Stable recombinant production of codon-scrambled lubricin and mucin in human cells

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition. The codon-scrambling strategy was applied to generate synonymous genes, or “synDNAs,” for two mucins of commercial interest: lubricin and mucin 1. Stable, long-term recombinant production in suspension-adapted human 293-F cells was demonstrated for the synonymous lubricin complementary DNA (cDNA), which we refer to as SynLubricin. Under optimal conditions, a 293-F subpopulation produced recombinant SynLubricin at more than 200 mg/L of media and was stable throughout 2 months of continuous culture. Functionality tests confirmed that the recombinant lubricin could effectively inhibit cell adhesion and lubricate cartilage explants. Together, our work provides a viable workflow for cDNA design and stable mucin production in mammalian host production systems.     

系统生物学和合成生物学研究在生物燃料生产菌株改造中的应用

基于生物质资源生产环境友好的生物燃料,对经济和社会的可持续发展具有重要意义,但其生产成本高的问题十分突出,而高效生产菌株的获得是解决这一问题的根本出路。以下综述了利用系统生物学研究所获得的信息进行菌种改造的过程,重点论述了生产菌株胁迫耐受性方面的研究进展,并讨论了系统生物学、合成生物学和代谢工程技术在改造生物燃料生产菌株中的应用,展望了合成生物学在构建高效生物能源生产菌株方面应用的前景。     

Development of GRAS strains for nutraceutical production using systems and synthetic biology approaches: advances and prospects

Nutraceuticals are food substances with medical and health benefits for humans. Limited by complicated procedures, high cost, low yield, insufficient raw materials, resource waste, and environment pollution, chemical synthesis and extraction are being replaced by microbial synthesis of nutraceuticals. Many microbial strains that are generally regarded as safe (GRAS) have been identified and developed for the synthesis of nutraceuticals, and significant nutraceutical production by these strains has been achieved. In this review, we systematically summarize recent advances in nutraceutical research in terms of physiological effects on health, potential applications, drawbacks of traditional production processes, characteristics of production strains, and progress in microbial fermentation. Recent advances in systems and synthetic biology techniques have enabled comprehensive understanding of GRAS strains and its wider applications. Thus, these microbial strains are promising cell factories for the commercial production of nutraceuticals.     

Stapled NRPS enhances the production of valinomycin in Escherichia coli

Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full biosynthetic pathway in a host often leads to low productivity, which is at least in part due to the low efficiency of enzyme interaction in vivo except for the well-known reasons of metabolic burden (e.g., expression of large NRP synthetases—NRPSs with molecular weights of >100 kDa) and cellular toxicity on host cells. To enhance the catalytic efficiency of large NRPSs in vivo, here we propose to staple NRPS enzymes by using short peptide/protein pairs (e.g., SpyTag/SpyCatcher) for enhanced NRP production. We achieve this goal by introducing a stapled NRPS system for the biosynthesis of the antibiotic NRP valinomycin in E. coli. The results indicate that stapled valinomycin synthetase (Vlm1 and Vlm2) enables higher product accumulation than those two free enzymes (e.g., the maximum improvement is nearly fourfold). After further optimization by strain and bioprocess engineering, the final valinomycin titer maximally reaches about 2800 µg/L, which is 73 times higher than the initial titer of 38 µg/L. We expect that stapling NRPS enzymes will be a promising catalytic strategy for high-level biosynthesis of NRP natural products.     

A hybrid systems framework for cellular processes

With the availability of technologies that allow us to obtain stimulus-response time series data for modeling and system identification, there is going to be an increasing need for conceptual frameworks in which to formulate and test hypotheses about intra- and inter-cellular dynamics, in general and not just dependent on a particular cell line, cell type, organism, or technology. While the semantics can be quite different, biologists and systems scientists use in many cases a similar language (notion of feedback, regulation, etc.). A more abstract system-theoretic framework for signals, systems, and control could provide the biologist with an interface between the domains. Apart from recent examples to identify functional elements and describing them in engineering terms, there have been various more abstract developments to describe dynamics at the cell level in the past. This includes Rosen's (M,R)-systems. This paper presents an abstract and general compact mathematical framework of intracellular dynamics, regulation and regime switching inspired by (M,R)-theory and based on hybrid automata.     

The chemical formula of a magnetotactic bacterium

Elucidation of the chemical logic of life is one of the grand challenges in biology, and essential to the progress of the upcoming field of synthetic biology. Treatment of microbial cells explicitly as a "chemical" species in controlled reaction (growth) environments has allowed fascinating discoveries of elemental formulae of a few species that have guided the modern views on compositions of a living cell. Application of mass and energy balances on living cells has proved to be useful in modeling of bioengineering systems, particularly in deriving optimized media compositions for growing microorganisms to maximize yields of desired bio-derived products by regulating intra-cellular metabolic networks. In this work, application of elemental mass balance during growth of Magnetospirillum gryphiswaldense in bioreactors has resulted in the discovery of the chemical formula of the magnetotactic bacterium. By developing a stoichiometric equation characterizing the formation of a magnetotactic bacterial cell, coupled with rigorous experimental measurements and robust calculations, we report the elemental formula of M. gryphiswaldense cell as CH(2.06)O(0.13)N(0.28)Fe(1.74×10(-3)). Remarkably, we find that iron metabolism during growth of this magnetotactic bacterium is much more correlated individually with carbon and nitrogen, compared to carbon and nitrogen with each other, indicating that iron serves more as a nutrient during bacterial growth rather than just a mineral. Magnetotactic bacteria have not only invoked some interest in the field of astrobiology for the last two decades, but are also prokaryotes having the unique ability of synthesizing membrane bound intracellular organelles. Our findings on these unique prokaryotes are a strong addition to the limited repertoire, of elemental compositions of living cells, aimed at exploring the chemical logic of life.     

A day of systems and synthetic biology for non‐experts

From understanding ageing to the creation of artificial membrane‐bounded ‘organisms’, systems biology and synthetic biology are seen as the latest revolutions in the life sciences. They certainly represent a major change of gear, but paradigm shifts? This is open to debate, to say the least. For scientists they open up exciting ways of studying living systems, of formulating the ‘laws of life’, and the relationship between the origin of life, evolution and artificial biological systems. However, the ethical and societal considerations are probably indistinguishable from those of human genetics and genetically modified organisms. There are some tangible developments just around the corner for society, and as ever, our ability to understand the consequences of, and manage, our own progress lags far behind our technological abilities. Furthermore our educational systems are doing a bad job of preparing the next generation of scientists and non‐scientists.     

Application of imaging tools to plant cell culture: Relationship between plant cell aggregation and flavonoid production

Summary Image analysis tools were developed to measure biomass concentration, aggregate size and distribution, and pigmentation from anthocyanin-producing cell suspension cultures of ohelo (Vaccinium pahalae). The ex situ imaging system could image cell aggregates from 30 μm to 2 mm in diameter. The image analysis algorithm was based on extracted geometric features and morphological methods for biomass volume estimates, and hue, saturation, and intensity color characteristics for pigmentation estimates. Detailed information available from sampled cell culture images was validated by comparison to standard destructive manual measurements. Image analysis measurements revealed that pigment accumulation was negatively correlated with aggregate size. Although a substantial proportion of small aggregates remained colorless, the highly-pigmented small aggregates, 18 to 238 μm in breadth, contributed over 70% of the culture anthocyanin production (mg L⁻¹), despite their minor contribution to the overall biomass. The relative frequency of pigmented aggregates was higher in large-size aggregate classes; however, the pigmented sectors were mostly confined to only the periphery of the aggregates. As a result, large aggregate classes had only a minor contribution to overall culture anthocyanin yield.     

Opto-thermal technologies for microscopic analysis of cellular temperature-sensing systems

Could enzymatic activities and their cooperative functions act as cellular temperature-sensing systems? This review introduces recent opto-thermal technologies for microscopic analyses of various types of cellular temperature-sensing system. Optical microheating technologies have been developed for local and rapid temperature manipulations at the cellular level. Advanced luminescent thermometers visualize the dynamics of cellular local temperature in space and time during microheating. An optical heater and thermometer can be combined into one smart nanomaterial that demonstrates hybrid function. These technologies have revealed a variety of cellular responses to spatial and temporal changes in temperature. Spatial temperature gradients cause asymmetric deformations during mitosis and neurite outgrowth. Rapid changes in temperature causes imbalance of intracellular Ca²⁺ homeostasis and membrane potential. Among those responses, heat-induced muscle contractions are highlighted. It is also demonstrated that the short-term heating hyperactivates molecular motors to exceed their maximal activities at optimal temperatures. We discuss future prospects for opto-thermal manipulation of cellular functions and contributions to obtain a deeper understanding of the mechanisms of cellular temperature-sensing systems.     

复杂天然产物合成人工生物系统的设计与构建

天然产物是人类疾病预防和治疗药物的最重要来源。合成生物学技术的蓬勃发展为天然产物的开发注入了全新的活力。文中重点介绍了如何利用合成生物技术进行复杂天然产物合成人工生物系统的设计与构建,包括与此相关的生物元件理性设计、生物元件挖掘、途径装配与集成,模块的组装与系统的适配等内容。     

A modular modulation method for achieving increases in metabolite production

Increasing the production of overproducing strains represents a great challenge. Here, we develop a modular modulation method to determine the key steps for genetic manipulation to increase metabolite production. The method consists of three steps: (i) modularization of the metabolic network into two modules connected by linking metabolites, (ii) change in the activity of the modules using auxiliary rates producing or consuming the linking metabolites in appropriate proportions and (iii) determination of the key modules and steps to increase production. The mathematical formulation of the method in matrix form shows that it may be applied to metabolic networks of any structure and size, with reactions showing any kind of rate laws. The results are valid for any type of conservation relationships in the metabolite concentrations or interactions between modules. The activity of the module may, in principle, be changed by any large factor. The method may be applied recursively or combined with other methods devised to perform fine searches in smaller regions. In practice, it is implemented by integrating to the producer strain heterologous reactions or synthetic pathways producing or consuming the linking metabolites. The new procedure may contribute to develop metabolic engineering into a more systematic practice. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:656–667, 2015     

New tools and new biology: Recent miniaturized systems for molecular and cellular biology

Recent advances in applied physics and chemistry have led to the development of novel microfluidic systems. Microfluidic systems allow minute amounts of reagents to be processed using μm-scale channels and offer several advantages over conventional analytical devices for use in biological sciences: faster, more accurate and more reproducible analytical performance, reduced cell and reagent consumption, portability, and integration of functional components in a single chip. In this review, we introduce how microfluidics has been applied to biological sciences. We first present an overview of the fabrication of microfluidic systems and describe the distinct technologies available for biological research. We then present examples of microsystems used in biological sciences, focusing on applications in molecular and cellular biology.     

构建酿酒酵母工程菌合成香紫苏醇

香紫苏醇是一种来源于植物的双环二萜醇,常用于香味成分且具有重要生物学活性。为实现香紫苏醇的微生物生产,以酿酒酵母为宿主,表达焦磷酸赖百当烯二醇酯合酶和香紫苏醇合酶,构建香紫苏醇的人工生物合成途径。发现过表达前体代谢关键酶、蛋白质融合增强底物通道效应及去除异源蛋白信号肽等,有利于香紫苏醇合成。在摇瓶培养条件下,组合优化得到的工程菌株S6的香紫苏醇产量达到8.96 mg/L。研究结果对其他萜类化合物的异源生物合成具有参考价值。     

Directed evolution of LuxI for enhanced OHHL production

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.     

The importance and future of biochemical engineering

Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on “The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes”. On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.     

Road to the future of systems biotechnology: CRISPR-Cas-mediated metabolic engineering for recombinant protein production

The rising potential for CRISPR–Cas-mediated genome editing has revolutionized our strategies in basic and practical bioengineering research. It provides a predictable and precise method for genome modification in a robust and reproducible fashion. Emergence of systems biotechnology and synthetic biology approaches coupled with CRISPR–Cas technology could change the future of cell factories to possess some new features which have not been found naturally. We have discussed the possibility and versatile potentials of CRISPR–Cas technology for metabolic engineering of a recombinant host for heterologous protein production. We describe the mechanisms involved in this metabolic engineering approach and present the diverse features of its application in biotechnology and protein production.     

Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.     

系统生物学与细胞发生系统动力学

系统生物学是系统理论和实验生物技术、计算机数学模型等方法整合的生物系统研究,系统遗传学研究基因组的稳态与进化、功能基因组和生物性状等复杂系统的结构、动态与发生演变等。合成生物学是系统生物学的工程应用,采用工程学方法、基因工程和计算机辅助设计等研究人工生物系统的生物技术。系统与合成生物学的结构理论,序列标志片段显示分析与微流控生物芯片,广泛用于研究细胞代谢、繁殖和应激的自组织进化、生物体形态发生等细胞分子生物系统原理等。     

Switching control of an essential gene for reprogramming of cellular phenotypes in Escherichia coli

Biological systems designs require various dynamic controllers capable of modulating cellular phenotypes to adapt to changing environments. Cellular phenotypes are simultaneously affected by combinations of multiple genes that are controlled by global regulators. However, it is difficult to intentionally control the expression of these global regulators dynamically because they are essential for cell survival and are involved in regulatory networks clustered in operons. Here, we designed a platform that allows dynamic modulation of the expression of an essential gene. Using this system, comprising of on/off switches that respond to an extracellular stimulus, we successfully demonstrated the switching control of the expression of fusA encoding elongation factor G (EF-G). An additional control module in this system that responds to changed external signals was shown to provide the capacity to "switch gears" and reprogram cellular phenotypes with desired timing.