Strategies for systems-level metabolic engineering

Bio-based production of chemicals, fuels and materials is becoming more and more important due to the increasing environmental problems and sharply increasing oil price. To make these biobased processes economically competitive, the biotechnology industry explores new ways to improve the performance of microbial strains in fermentation processes. In contrast to the random mutagenesis and/or intuitive local metabolic engineering practiced in the past, we are now moving towards global-scale metabolic engineering, aided by various experimental and computational tools. This has recently led to some remarkable achievements for the overproduction of valueadded products. In this review, we highlight several relevant gene manipulation tools and computational tools using genome-scale stoichiometric models, and provide useful strategies for successful metabolic engineering along with selected exemplary studies.     

Dynamic metabolic engineering: New strategies for developing responsive cell factories

Metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable small molecules produced naturally in microorganisms, as well as those produced via heterologous pathways. Typically, the approaches have been focused on up‐ and downregulation of genes to redistribute steady‐state pathway fluxes, but more recently a number of groups have developed strategies for dynamic regulation, which allows rebalancing of fluxes according to changing conditions in the cell or the fermentation medium. This review highlights some of the recently published work related to dynamic metabolic engineering strategies and explores how advances in high‐throughput screening and synthetic biology can support development of new dynamic systems. Dynamic gene expression profiles allow trade‐offs between growth and production to be better managed and can help avoid build‐up of undesired intermediates. The implementation is more complex relative to static control, but advances in screening techniques and DNA synthesis will continue to drive innovation in this field.     

Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates

Implementing several metabolic engineering strategies, either individually or in combination, it is possible to construct microbial plastic factories to produce a variety of polyhydroxyalkanoate (PHA) biopolymers with desirable structures and material properties. Approaches include external substrate manipulation, inhibitor addition, recombinant gene expression, host cell genome manipulation and, most recently, protein engineering of PHA biosynthetic enzymes. In addition, mathematical models and molecular methods can be used to elucidate metabolically engineered systems and to identify targets for performance improvement.     

Recent trends in metabolic engineering of microbial chemical factories

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MicroRNAs as targets for engineering of CHO cell factories

MicroRNAs (miRNAs) are strongly implicated in the global regulation of gene expression, and, in this regard, they consequently affect metabolic pathways on every regulatory level in different species. This characteristic makes miRNAs a promising target for cell engineering, and they could have multiple applications in medicine and biotechnology. However, a more profound, mechanistic understanding of miRNA action is needed for their potential to be translated into the development of industrially relevant cell factories with novel features. Here, we highlight the potential of miRNAs for the engineering of Chinese hamster ovary (CHO) cells, these being the most prevalent cell factory system for biopharmaceutical production. A key advantage of miRNAs, in contrast to most cell-engineering approaches that rely on overexpression of regulatory proteins, is that they do not compete for the translational machinery that is required to express the recombinant product. However, we also summarize the limitations and challenges that will have to be overcome to exploit fully miRNA technology.     

Modular co-culture engineering,a new approach for metabolic engineering

With the development of metabolic engineering, employment of a selected microbial host for accommodation of a designed biosynthetic pathway to produce a target compound has achieved tremendous success in the past several decades. Yet, increasing requirements for sophisticated microbial biosynthesis call for establishment and application of more advanced metabolic engineering methodologies. Recently, important progress has been made towards employing more than one engineered microbial strains to constitute synthetic co-cultures and modularizing the biosynthetic labor between the co-culture members in order to improve bioproduction performance. This emerging approach, referred to as modular co-culture engineering in this review, presents a valuable opportunity for expanding the scope of the broad field of metabolic engineering. We highlight representative research accomplishments using this approach, especially those utilizing metabolic engineering tools for microbial co-culture manipulation. Key benefits and major challenges associated with modular co-culture engineering are also presented and discussed.     

Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories

The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine-tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l⁻¹ (88% yield) was reached in an optimized fed-batch bioreactor. This precursor-directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant tabersonine resources.     

Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.     

Recent advances in improving metabolic robustness of microbial cell factories

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Yeast cell permeabilizing beta-1,3-glucanases: A tool for the integration of downstream processes and metabolic engineering applications to yeast

In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains. We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast.     

应用基因组工程构建新型大肠杆菌细胞工厂

基因组工程（genome engineering）是指为了实现某一目标对复制系统进行有意地、广泛地遗传修饰。大肠杆菌作为常用细胞工厂之一,在生物制造领域应用广泛,已成为合成生物学的主要研究对象。应用基因组工程改造大肠杆菌可以进一步拓展其应用范围。介绍了基因组工程最新技术发展,如基因组整合、染色体进化、多元自动化基因组工程、可寻迹多元重组工程等,及其在改造大肠杆菌提高其生产能力、稳定性及环境耐受能力等方面的研究进展。     

Homogenizing bacterial cell factories: Analysis and engineering of phenotypic heterogeneity

In natural habitats, microbes form multispecies communities that commonly face rapidly changing and highly competitive environments. Thus, phenotypic heterogeneity has evolved as an innate and important survival strategy to gain an overall fitness advantage over cohabiting competitors. However, in defined artificial environments such as monocultures in small- to large-scale bioreactors, cell-to-cell variations are presumed to cause reduced production yields as well as process instability. Hence, engineering microbial production toward phenotypic homogeneity is a highly promising approach for synthetic biology and bioprocess optimization.In this review, we discuss recent studies that have unraveled the cell-to-cell heterogeneity observed during bacterial gene expression and metabolite production as well as the molecular mechanisms involved. In addition, current single-cell technologies are briefly reviewed with respect to their applicability in exploring cell-to-cell variations. We highlight emerging strategies and tools to reduce phenotypic heterogeneity in biotechnological expression setups. Here, strain or inducer modifications are combined with cell physiology manipulations to achieve the ultimate goal of equalizing bacterial populations. In this way, the majority of cells can be forced into high productivity, thus reducing less productive subpopulations that tend to consume valuable resources during production. Modifications in uptake systems, inducer molecules or nutrients represent valuable tools for diminishing heterogeneity.Finally, we address the challenge of transferring homogeneously responding cells into large-scale bioprocesses. Environmental heterogeneity originating from extrinsic factors such as stirring speed and pH, oxygen, temperature or nutrient distribution can significantly influence cellular physiology. We conclude that engineering microbial populations toward phenotypic homogeneity is an increasingly important task to take biotechnological productions to the next level of control.     

Recent advances in engineering of microbial cell factories for intelligent pH regulation and tolerance

pH regulation is a serious concern in the industrial fermentation process as pH adjustment heavily utilizes acid/base and pollutes the environment. Under pH-stress conditions, microbial growth and production of valuable target products may be severely affected. Furthermore, some strains generating acidic or alkaline products require self pH regulation and increased tolerance against pH-stress. For pH control, synthetic biology has provided advanced engineering approaches to construct robust and more intelligent microbial strains, exhibiting tolerance to pH-stress to cope with limitations of pH regulation. This study reviewed the current progress of advanced strain evolution strategies to engineer pH-stress tolerant strains via synthetic biology. In addition, a large number of pH-responsive elements, including promoters, riboswitches, and some proteins have been investigated and applied for construction of pH-responsive genetic circuits and intelligent pH-responsive microbial strains.