme-IL interactions as well as strategies for improving enzyme activity in IL are discussed, with specific emphases on biocompatible ILs, thermostable and IL-tolerant enzymes, enzyme immobilization, and surface charge engineering. Also reviewed is the protein engineering toolsets (directed evolution and rational design) to improve the biocatalysts' activity, stability and product selectivity in IL systems. The alliance between IL and LDEs offers a great opportunity for developing a biocatalytic route for lignin valorization.     

Protein and Genome Evolution in Mammalian Cells for Biotechnology Applications

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.     

Fiber-based tissue engineering: Progress,challenges, and opportunities

Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the abovementioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice.     

蛋白质定向进化及其在微生物代谢调控中的应用

蛋白质定向进化是非理性改造蛋白质的一种有效方法。利用蛋白质定向进化技术可以改变代谢流,扩展或构建新的代谢途径,弱化或消除不必要或有害的代谢途径,从而达到提高某种代谢产物产率或降解有害物质的目的。蛋白质定向进化技术在代谢调控中的应用有效拓宽了代谢工程的应用范围。本文介绍了主要的蛋白质定向进化技术如易错PCR技术、DNA改组技术、交错延伸技术和临时模板随机嵌合技术等,评述了蛋白质定向进化技术对微生物细胞代谢中的关键蛋白进行定向改造,从而改善其代谢能力,调控微生物代谢等的应用。     

The state-of-the-art strategies of protein engineering for enzyme stabilization

Enzymes generated by natural recruitment and protein engineering have greatly contribute in various sets of applications. However, their insufficient stability is a bottleneck that limit the rapid development of biocatalysis. Novel approaches based on precise and global structural dissection, advanced gene manipulation, and combination with the multidisciplinary techniques open a new horizon to generate stable enzymes efficiently. Here, we comprehensively introduced emerging advances of protein engineering strategies for enzyme stabilization. Then, we highlighted practical cases to show importance of enzyme stabilization in pharmaceutical and industrial applications. Combining computational enzyme design with molecular evolution will hold considerable promise in this field.     

Novel opportunities and challenges offered by nanobiomaterials in tissue engineering

Over the last decades, tissue engineering has demonstrated an unquestionable potential to regenerate damaged tissues and organs. Some tissue-engineered solutions recently entered the clinics (eg, artificial bladder, corneal epithelium, engineered skin), but most of the pathologies of interest are still far from being solved. The advent of stem cells opened the door to large-scale production of “raw living matter” for cell replacement and boosted the overall sector in the last decade. Still reliable synthetic scaffolds fairly resembling the nanostructure of extracellular matrices, showing mechanical properties comparable to those of the tissues to be regenerated and capable of being modularly functionalized with biological active motifs, became feasible only in the last years thanks to newly introduced nanotechnology techniques of material design, synthesis, and characterization. Nanostructured synthetic matrices look to be the next generation scaffolds, opening new powerful pathways for tissue regeneration and introducing new challenges at the same time. We here present a detailed overview of the advantages, applications, and limitations of nanostructured matrices with a focus on both electrospun and self-assembling scaffolds.     

蛋白质定向进化的研究技术及应用

蛋白质定向进化是在人类改造蛋白质的过程中产生的。蛋白质定向进化通常分三步进行,即随机诱变、体外重组和筛选。每一步都有多种研究技术。蛋白质定向进化大大加速了人类改造蛋白质的步伐,在实际应用研究和基础理论研究中都具有重要意义。     

Nuclear and plastid genetic engineering of plants: Comparison of opportunities and challenges

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to ‘bioreactor’ plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.     

Directed polymerase evolution

Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity.     

Genetic engineering of wheat--current challenges and opportunities

Wheat is one of the major staple food crops grown worldwide; however, productivity in cereal crops has not kept pace with the world population growth. A significant increase in wheat production (>40% by 2020) is needed simply to keep up with the growing demand. This increase is unlikely to be achieved by conventional plant breeding methods because of the limited gene pool available. The application of recombinant techniques to improve wheat quality and yield is not only desirable but also has potential to open up new opportunities. Although there has been significant progress in developing gene-transformation technologies for improving these traits, this remains an important challenge for plant biotechnology. Obstacles to translate the full potential of the genomic era to wheat breeding include the need to develop elite wheat varieties without selectable markers, introducing minimal or nil intergenic DNA and social and market issues concerning genetically engineered food products.     

Protein domain library generation by overlap extension (PDLGO): a tool for enzyme engineering

We introduce an upgraded version of the error-prone polymerase chain reaction (epPCR) comprising three DNA polymerase-catalyzed steps. It improves the common epPCR strategy such that random mutations can be confined exactly to a distinct, but freely selectable, sequence region within a gene without the need for flanking restriction endonuclease sites. The new method is called protein domain library generation by overlap extension (PDLGO). To validate PDLGO, we generated a random library of EstE, a multidomain esterase from Xanthomonas vesicatoria. It was demonstrated that random mutations appear exclusively within the catalytic domains as intended. The domains of EstE flanking the catalytic domains are required for transport of EstE to the cell envelope and remain unaltered. Microplates with integrated pH sensors, providing a substrate-independent high-throughput screening tool, were used to analyze whole cells of E. coli expressing the variants of the EstE library. A variant (P286H) with substantially increased catalytic activity was identified. Our results indicate that combining PDLGO with microplates containing integrated pH sensors provides a simple and rapid toolbox for directed evolution of esterases.     

蛋白质突变体基因库构建方法的研究进展

体外定向进化是蛋白质工程中一个非常有效的设计策略。最近几年,在过去常用的寡核苷酸介导的随机突变、易错PCR和DNA改组等方法的基础上又出现了一些新的定向进化方法。本文对这些方法及其特点加以总结,为解决特定问题选取何种方法提供一定依据。最近研究表明：定向进化和理性设计相结合、定向进化和以结构为基础的计算设计方法相结合正成为蛋白质工程中两个新的发展方向。     

蛋白质体外进化建库技术研究

蛋白质体外进化技术是蛋白质工程发展的一个里程碑,也是改造蛋白质的一种有效工具。它不仅具有重要的应用价值,而且有助于蛋白质结构与功能的研究。通过蛋白质体外进化技术已成功地改造了许多蛋白质,有些已应用于工农业生产。体外进化技术分为两步:建库和筛选。本文主要对蛋白质体外进化策略及对体外随机突变技术、DNA重组技术、利用活细胞自身修复系统构建突变文库等几种定向进化突变文库建立技术进行了介绍与论述,同时还对蛋白质体外进化技术的应用及与其它学科结合的研究前景进行了分析,为获得具有改进功能或全新功能的蛋白质提供理论基础。     

bPE toolkit: toolkit for computational protein engineering

We present a computational toolkit consisting of five utility tools, for performing basic operations on a protein structure file in PDB format. The toolkit consists of five different programs which can be integrated as part of a pipeline for computational protein structure characterization or as a standalone analysis package. The programs include tools for chirality check for amino acids (ProChiral), contact map generation (CoMa), data redundancy (DaRe), hydrogen bond potential energy (HyPE) and electrostatic interaction energy (EsInE). All programs in the toolkit can be accessed and downloaded through the following link: http://www.iitg.ac.in/bpetoolkit/.     

A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica

Development of a high-throughput eukaryotic screening procedure is important to increase success in obtaining improved enzymes through directed enzyme evolution. This procedure was developed for the yeast Yarrowia lipolytica which becomes the second eukaryotic host for this purpose. The extracellular lipase Lip2 was used as expressed enzyme but this system will be easily adjusted for other enzymes. We adapted and optimized the protocol for protein expression by Y. lipolytica in 96-well microplates. Yeast transformation efficiency and expression cassette insertion were increased by constructing a strain containing a zeta docking platform for targeted integration into the genome. The coefficient of variance of the full process was reduced from 36.3% to 18.9%. The main part of the variability (11.7%) arises from the specific lipase enzyme assay whereas the coefficient of variance concerning transformation, growth and expression steps represents only 7.2%. The rate of clone with no activity was reduced from 5.8% to 0.2%. Both transformation efficiency and variability are then compatible with high-throughput screening in the yeast Y. lipolytica.