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。     

Systems metabolic engineering: Genome-scale models and beyond

The advent of high throughput genome-scale bioinformatics has led to an exponential increase in available cellular system data. Systems metabolic engineering attempts to use data-driven approaches – based on the data collected with high throughput technologies – to identify gene targets and optimize phenotypical properties on a systems level. Current systems metabolic engineering tools are limited for predicting and defining complex phenotypes such as chemical tolerances and other global, multigenic traits. The most pragmatic systems-based tool for metabolic engineering to arise is the in silico genome-scale metabolic reconstruction. This tool has seen wide adoption for modeling cell growth and predicting beneficial gene knockouts, and we examine here how this approach can be expanded for novel organisms. This review will highlight advances of the systems metabolic engineering approach with a focus on de novo development and use of genome-scale metabolic reconstructions for metabolic engineering applications. We will then discuss the challenges and prospects for this emerging field to enable model-based metabolic engineering. Specifically, we argue that current state-of-the-art systems metabolic engineering techniques represent a viable first step for improving product yield that still must be followed by combinatorial techniques or random strain mutagenesis to achieve optimal cellular systems.     

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。     

全局转录调控及其在代谢工程中的应用

全局转录调控是一种全新的改进细胞表型的定向进化方法,通过error-prone PCR、DNA shuffling等技术对细胞中的σ因子和其他转录元件进行多轮突变修饰,改变RNA聚合酶的转录效率和对启动子的亲和能力,使细胞的转录在整体水平上发生改变,导致许多由多种基因控制的细胞表型得以改进。全局转录调控可以对代谢途径快速优化,在代谢工程中已被成功地应用于各种代谢产物的生物合成中。随着全局转录调控理论的不断完善,其应用前景也将越来越广阔。     

启动子和细胞全局转录机制的定向进化在微生物代谢工程中的应用

通过随机突变和定向选择而进行的定向进化(又称分子进化或人工进化)在改造酶的催化特性和稳定性、扩展酶的底物范围等方面具有广泛的应用。近年来,定向进化也开始应用在对结构基因的启动子区域和具有调节功能的蛋白如转录因子等进行代谢工程改造,并成功选育了对环境胁迫因素具有较强耐受性,以及发酵效率提高的微生物菌种。以下着重介绍近年来启动子的定向进化,包括启动子的强度和调节功能的分子进化,以及细胞全局转录工程等技术在微生物代谢工程中的应用,这些定向进化技术使人们可以更精细地调节基因表达水平,并可同时改变细胞内多个基因的转录水平,是代谢工程研究新的有力工具。     

Using continuous directed evolution to improve enzymes for plant applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme’s activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the yeast (Saccharomyces cerevisiae) OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt (“plantize”) enzymes from prokaryotes—especially exotic prokaryotes—to function well in mild, plant-like conditions.

Continuous directed evolution using the yeast OrthoRep system is a powerful way to improve enzymes for use in plant engineering as illustrated by “plantizing” a bacterial thiamin synthesis enzyme.     

In silico design and adaptive evolution of Escherichia coli for production of lactic acid

The development and validation of new methods to help direct rational strain design for metabolite overproduction remains an important problem in metabolic engineering. Here we show that computationally predicted E. coli strain designs, calculated from a genome-scale metabolic model, can lead to successful production strains and that adaptive evolution of the engineered strains can lead to improved production capabilities. Three strain designs for lactate production were implemented yielding a total of 11 evolved production strains that were used to demonstrate the utility of this integrated approach. Strains grown on 2 g/L glucose at 37 degrees C showed lactate titers ranging from 0.87 to 1.75 g/L and secretion rates that were directly coupled to growth rates.     

Adaptive laboratory evolution--harnessing the power of biology for metabolic engineering

Adaptive laboratory evolution (ALE) strategies allow for the metabolic engineering of microorganisms by combining genetic variation with the selection of beneficial mutations in an unbiased fashion. These ALE strategies have been proven highly effective in the optimization of production strains. In contrast to rational engineering strategies and directed modification of specific enzymes, ALE has the advantage of letting nonintuitive beneficial mutations occur in many different genes and regulatory regions in parallel. So far, the majority of applications of ALE in metabolic engineering have used well-characterized platform organisms such as Saccharomyces cerevisiae and Escherichia coli; however, applications for other microorganisms are on the rise. This review will focus on current applications of ALE as a tool for metabolic engineering and discuss advancements and achievements that have been made in this field.     

Directed evolution of biocatalytic processes

The benefits of applying biocatalysts to organic synthesis, such as their high chemo-, regio-, and enantio-specificity and selectivity, must be seriously considered, especially where chemical routes are unavailable, complex or prohibitively expensive. In cases where a potential biocatalytic route is not yet efficient enough to compete with chemical synthesis, directed evolution, and/or process engineering could be implemented for improvements. While directed evolution has demonstrated great potential to enhance enzyme properties, there will always be some aspects of biocatalytic processes that it does not address. Even where it can be successfully applied, the resources required for its implementation must currently be weighed against the feasibility of, and resources available for developing a chemical synthesis route. Here, we review the potential of combining directed evolution with process engineering, and recent developments to improve their implementation. Favourable targets for the directed evolution of new biocatalysts are the syntheses of highly complex molecules, especially where chemistry, metabolic engineering or recombineering provide a partial solution. We also review some of the recent advances in the application of these approaches alongside the directed evolution of biocatalysts.     

Metabolic engineering in dark fermentative hydrogen production; theory and practice

Dark fermentation is an attractive option for hydrogen production since it could use already existing reactor technology and readily available substrates without requiring a direct input of solar energy. However, a number of improvements are required before the rates and yields of such a process approach those required for a practical process. Among the options for achieving the required advances, metabolic engineering offers some powerful tools for remodeling microbes to increase product production rates and molar yields. Here we review the current metabolic engineering tool box that is available, discuss the current status of engineering efforts as applied to dark hydrogen production, and suggest areas for future improvements.     

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature’s biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

     

Engineered enzymes for chemical production

In order to enable competitive manufacturing routes, most biocatalysts must be tailor-made for their processes. Enzymes from nature rarely have the combined properties necessary for industrial chemical production such as high activity and selectivity on non-natural substrates and toleration of high concentrations of organic media over the wide range of conditions (decreasing substrate, increasing product concentrations, solvents, etc.,) that will be present over the course of a manufacturing process. With the advances in protein engineering technologies, a variety of enzyme properties can be altered simultaneously, if the appropriate screening parameters are employed. Here we discuss the process of directed evolution for the generation of commercially viable biocatalysts for the production of fine chemicals, and how novel approaches have helped to overcome some of the challenges.     

Directed evolution of novel protein functions

Directed evolution has been successfully used to engineer proteins for basic and applied biological research. However, engineering of novel protein functions by directed evolution remains an overwhelming challenge. This challenge may come from the fact that multiple simultaneously or synergistic mutations are required for the creation of a novel protein function. Here we review the key developments in engineering of novel protein functions by using either directed evolution or a combined directed evolution and rational or computational design approach. Specific attention will be paid to a molecular evolution model for generation of novel proteins. The engineered novel proteins should not only broaden the range of applications of proteins but also provide new insights into protein structure-function relationship and protein evolution.     

Automated engineering of synthetic metabolic pathways for efficient biomanufacturing

Metabolic engineering involves the engineering and optimization of processes from single-cell to fermentation in order to increase production of valuable chemicals for health, food, energy, materials and others. A systems approach to metabolic engineering has gained traction in recent years thanks to advances in strain engineering, leading to an accelerated scaling from rapid prototyping to industrial production. Metabolic engineering is nowadays on track towards a truly manufacturing technology, with reduced times from conception to production enabled by automated protocols for DNA assembly of metabolic pathways in engineered producer strains. In this review, we discuss how the success of the metabolic engineering pipeline often relies on retrobiosynthetic protocols able to identify promising production routes and dynamic regulation strategies through automated biodesign algorithms, which are subsequently assembled as embedded integrated genetic circuits in the host strain. Those approaches are orchestrated by an experimental design strategy that provides optimal scheduling planning of the DNA assembly, rapid prototyping and, ultimately, brings forward an accelerated Design-Build-Test-Learn cycle and the overall optimization of the biomanufacturing process. Achieving such a vision will address the increasingly compelling demand in our society for delivering valuable biomolecules in an affordable, inclusive and sustainable bioeconomy.     

Metabolic engineering of aroma components in fruits

Plants have the ability to produce a diversity of volatile metabolites, which attract pollinators and seed dispersers and strengthen plant defense responses. Selection by plant breeders of traits such as rapid growth and yield leads, in many cases, to the loss of flavor and aroma quality in crops. How the aroma can be improved without affecting other fruit attributes is a major unsolved issue. Significant advances in metabolic engineering directed at improving the set of volatiles that the fruits emit has been aided by the characterization of enzymes involved in the biosynthesis of flavor and aroma compounds in some fruits. However, before this technology can be successfully applied to modulate the production of volatiles in different crops, further basic research is needed on the mechanisms that lead to the production of these compounds in plants. Here we review the biosynthesis and function of volatile compounds in plants, and the attempts that have been made to manipulate fruit aroma biosynthesis by metabolic engineering. In addition, we discuss the possibilities that molecular breeding offers for aroma enhancement and the implications of the latest advances in biotechnological modification of fruit flavor and aroma.     

Starch and α-glucan acting enzymes, modulating their properties by directed evolution

Starch is the major food reserve in plants and forms a large part of the daily calorie intake in the human diet. Industrially, starch has become a major raw material in the production of various products including bio-ethanol, coating and anti-staling agents. The complexity and diversity of these starch based industries and the demand for high quality end products through extensive starch processing, can only be met through the use of a broad range of starch and α-glucan modifying enzymes. The economic importance of these enzymes is such that the starch industry has grown to be the largest market for enzymes after the detergent industry. However, as the starch based industries expand and develop the demand for more efficient enzymes leading to lower production cost and higher quality products increases. This in turn stimulates interest in modifying the properties of existing starch and α-glucan acting enzymes through a variety of molecular evolution strategies. Within this review we examine and discuss the directed evolution strategies applied in the modulation of specific properties of starch and α-glucan acting enzymes and highlight the recent developments in the field of directed evolution techniques which are likely to be implemented in the future engineering of these enzymes.     

In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection

Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process. For cultivation of library clones we employed optically clear gel-like microcarriers of nanoliter volume (NLRs) as reaction vessels and used fluorescence-assisted particle sorting to estimate the growth behavior of each of the gel-embedded clones in a highly parallelized fashion. We demonstrate that the growth behavior correlates with the desired improvements in enzyme performance and that we can fine-tune selection stringency by including an antimetabolite in the assay. As a model enzyme reaction, we improve the racemization of ornithine, a possible starting block for the large-scale synthesis of sulphostin, by a broad-spectrum amino acid racemase and confirm the discriminatory power by showing that even moderately improved enzyme variants can be readily identified.