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The importance of engineering physiological functionality into microbes
Opinion
The importance of engineering physiological functionality into microbes Yanping Zhang1, Yan Zhu1, Yang Zhu2,3 and Yin Li1 1
Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China TNO Quality of Life, Department of Biosciences, PO Box 360, 3700 AJ Zeist, the Netherlands 3 Bioprocess Engineering Group, Wageningen University and Research Centre, PO Box 8129, 6700 EV Wageningen, the Netherlands 2
Good physiological performance of industrial microbes is crucial for successful bioprocesses. Conventional metabolism-oriented engineering strategies often fail to obtain expected phenotypes owing to focusing narrowly on targeted metabolic capabilities while neglecting microbial physiological responses to environmental stresses. To meet the new challenges posed by the biotechnological production of fuels, chemicals and materials, microbes should exert strong physiological robustness and fitness, in addition to strong metabolic capabilities, to enable them to work efficiently in actual bioprocesses. Here, we address the importance of engineering physiological functionalities into microbes and illustrate the operation procedure. We believe that this physiology-oriented engineering strategy is a promising approach for improving the physiological performance of industrial microbes for efficient bioprocesses. Developments in engineering microbes and new challenges Microbes are widely used for the production of pharmaceuticals, enzymes and chemicals (Figure 1) [1,2]. Given that the productivity of microbes isolated from nature is generally low, genetic and metabolic engineering strategies have been used increasingly to either modify or introduce new cellular metabolic capabilities. Since the 1990s, metabolically engineered microbes have been applied extensively in the production of organic acid, amino acids [3], sugar alcohols [4], biofuels [5] and pharmaceuticals [6]. Recent developments in ‘-omics’ technologies have powered metabolic engineering strategies from the level of a local pathway to that of the global metabolic network. Advances in metabolic engineering combined with various ‘-omics’ approaches [7] have contributed to improving cellular metabolic activities to achieve a more efficient biotechnological production of target products [8– 10]. Previous efforts to engineer microbes were mainly metabolism oriented. Scientists attempted to improve product yield and to decrease costs by modifying microbial metabolic pathways and substrate transport systems, predominantly by using gene-by-gene modifications. These Corresponding author: Li, Y. ([email protected])
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strategies are more effective compared with conventional breeding approaches that often randomly alter the metabolism of the target microbes [2]. Nevertheless, metabolismoriented engineering approaches often cause unwanted cellular stresses that are induced by overproduction of foreign proteins [11], or by the accumulation of toxic intermediates [12], resulting in a decrease in overall cell fitness. In addition, single- or multiple-genetic modification of target metabolic pathways frequently result in the alteration of the intracellular micro-environment (e.g. internal redox state) within the engineered microbes, leading to changes of in situ enzyme activities [13,14] and thus an unexpected phenotype. Moreover, engineering the performance of industrial microbes should not only rely on engineering the metabolic capability, as this is often affected by external circumstances, such as the redox potential of the environment [15]. The solution to these problems requires a comprehensive understanding of the complex metabolic and regulatory networks of microbes. As the entire microbial cell, rather than a specific pathway, functions in actual bioprocesses, it is crucial to modify the strain globally as opposed to only engineering a few specific pathways. There are several obstacles in the biotechnological production of fuels, chemicals and materials that demonstrate the urgency behind developing an alternative engineering approach (Table 1). First, unavoidable perturbations are often encountered in actual industrial bioprocesses owing to the non-uniformity of large-scale fermentation systems. This leads to the exposure of industrial strains to physical stresses, such as unsuitable pH, temperature [16] and dissolved oxygen [17], as well as osmotic pressure [18]. The physiological characteristics of microbes thus need to be modified to render them sufficiently robust to be able to work efficiently in industrial bioprocesses. Second, it is always desirable to achieve the highest final titer of the target metabolite; however, a prerequisite for this goal is that microbes must tolerate high concentrations of metabolites and substrates [19,20]. Third, microbes are expected to grow and produce target metabolite as efficiently as possible to achieve the highest productivity. However, the fitness of microbes needs improvement to enable them to work efficiently throughout the entire production process, particularly during the final stages in which metabolites
0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.08.006 Available online 28 September 2009
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Figure 1. Recent advances in the engineering of microbes. Before 1970, microbes were used mainly in the traditional brewing and fermentation industries, such as the production of soy sauce, cheese, alcohol, antibiotics and other natural products. The production strains used were either selected from nature or mutated physically and chemically. Since the 1970s, with the development of recombinant DNA technology, scientists began to engineer microbes to meet desired requirements using genetic engineering, which enabled the introduction of novel microbial metabolic pathways. Among the products produced biotechnologically were recombinant pharmaceuticals and their precursors, antibiotics, amino acids and enzymes. Since the 1990s, the concept of metabolic engineering has enabled further improvements in microbial engineering and facilitated the broadening of substrates spectra, enabling improved titers and yields, as well as the synthesis of new bioproducts. Metabolically engineered microbes have driven the rapid development of the biotechnological production of fine-chemicals, amino acids and biodegradable polymers. More recently, increasing food demands together with impending energy crises and environmental pollution, have driven the application of microbes for the production of biofuels, bulk-chemicals and biomaterials from renewable non-food biomass, which requires further improvements of the microbes used to increase product titer and productivity and to decrease the incurring production cost. As discussed in the main text, we argue that physiological engineering aimed at introducing physiological functionalities into microbes will be able to meet these new challenges in the future.
are largely accumulated, while nutrients are in shortage and cell density is high. Lastly, industrial strains are used increasingly for the conversion of cellulosic biomass to bulk chemicals and to value-added metabolites, which requires their resistance to inhibitors present in lignocellulosic hydrolysates [21]. In addition to these challenges, industrial strains also need to meet the challenges of actual industrial bioprocesses, such as tolerance to high temperature [22] and strong acidic conditions [23]. The metabolic activity of microbes reflects their physiological responses to their habitats or specific environment. The microbial physiological performance, which determines the applicability of an industrial strain, depends on the modification and the integration of intracellular components, and is influenced by the extracellular environment, such as fermentation conditions. Compared with their metabolic capability, other physiological characteristics of microbes (mainly their fitness and robustness) affect their industrial performance, and thus should also be engineered to ensure industrially valuable strains. To this end, a physiology-oriented engineering approach, aimed at improving the physiological performances of industrial strains, is important to realize efficient bioprocesses.
The concept of engineering physiological functionalities into microbes Engineering of microbial physiological functionalities can be achieved by, but is not limited to, strain adaptation, strain evolution and genetic modification. The main physiological characteristics related to industrial applications include microbial metabolic capability, insensitivity of pathway key enzymes to end-product inhibition or feedback repression, robustness under adverse environmental perturbations, tolerance of high concentration substrates or metabolites, and fitness throughout the entire production processes. Different from the conventional metabolism-oriented engineering strategy, such a strategy focuses primarily on the physiological status of microbial cells and on the physiological functionality related to the actual biotechnological production processes. Therefore, this strategy aims not only to improve microbial metabolic activities at a specific physiological status, but also to further investigate the molecular mechanisms underpinning the desired physiological characteristics. To engineer microbial functionalities successfully, it is important to understand how cells sense and subsequently adapt to environmental changes and what contributes to 665
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Table 1. Physiological characteristics important for industrial microbes Requirement of bioprocess
Corresponding physiological characteristics Metabolic capability: selective conversion of substrate to target product with reduced generation of by-products
Host
Strategy
Klebsiella pneumoniae
Elimination of ethanol production
Results
1,3-proanediol yield = 0.7 mol mol-1 Bacillus subtilis Elimination of lactate and butanediol Ethanol yield = 0.51 g g-1 pathway; introduction of ethanol pathways Thermoanaerobacterium Inactivation of pathways for Ethanol titer = 37 g L-1 saccharolyticum organic acid formation Lactococcus lactis Inactivation of lactate Homolactic acid dehydrogenase and introduction fermentation shifted of alanine dehydrogenase to homoalanine fermentation Succinic acid yield Mannheimia Inactivation of metabolic = 0.97 g g-1 succiniciproducens pathways leading to by-product formation by disrupting the ldhA, pflB, pta and ackA genes Pseudomonas putida Expression of xylose isomerase Growth yield on Metabolic capability: Inexpensive and xylulokinase xylose (52%), arabinose wide substrate spectra medium (52%) and glucose (55%) and simple growth formulation requirements Zymomonas mobilis Insertion of essential genes Co-fermentation of for pentose utilization glucose, xylose and arabinose; ethanol yield = 84%, ethanol titer = 42 g L-1 Corynebacterium Heterologous expression Oxygen-deprived glutamicum of xylA and xylB genes strain CRX2 cells from E. coli simultaneously consumed 5% glucose and 2.5% xylose in a mineral medium Clostridium Overexpression of Butanol titer Tolerance of high High titer acetobutylicum groESL genes increased by 40% concentration of Lactobacillus Overproduction of three Enhanced survival in metabolites and plantarum WCFS1 small heat shock proteins the presence of substrates 12% ethanol Saccharomyces Mutagenesis of transcription Volumetric production cerevisiae factor Spt15p and selection of ethanol increased for increased ethanol tolerance by 69% Fitness throughout Escherichia coli Balancing of carbon Fermentation of high High the entire production flux: fermentative pathway concentrations of productivity process and glyoxylate pathway glucose; 24 h; increased succinic acid yield (1.6 mol mol-1) High resistance Pichia stipitis Adaptation: enhanced Ethanol yield = 0.45 g g-1 Growth with to inhibitors sulfate and furfural tolerance in NaOH-neutralized rice undiluted straw hydrolysate without cellulosic detoxification hydrolysates Fitness at high Bacillus Isolated from nature 55oC; L-lactic acid Low probability sp. strain 2-6 = 182 g L-1; productivity of contamination temperature or acidic pH conditions = 3 g L-1 h-1 in open fermentation without sterilization Bacillus coagulans Isolated from nature 50oC; pH 5.0; conversion of biomass-derived sugars to L-lactic acid Lactobacillus Genome shuffling Ability to grow at a strain LB-WT lower pH; production of lactic acid increased threefold Lactococcus lactis Glutathione taken up from Improved resistance to Robustness to Strain stability medium or produced by oxidative and acid stress environmental metabolic engineering perturbations and high genetic stability Escherichia coli K-12 A multiple-deletion series, Accurate propagation reduction in genome of recombinant genes size of up to 15% Genetic stability increased Escherichia coli Chemical-induced chromosomal tenfold; growth-phase evolution (CIChE), using recA productivity of homologous recombination to poly-3-hydroxybutyrate evolve a chromosome with increased fourfold multiple tandem copies of recombinant genes
High yield of target product
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Refs
[34] [58]
[22] [59]
[9]
[60]
[38]
[61]
[62] [63]
[24]
[64]
[65]
[66]
[67]
[68]
[69,70]
[71]
[72]
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Table 1 (Continued ) Requirement of bioprocess Ease of separation or downstream processing
Corresponding physiological characteristics Cell size and flocculation
Host
Strategy
Results
Refs
Saccharomyces cerevisiae
Introduction of different combinations of flocculation genes and promoters
Flocculation behavior of yeast strains can be tightly controlled and fine tuned
[73]
these adaptations. Furthermore, it is crucial to elucidate why some species have specific physiological characteristics, such as their ability to survive high concentrations of solvents, high temperature and high osmotic pressure, which is useful for their efficient engineering. Therefore, microbial physiological analysis and engineering approaches must be taken into account when attempting to engineer physiological functionalities into microbes. A general strategy for engineering physiological functionalities A generally feasible strategy for strain improvement by means of physiological engineering (Box 1) relies on integration of knowledge of physiological functionality and efficient engineering approaches. Basic procedures and approaches that need to be considered are illustrated in Figure 2. However, successful engineering of physiological functionality is not necessarily confined to the procedure and techniques presented here. First step: defining the desired physiological characteristics of target strains The decision about which are the desired physiological characteristics of a target strain is usually based on the respective bioprocess conditions, which depend on different factors, such as market requirements for the microbial products, convergence of upstream and downstream processes and the cost of the whole bioprocess. For example, to produce ethanol from corn straw, a promising process is to ferment corn straw that has been hydrolyzed by sulfuric acid to ethanol using strains that
Box 1. Physiological engineering: an evolving technology for strain improvement The term ‘physiological engineering’ was first used by Jens Nielsen in 1997 in relation to penicillin production by Penicillium chrysogenum [57]. At that time, physiological engineering referred to understanding the function of important pathways in microorganisms by using an integrated approach of microbial physiology and bioreaction engineering. It involved metabolic flux analysis, metabolic control analysis and kinetic modeling to generate fundamental knowledge for metabolic engineering that was based on reproducible cultivation experiments and reliable measurements. During the past decade, metabolic engineering has become the central approach for strain improvement, mainly targeted at improving specific metabolic capabilities. However, other physiological functionalities, including adaptation ability, fitness, robustness and stress tolerance, are also important for efficient biotechnological production of fuels, chemicals, and materials. In this context, an evolved concept of physiological engineering, in the sense it is used here, refers to a strategy of strain improvement with the aim of either improving existing or engineering novel functionalities into microbes to meet new (industrial) challenges.
could convert both hexose and pentose in the hydrolysate while simultaneously tolerating high concentrations of ethanol [24] and of any inhibitors present in the straw hydrolysate, such as acetate and furfural [25]. Furthermore, to avoid contamination and to save costs associated with cooling, acidic fermentation at high temperature is desirable [22]. Moreover, to increase productivity, the microbial cells should also be able to convert the sugars to ethanol at high speed [26]. Therefore, the ideal candidate strains for a process converting lignocellulose to ethanol should have at least the following physiological characteristics: (i) efficient conversion of hexose and pentose to ethanol; (ii) high tolerance of ethanol and inhibitors; (iii) active metabolism under high temperature or acidic conditions (or both); (iv) fitness throughout the entire production process; and (v) characteristics suitable for down-stream processes, such as cell size and their flocculation capability. Second step: screening candidate strains that have the target physiological characteristics Strains that have one or more of the specific desired physiological characteristics can be obtained by directed evolution, random or rational mutation, and stressinduced adaptation [27,28]. The resulting strains, as well as any naturally occurring thermophiles, acidophiles, alkaliphiles, halophiles, or solvent-tolerant strains, constitute a suitable microbial starting library for the further screening of candidate strains. The library might thus contain strains from culture collections, or those isolated from nature or mutated and/or engineered artificially. In reality, many native microorganisms that have desirable physiological characteristics cannot necessarily be cultured in the laboratory. In such cases, Escherichia coli clones of a metagenomic library carrying large DNA fragments can be added to the microbial library for screening. Once the starting microbial library is established, the efficiency of the screening process will rely on available high-throughput screening methods. Generally speaking, through targeted selection and directed mutation and/or evolution, potential strains that exhibit at least some of the desired physiological characteristics could already be obtained at this stage. In the case of ethanol production from corn straw hydrolysate at high temperature, some useful starting strains are already known that can utilize xylose, such as Pichia stipitis [29], or are able to ferment glucose to ethanol efficiently with high ethanol tolerance, such as Saccharomyces cerevisiae [24]. Other suitable starting strains include Thermoanaerobacter ethanolicus, which can ferment glucose and xylose to ethanol at 67–70oC [30], and Actinobacillus succinogenes, which is able to tolerate hydrolytic inhibitors 667
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Figure 2. General procedure for strain improvement by physiological engineering. In the first step, the desired physiological characteristics of the target microbes need to be defined, including their desired metabolic capability, robustness and fitness. During the second step, candidate strains, which have one or more of the desired characteristics, are selected and further screened. Candidate strains can be chosen from known extremophiles and solvent-tolerant strains, as well as from any characterized random or rational mutant strains. In the third step, the final host strain is selected, based on the physiological functionalities of the candidate strains and their underlying complexity, which affects their potential to introduce additional individual physiological characteristic. In the fourth step, the remaining desired physiological characteristics are introduced into the host strain via two possible routes: either by introducing metabolic capabilities into host strains with low metabolic capability (Route 1), or by other evolutionary engineering approaches for strains with low robustness and fitness (Route 2), as shown here. When the engineering in step 4 does not achieve expected results, the procedure should restart from step 2 and be repeated until the desired results are achieved.
[31]. Nevertheless, a combination of these desired physiological characteristics will need to be engineered into a single host strain as discussed below. Third step: selecting the host strain Once candidate strains are obtained with one or more desired physiological characteristics, the next step defines the candidate strain that should be used as a host for acquiring the remaining physiological characteristics. To achieve this, further knowledge of the microbial physiological functionality is needed, including an understanding of why these microbes have the specific physiological features, how they acquired these naturally and which cellular components contribute to these. Among the desired physiological characteristics, metabolic capability 668
(metabolic pathways and transport systems) has been studied extensively [1,3,32–35] and the corresponding genes encoding (putative) metabolic capabilities can be identified from genome-wide annotations [2,9,29]. However, other physiological characteristics concerning microbial fitness and robustness are less well understood and probably depend on more complex cellular components. Thus, different physiological characteristics have a different degree of underlying complexity, which will need to be considered when designing engineering strategies. Studies of the natural evolution of microbes might be able to reveal which of the physiological characteristics are most complex and, thus, most difficult to acquire during evolution. In the case of fermenting lignocellulose to ethanol, the tolerance of inhibitors present in
Opinion lignocellulosic hydrolysate is the most complex functionality that could be acquired through evolution. For example, Zymomonas mobilis can produce ethanol with high yield and efforts have been made to broaden its spectrum of substrates [32,36]. However, this strain is not well suited for cellulosic ethanol production owing to its intolerance of acetic acid present in the lignocellulosic hydrolysate [37,38]. The candidate strain that exhibits the most complex physiological characteristics and that is also genetically accessible can then be selected as a suitable host strain, as engineering the remaining and less complex physiological characteristics into this strain will be easier. Fourth step: engineering desired physiological functionalities into the selected host strain As the resulting candidate host strain typically has only some of the desired physiological characteristics, the remaining desired physiological functionalities need to be introduced by engineering approaches. If the host microbial cells are robust and fit, the engineering strategy should focus on introducing the target metabolic capabilities (including necessary pathways, efficient transport and cofactor regeneration systems) into the host strain lacking the metabolic activity for production of the desired product (Route 1, Step 4, Figure 2). Microbial metabolic capability has been studied extensively, and successful application of genetic engineering, metabolic engineering and genome shuffling has been reported [1,3,9,10,32–35,38,39]. If the host strains are genetically accessible, this approach can therefore be implemented relatively easily in practice by using techniques developed from metabolic engineering. Another approach can be designed for microbial cells that are highly metabolically active, but are not sufficiently robust and fit. A promising route to increase the fitness and robustness of microbes is to decipher the natural evolutionary program of extremophiles and other stress-tolerant strains, with the underlying rationale that target physiological characteristics in the host strain could be obtained by stress-induced adaptation, directed evolution of regulation factors or DNA manipulation genes, or genome shuffling. Recent studies of microbial natural evolution have revealed that natural adaptation and evolution are assisted by several master regulators, including alternative sigma factors (a prokaryotic transcription initiation factor that enables specific binding of RNA polymerase to gene promoters), small molecule effectors, gene repressors and some inorganic molecules [27]. Engineering these evolutionary master regulators would therefore constitute a promising evolutionary approach to reprogramming physiological functionality [27] as illustrated in Route 2 of Step 4 in Figure 2. Indeed, by engineering sigma factors, a novel method termed ‘global transcription machinery engineering’ was developed, which resulted in a significantly improved ethanol tolerance by Saccharomyces cerevisiae and a 69% increase in the volumetric productivity of ethanol [24]. Small molecule effectors and gene repressors have also been targeted to generate genetic diversity by fine-tuning transcription or base substitutions and have resulted in an E. coli mutant with improved rifampicin resistance [27]. Microbial evolution studies have also
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revealed that the evolution of microbes is always directed towards their own benefit [40]. Therefore, the key to success is to determine the appropriate evolutionary factors controlling the expected physiological functionalities and to engineer these subsequently. In actual practice, most host strains will need to be modified with regard to both, metabolic capability and their fitness and robustness. The two engineering routes pursued in Step 4 of Figure 2 will thus need to be integrated to engineer a desired industrially valuable strain. Moreover, in an improvement process involving selected strains, approaches for acquiring ‘high robustness and fitness’ are likely to be more complex than those required to introduce high metabolic capabilities. Thus, all steps proposed here might need to be repeated until both desired characteristics are achieved. Specifically, if the engineering attempts of step 4 are unable to achieve the expected results with regard to increasing robustness and fitness, the engineering strategy should restart from step 2 and repeated until desired strains are obtained (Figure 2). Pioneering studies of engineering industrial microbes The past decade has witnessed rapid progress in improving microbial metabolic capability with the development of several novel approaches to engineering microbes, including cofactor engineering to shift metabolite profile [41], systems metabolic engineering for improved amino acid production [10], ribosome engineering for improved antibiotics production [42] and global transcription machinery engineering for improved ethanol productivity [24]. However, only a few engineered strains have been used on an industrial scale successfully. Two of these are further discussed below: an E. coli strain producing 1,3propanediol, and bacteria or yeast strains for the production of ethanol from lignocellulosic materials. As an important chemical and raw material, the biotechnological production of 1,3-propanediol has attracted considerable attention. Much effort has been made to engineer the wild types of 1,3-propanediol-producing strains, such as Klebsiella pneumoniae, Citrobacter freundii and Clostridium butyricum, but the maximum titer of 1,3-propanediol that could be achieved in batch and fedbatch fermentation processes reached only 78 g L-1 [12,43,44]. This was due to the intolerance of E. coli to 1,3-propanediol and an imbalance of the metabolic activities of the glycerol dehydratase and 1,3-propanediol oxidoreductase enzymes that are involved in the 1,3-propanediol production pathway. Escherichia coli has several advantages as a biocatalyst for 1,3-propanediol production, including its ability to ferment a range of sugars, the lack of requirements for complex growth factors, and its proven industrial use (e.g. for production of recombinant proteins), despite the fact that it lacks the necessary pathways of converting sugars to 1,3-propanediol. Through a series of engineering events, including the deletion, overexpression or fine-tuning of 25 targeted genes, DuPont (http:// www.dupont.com/) and Genencor (http://www.genencor. com) successfully obtained an engineered E. coli strain that was able to produce a 1,3-propanediol titer of 130 g L-1 using glucose as feedstock [45]. This success constitutes an elegant bench mark of metabolic engineering. However, 669
Opinion using physiological engineering, the number of the metabolic genes that need to be introduced, disrupted or modified could be reduced significantly. The expected high titer and productivity of 1,3-propandiol might be relatively easily obtained through engineering on transcription levels only. The second example concerns recent advances in engineering strains that are able to produce ethanol from lignocellulosic materials. Here, E. coli [46,47], Z. mobilis [32,36–38,48], which produces ethanol from a specific sugar, and S. cerevisiae [24,49,50], which shows a wide spectrum of sugar substrates, have been used extensively as host strains. To improve ethanol yield, genes involved in either xylose metabolism or ethanol production were successfully expressed, while competing branches were simultaneously eliminated [51]. Subsequently, ethanolinduced adaptation [47,49], or evolution of master regulators (e.g. global transcription machinery engineering [24]) were carried out with the goal to meet the major criteria for cellulosic ethanol producing strains: the tolerance to ethanol and resistance to inhibitors [51,52]. Several isolates showing significant improvements could be obtained and demonstrated great potential for industrial application, although they still exhibited some deficiencies that might limit their application in actual bioprocesses. For E. coli LY01, these included the narrow usable pH range and the high sensitivity to a mixture of inhibitors [47], whereas Z. mobilis AX101 was intolerant to acetic acid [37,38]. Another resulting strain, S. cerevisiae INVSC1, showed a low resistance to low pH and inhibitors [53]. We believe that by implementing the approaches and procedures of physiological engineering as illustrated in Figure 2, further breakthroughs can be anticipated in overcoming microbial physiological limits of these host strains. Alternatively, directly engineering the ethanol production ability into a new robust host strain that is tolerant of inhibitors in hydrolysate and high concentration of ethanol, might be able to meet the demand of cellulosic-based ethanol production. Conclusions and future perspectives Increasing demands on biotechnology to provide solutions for renewable energies, global food security and environmental pollution are the main driving forces for the engineering of novel microbes at a systems level that are able to produce biofuels, bulk-chemicals and other biomaterials [54–56]. Compared with their metabolic capability, the physiological status and the resulting physiological functionalities of microbes, including adaptation and robustness to harsh environments, stress tolerance and fitness throughout the entire production process are becoming increasingly relevant for strains that are to be applied in industrial bioprocesses. To obtain these desired industrial strains, we must define the desired engineering target, obtain strains with various desired characteristics, select the right host strain and engineer desired physiological functionalities into microbes by efficient approaches. The concepts and procedures of engineering physiological functionality into microbes discussed here aim to provide incentives for further strain improvements using engineering approaches. 670
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Apart from the example of lignocellulosic ethanol process described, there is increasing demand from chemical companies to produce other bulk chemicals using biotechnological process. These include 1,3-propanediol, acrylic acid, lactic acid, butanol, 1,4-butanediol, succinic acid, isoprene, isoamyl alcohol, adipic acid and citric acid. The ultimate success of biotechnological routes over conventional petrochemical processes will depend on the one hand on whether biomass-derived sugars can be used as the main carbon source to reduce the raw material cost and, on the other hand, on whether the final product titer can be improved enough to decrease the downstream separation cost. These principles can also be applied to improving the efficiency of biotechnological production of other fermentation products, such as amino acids, organic acids, antibiotics, sugar alcohols, polymers, vitamins and enzymes. Four major obstacles for a wide implementation of physiological engineering currently exist: (i) Engineering targets: as the microbial stress response is a complex system, defining a target for physiological engineering can be difficult even with the assistance of systems biology. Computational tools are thus needed to assist the screening and discovery of potential targets by constructing a virtual cell model and in silico analysis; (ii) Evolution pathway: the presence of extremophiles in extreme environments is a consequence of their longterm evolution and/or adaptation. A comprehensive understanding of the mechanisms underlying the evolution pathway can help to design an artificial evolution pathway to facilitate engineering physiological functionalities; (iii) Genetic instability of the engineered strains: engineered cells can be considered as ‘ill’ rather than ‘normal’ cells with the consequence that microbes might tend to return to the ‘normal’ state owing to the robustness of their metabolic and regulatory networks. The construction of microbes with reduced genome size by a synthetic biology approach might help in this regard, and examples demonstrating this possibility have recently emerged (Table 1); (iv) Population behavior: microbes do not exist alone in nature and microflora composed of single strains or a single species are usually less resistant to environmental perturbations and stresses compared with multi-species population. Knowledge from microbial population behaviors will be helpful in designing an efficient single-species biocatalyst, with the ultimate goal to develop bioprocesses that are based on microbial populations. Taken together, physiological engineering, which expands beyond traditional metabolic engineering paradigms, will become increasingly important in strain improvement approaches. Future insights into microbial physiological functionalities and evolutionary programs are likely to provide increasingly powerful techniques for engineering physiological functionalities into microbes to obtain a highly metabolically active, physiologically robust and fit strain, and therefore ensure their successful applications in biotechnology industries.
Opinion Acknowledgements Funding for our research was provided by the National Natural Science Foundation of China (30870040), National Basic Research Program of China (973 Project, 2007CB707803 and 2007CB714301), National High Technology Research and Development Program of China (863 Project, 2006AA020103 and 2006AA02Z237) and Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-G-005, KSCX2-YW-G007, and KSCX1-YW-11C3). Y.L. is supported by the Hundred Talents Program of the Chinese Academy of Sciences. We thank Linjiang Zhu for helpful discussions.
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23 Ka´da´r, Z. et al. (2007) Ethanol fermentation of various pretreated and hydrolysed substrates at low initial pH. Appl. Biochem. Biotechnol. 137–140, 847–858 24 Alper, H. et al. (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314, 1565–1568 25 Lin, F.M. et al. (2009) Comparative proteomic analysis of tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to furfural, a lignocellulosic inhibitory compound. Appl. Environ. Microbiol. 75, 3765–3776 26 Wisselink, H.W. et al. (2009) Novel evolutionary engineering approach for accelerated utilization of glucose, xylose, and arabinose mixtures by engineered Saccharomyces cerevisiae strains. Appl. Environ. Microbiol. 75, 907–914 27 Foster, P.L. (2007) Stress-induced mutagenesis in bacteria. Crit. Rev. Biochem. Mol. Biol. 42, 373–397 28 Galhardo, R.S. et al. (2007) Mutation as a stress response and the regulation of evolvability. Crit. Rev. Biochem. Mol. Biol. 42, 399–435 29 Jeffries, T.W. et al. (2007) Genome sequence of the lignocellulosebioconverting and xylose-fermenting yeast Pichia stipitis. Nat. Biotechnol. 25, 319–326 30 Lacis, L. and Lawford, H. (1989) Analysis of the variation in ethanol yield from glucose or xylose with continuously grown Thermoanaerobacter ethanolicus. Appl. Biochem. Biotechnol. 20–21, 479 31 Zheng, P. et al. (2009) Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes. Biores. Technol. 100, 2425–2429 32 Zhang, M. et al. (1995) Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science 267, 240–243 33 Alper, H. et al. (2005) Construction of lycopene-overproducing strains by combining systematic and combinatorial gene knockout targets. Nat. Biotechnol. 23, 612–616 34 Zhang, Y. et al. (2006) Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae. Metab. Eng. 8, 578–586 35 de Vos, W.M. and Hugenholtz, J. (2004) Engineering metabolic highways in Lactococci and other lactic acid bacteria. Trends Biotechnol. 22, 72–79 36 Joachimsthal, E.L. and Rogers, P.L. (2000) Characterization of a highproductivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures. Appl. Biochem. Biotechnol. 84–86, 343–356 37 Lawford, H.G. and Rousseau, J.D. (2002) Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose, xylose, and arabinose. Appl. Biochem. Biotechnol. 98–100, 429–448 38 Mohagheghi, A. et al. (2002) Cofermentation of glucose, xylose, and arabinose by genomic DNA-integrated xylose/arabinose fermenting strain of Zymomonas mobilis AX101. Appl. Biochem. Biotechnol. 98– 100, 885–898 39 Hida, H. et al. (2007) Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid. Appl. Microbiol. Biotechnol. 73, 1387–1393 40 Robleto, E.A. et al. (2007) Stationary phase mutagenesis in B. subtilis: a paradigm to study genetic diversity programs in cells under stress. Crit. Rev. Biochem. Mol. Biol. 42, 327–339 41 Lopez de Felipe, F. et al. (1998) Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. J. Bacteriol. 180, 3804–3808 42 Wang, G. et al. (2008) Dramatic activation of antibiotic production in Streptomyces coelicolor by cumulative drug resistance mutations. Appl. Environ. Microbiol. 74, 2834–2840 43 Hao, J. et al. (2008) Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol production by over-expressing dhaT gene in Klebsiella pneumoniae TUAC01. J. Ind. Microbiol. Biotechnol. 35, 735–741 44 Nakamura, C.E. and Whited, G.M. (2003) Metabolic engineering for the microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459 45 Emptage, M. et al. E I DuPont de Nemours and Company (Wilmington, USA). Process for the biological production of 1,3–propanediol with high titer. US Patent 6514733 46 Ohta, K. et al. (1991) Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Appl. Environ. Microbiol. 57, 893–900
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The importance of engineering physiological functionality into microbes Yanping Zhang1, Yan Zhu1, Yang Zhu2,3 and Yin Li1 1
Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China TNO Quality of Life, Department of Biosciences, PO Box 360, 3700 AJ Zeist, the Netherlands 3 Bioprocess Engineering Group, Wageningen University and Research Centre, PO Box 8129, 6700 EV Wageningen, the Netherlands 2
Good physiological performance of industrial microbes is crucial for successful bioprocesses. Conventional metabolism-oriented engineering strategies often fail to obtain expected phenotypes owing to focusing narrowly on targeted metabolic capabilities while neglecting microbial physiological responses to environmental stresses. To meet the new challenges posed by the biotechnological production of fuels, chemicals and materials, microbes should exert strong physiological robustness and fitness, in addition to strong metabolic capabilities, to enable them to work efficiently in actual bioprocesses. Here, we address the importance of engineering physiological functionalities into microbes and illustrate the operation procedure. We believe that this physiology-oriented engineering strategy is a promising approach for improving the physiological performance of industrial microbes for efficient bioprocesses. Developments in engineering microbes and new challenges Microbes are widely used for the production of pharmaceuticals, enzymes and chemicals (Figure 1) [1,2]. Given that the productivity of microbes isolated from nature is generally low, genetic and metabolic engineering strategies have been used increasingly to either modify or introduce new cellular metabolic capabilities. Since the 1990s, metabolically engineered microbes have been applied extensively in the production of organic acid, amino acids [3], sugar alcohols [4], biofuels [5] and pharmaceuticals [6]. Recent developments in ‘-omics’ technologies have powered metabolic engineering strategies from the level of a local pathway to that of the global metabolic network. Advances in metabolic engineering combined with various ‘-omics’ approaches [7] have contributed to improving cellular metabolic activities to achieve a more efficient biotechnological production of target products [8– 10]. Previous efforts to engineer microbes were mainly metabolism oriented. Scientists attempted to improve product yield and to decrease costs by modifying microbial metabolic pathways and substrate transport systems, predominantly by using gene-by-gene modifications. These Corresponding author: Li, Y. ([email protected])
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strategies are more effective compared with conventional breeding approaches that often randomly alter the metabolism of the target microbes [2]. Nevertheless, metabolismoriented engineering approaches often cause unwanted cellular stresses that are induced by overproduction of foreign proteins [11], or by the accumulation of toxic intermediates [12], resulting in a decrease in overall cell fitness. In addition, single- or multiple-genetic modification of target metabolic pathways frequently result in the alteration of the intracellular micro-environment (e.g. internal redox state) within the engineered microbes, leading to changes of in situ enzyme activities [13,14] and thus an unexpected phenotype. Moreover, engineering the performance of industrial microbes should not only rely on engineering the metabolic capability, as this is often affected by external circumstances, such as the redox potential of the environment [15]. The solution to these problems requires a comprehensive understanding of the complex metabolic and regulatory networks of microbes. As the entire microbial cell, rather than a specific pathway, functions in actual bioprocesses, it is crucial to modify the strain globally as opposed to only engineering a few specific pathways. There are several obstacles in the biotechnological production of fuels, chemicals and materials that demonstrate the urgency behind developing an alternative engineering approach (Table 1). First, unavoidable perturbations are often encountered in actual industrial bioprocesses owing to the non-uniformity of large-scale fermentation systems. This leads to the exposure of industrial strains to physical stresses, such as unsuitable pH, temperature [16] and dissolved oxygen [17], as well as osmotic pressure [18]. The physiological characteristics of microbes thus need to be modified to render them sufficiently robust to be able to work efficiently in industrial bioprocesses. Second, it is always desirable to achieve the highest final titer of the target metabolite; however, a prerequisite for this goal is that microbes must tolerate high concentrations of metabolites and substrates [19,20]. Third, microbes are expected to grow and produce target metabolite as efficiently as possible to achieve the highest productivity. However, the fitness of microbes needs improvement to enable them to work efficiently throughout the entire production process, particularly during the final stages in which metabolites
0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.08.006 Available online 28 September 2009
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Figure 1. Recent advances in the engineering of microbes. Before 1970, microbes were used mainly in the traditional brewing and fermentation industries, such as the production of soy sauce, cheese, alcohol, antibiotics and other natural products. The production strains used were either selected from nature or mutated physically and chemically. Since the 1970s, with the development of recombinant DNA technology, scientists began to engineer microbes to meet desired requirements using genetic engineering, which enabled the introduction of novel microbial metabolic pathways. Among the products produced biotechnologically were recombinant pharmaceuticals and their precursors, antibiotics, amino acids and enzymes. Since the 1990s, the concept of metabolic engineering has enabled further improvements in microbial engineering and facilitated the broadening of substrates spectra, enabling improved titers and yields, as well as the synthesis of new bioproducts. Metabolically engineered microbes have driven the rapid development of the biotechnological production of fine-chemicals, amino acids and biodegradable polymers. More recently, increasing food demands together with impending energy crises and environmental pollution, have driven the application of microbes for the production of biofuels, bulk-chemicals and biomaterials from renewable non-food biomass, which requires further improvements of the microbes used to increase product titer and productivity and to decrease the incurring production cost. As discussed in the main text, we argue that physiological engineering aimed at introducing physiological functionalities into microbes will be able to meet these new challenges in the future.
are largely accumulated, while nutrients are in shortage and cell density is high. Lastly, industrial strains are used increasingly for the conversion of cellulosic biomass to bulk chemicals and to value-added metabolites, which requires their resistance to inhibitors present in lignocellulosic hydrolysates [21]. In addition to these challenges, industrial strains also need to meet the challenges of actual industrial bioprocesses, such as tolerance to high temperature [22] and strong acidic conditions [23]. The metabolic activity of microbes reflects their physiological responses to their habitats or specific environment. The microbial physiological performance, which determines the applicability of an industrial strain, depends on the modification and the integration of intracellular components, and is influenced by the extracellular environment, such as fermentation conditions. Compared with their metabolic capability, other physiological characteristics of microbes (mainly their fitness and robustness) affect their industrial performance, and thus should also be engineered to ensure industrially valuable strains. To this end, a physiology-oriented engineering approach, aimed at improving the physiological performances of industrial strains, is important to realize efficient bioprocesses.
The concept of engineering physiological functionalities into microbes Engineering of microbial physiological functionalities can be achieved by, but is not limited to, strain adaptation, strain evolution and genetic modification. The main physiological characteristics related to industrial applications include microbial metabolic capability, insensitivity of pathway key enzymes to end-product inhibition or feedback repression, robustness under adverse environmental perturbations, tolerance of high concentration substrates or metabolites, and fitness throughout the entire production processes. Different from the conventional metabolism-oriented engineering strategy, such a strategy focuses primarily on the physiological status of microbial cells and on the physiological functionality related to the actual biotechnological production processes. Therefore, this strategy aims not only to improve microbial metabolic activities at a specific physiological status, but also to further investigate the molecular mechanisms underpinning the desired physiological characteristics. To engineer microbial functionalities successfully, it is important to understand how cells sense and subsequently adapt to environmental changes and what contributes to 665
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Table 1. Physiological characteristics important for industrial microbes Requirement of bioprocess
Corresponding physiological characteristics Metabolic capability: selective conversion of substrate to target product with reduced generation of by-products
Host
Strategy
Klebsiella pneumoniae
Elimination of ethanol production
Results
1,3-proanediol yield = 0.7 mol mol-1 Bacillus subtilis Elimination of lactate and butanediol Ethanol yield = 0.51 g g-1 pathway; introduction of ethanol pathways Thermoanaerobacterium Inactivation of pathways for Ethanol titer = 37 g L-1 saccharolyticum organic acid formation Lactococcus lactis Inactivation of lactate Homolactic acid dehydrogenase and introduction fermentation shifted of alanine dehydrogenase to homoalanine fermentation Succinic acid yield Mannheimia Inactivation of metabolic = 0.97 g g-1 succiniciproducens pathways leading to by-product formation by disrupting the ldhA, pflB, pta and ackA genes Pseudomonas putida Expression of xylose isomerase Growth yield on Metabolic capability: Inexpensive and xylulokinase xylose (52%), arabinose wide substrate spectra medium (52%) and glucose (55%) and simple growth formulation requirements Zymomonas mobilis Insertion of essential genes Co-fermentation of for pentose utilization glucose, xylose and arabinose; ethanol yield = 84%, ethanol titer = 42 g L-1 Corynebacterium Heterologous expression Oxygen-deprived glutamicum of xylA and xylB genes strain CRX2 cells from E. coli simultaneously consumed 5% glucose and 2.5% xylose in a mineral medium Clostridium Overexpression of Butanol titer Tolerance of high High titer acetobutylicum groESL genes increased by 40% concentration of Lactobacillus Overproduction of three Enhanced survival in metabolites and plantarum WCFS1 small heat shock proteins the presence of substrates 12% ethanol Saccharomyces Mutagenesis of transcription Volumetric production cerevisiae factor Spt15p and selection of ethanol increased for increased ethanol tolerance by 69% Fitness throughout Escherichia coli Balancing of carbon Fermentation of high High the entire production flux: fermentative pathway concentrations of productivity process and glyoxylate pathway glucose; 24 h; increased succinic acid yield (1.6 mol mol-1) High resistance Pichia stipitis Adaptation: enhanced Ethanol yield = 0.45 g g-1 Growth with to inhibitors sulfate and furfural tolerance in NaOH-neutralized rice undiluted straw hydrolysate without cellulosic detoxification hydrolysates Fitness at high Bacillus Isolated from nature 55oC; L-lactic acid Low probability sp. strain 2-6 = 182 g L-1; productivity of contamination temperature or acidic pH conditions = 3 g L-1 h-1 in open fermentation without sterilization Bacillus coagulans Isolated from nature 50oC; pH 5.0; conversion of biomass-derived sugars to L-lactic acid Lactobacillus Genome shuffling Ability to grow at a strain LB-WT lower pH; production of lactic acid increased threefold Lactococcus lactis Glutathione taken up from Improved resistance to Robustness to Strain stability medium or produced by oxidative and acid stress environmental metabolic engineering perturbations and high genetic stability Escherichia coli K-12 A multiple-deletion series, Accurate propagation reduction in genome of recombinant genes size of up to 15% Genetic stability increased Escherichia coli Chemical-induced chromosomal tenfold; growth-phase evolution (CIChE), using recA productivity of homologous recombination to poly-3-hydroxybutyrate evolve a chromosome with increased fourfold multiple tandem copies of recombinant genes
High yield of target product
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Refs
[34] [58]
[22] [59]
[9]
[60]
[38]
[61]
[62] [63]
[24]
[64]
[65]
[66]
[67]
[68]
[69,70]
[71]
[72]
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Table 1 (Continued ) Requirement of bioprocess Ease of separation or downstream processing
Corresponding physiological characteristics Cell size and flocculation
Host
Strategy
Results
Refs
Saccharomyces cerevisiae
Introduction of different combinations of flocculation genes and promoters
Flocculation behavior of yeast strains can be tightly controlled and fine tuned
[73]
these adaptations. Furthermore, it is crucial to elucidate why some species have specific physiological characteristics, such as their ability to survive high concentrations of solvents, high temperature and high osmotic pressure, which is useful for their efficient engineering. Therefore, microbial physiological analysis and engineering approaches must be taken into account when attempting to engineer physiological functionalities into microbes. A general strategy for engineering physiological functionalities A generally feasible strategy for strain improvement by means of physiological engineering (Box 1) relies on integration of knowledge of physiological functionality and efficient engineering approaches. Basic procedures and approaches that need to be considered are illustrated in Figure 2. However, successful engineering of physiological functionality is not necessarily confined to the procedure and techniques presented here. First step: defining the desired physiological characteristics of target strains The decision about which are the desired physiological characteristics of a target strain is usually based on the respective bioprocess conditions, which depend on different factors, such as market requirements for the microbial products, convergence of upstream and downstream processes and the cost of the whole bioprocess. For example, to produce ethanol from corn straw, a promising process is to ferment corn straw that has been hydrolyzed by sulfuric acid to ethanol using strains that
Box 1. Physiological engineering: an evolving technology for strain improvement The term ‘physiological engineering’ was first used by Jens Nielsen in 1997 in relation to penicillin production by Penicillium chrysogenum [57]. At that time, physiological engineering referred to understanding the function of important pathways in microorganisms by using an integrated approach of microbial physiology and bioreaction engineering. It involved metabolic flux analysis, metabolic control analysis and kinetic modeling to generate fundamental knowledge for metabolic engineering that was based on reproducible cultivation experiments and reliable measurements. During the past decade, metabolic engineering has become the central approach for strain improvement, mainly targeted at improving specific metabolic capabilities. However, other physiological functionalities, including adaptation ability, fitness, robustness and stress tolerance, are also important for efficient biotechnological production of fuels, chemicals, and materials. In this context, an evolved concept of physiological engineering, in the sense it is used here, refers to a strategy of strain improvement with the aim of either improving existing or engineering novel functionalities into microbes to meet new (industrial) challenges.
could convert both hexose and pentose in the hydrolysate while simultaneously tolerating high concentrations of ethanol [24] and of any inhibitors present in the straw hydrolysate, such as acetate and furfural [25]. Furthermore, to avoid contamination and to save costs associated with cooling, acidic fermentation at high temperature is desirable [22]. Moreover, to increase productivity, the microbial cells should also be able to convert the sugars to ethanol at high speed [26]. Therefore, the ideal candidate strains for a process converting lignocellulose to ethanol should have at least the following physiological characteristics: (i) efficient conversion of hexose and pentose to ethanol; (ii) high tolerance of ethanol and inhibitors; (iii) active metabolism under high temperature or acidic conditions (or both); (iv) fitness throughout the entire production process; and (v) characteristics suitable for down-stream processes, such as cell size and their flocculation capability. Second step: screening candidate strains that have the target physiological characteristics Strains that have one or more of the specific desired physiological characteristics can be obtained by directed evolution, random or rational mutation, and stressinduced adaptation [27,28]. The resulting strains, as well as any naturally occurring thermophiles, acidophiles, alkaliphiles, halophiles, or solvent-tolerant strains, constitute a suitable microbial starting library for the further screening of candidate strains. The library might thus contain strains from culture collections, or those isolated from nature or mutated and/or engineered artificially. In reality, many native microorganisms that have desirable physiological characteristics cannot necessarily be cultured in the laboratory. In such cases, Escherichia coli clones of a metagenomic library carrying large DNA fragments can be added to the microbial library for screening. Once the starting microbial library is established, the efficiency of the screening process will rely on available high-throughput screening methods. Generally speaking, through targeted selection and directed mutation and/or evolution, potential strains that exhibit at least some of the desired physiological characteristics could already be obtained at this stage. In the case of ethanol production from corn straw hydrolysate at high temperature, some useful starting strains are already known that can utilize xylose, such as Pichia stipitis [29], or are able to ferment glucose to ethanol efficiently with high ethanol tolerance, such as Saccharomyces cerevisiae [24]. Other suitable starting strains include Thermoanaerobacter ethanolicus, which can ferment glucose and xylose to ethanol at 67–70oC [30], and Actinobacillus succinogenes, which is able to tolerate hydrolytic inhibitors 667
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Figure 2. General procedure for strain improvement by physiological engineering. In the first step, the desired physiological characteristics of the target microbes need to be defined, including their desired metabolic capability, robustness and fitness. During the second step, candidate strains, which have one or more of the desired characteristics, are selected and further screened. Candidate strains can be chosen from known extremophiles and solvent-tolerant strains, as well as from any characterized random or rational mutant strains. In the third step, the final host strain is selected, based on the physiological functionalities of the candidate strains and their underlying complexity, which affects their potential to introduce additional individual physiological characteristic. In the fourth step, the remaining desired physiological characteristics are introduced into the host strain via two possible routes: either by introducing metabolic capabilities into host strains with low metabolic capability (Route 1), or by other evolutionary engineering approaches for strains with low robustness and fitness (Route 2), as shown here. When the engineering in step 4 does not achieve expected results, the procedure should restart from step 2 and be repeated until the desired results are achieved.
[31]. Nevertheless, a combination of these desired physiological characteristics will need to be engineered into a single host strain as discussed below. Third step: selecting the host strain Once candidate strains are obtained with one or more desired physiological characteristics, the next step defines the candidate strain that should be used as a host for acquiring the remaining physiological characteristics. To achieve this, further knowledge of the microbial physiological functionality is needed, including an understanding of why these microbes have the specific physiological features, how they acquired these naturally and which cellular components contribute to these. Among the desired physiological characteristics, metabolic capability 668
(metabolic pathways and transport systems) has been studied extensively [1,3,32–35] and the corresponding genes encoding (putative) metabolic capabilities can be identified from genome-wide annotations [2,9,29]. However, other physiological characteristics concerning microbial fitness and robustness are less well understood and probably depend on more complex cellular components. Thus, different physiological characteristics have a different degree of underlying complexity, which will need to be considered when designing engineering strategies. Studies of the natural evolution of microbes might be able to reveal which of the physiological characteristics are most complex and, thus, most difficult to acquire during evolution. In the case of fermenting lignocellulose to ethanol, the tolerance of inhibitors present in
Opinion lignocellulosic hydrolysate is the most complex functionality that could be acquired through evolution. For example, Zymomonas mobilis can produce ethanol with high yield and efforts have been made to broaden its spectrum of substrates [32,36]. However, this strain is not well suited for cellulosic ethanol production owing to its intolerance of acetic acid present in the lignocellulosic hydrolysate [37,38]. The candidate strain that exhibits the most complex physiological characteristics and that is also genetically accessible can then be selected as a suitable host strain, as engineering the remaining and less complex physiological characteristics into this strain will be easier. Fourth step: engineering desired physiological functionalities into the selected host strain As the resulting candidate host strain typically has only some of the desired physiological characteristics, the remaining desired physiological functionalities need to be introduced by engineering approaches. If the host microbial cells are robust and fit, the engineering strategy should focus on introducing the target metabolic capabilities (including necessary pathways, efficient transport and cofactor regeneration systems) into the host strain lacking the metabolic activity for production of the desired product (Route 1, Step 4, Figure 2). Microbial metabolic capability has been studied extensively, and successful application of genetic engineering, metabolic engineering and genome shuffling has been reported [1,3,9,10,32–35,38,39]. If the host strains are genetically accessible, this approach can therefore be implemented relatively easily in practice by using techniques developed from metabolic engineering. Another approach can be designed for microbial cells that are highly metabolically active, but are not sufficiently robust and fit. A promising route to increase the fitness and robustness of microbes is to decipher the natural evolutionary program of extremophiles and other stress-tolerant strains, with the underlying rationale that target physiological characteristics in the host strain could be obtained by stress-induced adaptation, directed evolution of regulation factors or DNA manipulation genes, or genome shuffling. Recent studies of microbial natural evolution have revealed that natural adaptation and evolution are assisted by several master regulators, including alternative sigma factors (a prokaryotic transcription initiation factor that enables specific binding of RNA polymerase to gene promoters), small molecule effectors, gene repressors and some inorganic molecules [27]. Engineering these evolutionary master regulators would therefore constitute a promising evolutionary approach to reprogramming physiological functionality [27] as illustrated in Route 2 of Step 4 in Figure 2. Indeed, by engineering sigma factors, a novel method termed ‘global transcription machinery engineering’ was developed, which resulted in a significantly improved ethanol tolerance by Saccharomyces cerevisiae and a 69% increase in the volumetric productivity of ethanol [24]. Small molecule effectors and gene repressors have also been targeted to generate genetic diversity by fine-tuning transcription or base substitutions and have resulted in an E. coli mutant with improved rifampicin resistance [27]. Microbial evolution studies have also
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revealed that the evolution of microbes is always directed towards their own benefit [40]. Therefore, the key to success is to determine the appropriate evolutionary factors controlling the expected physiological functionalities and to engineer these subsequently. In actual practice, most host strains will need to be modified with regard to both, metabolic capability and their fitness and robustness. The two engineering routes pursued in Step 4 of Figure 2 will thus need to be integrated to engineer a desired industrially valuable strain. Moreover, in an improvement process involving selected strains, approaches for acquiring ‘high robustness and fitness’ are likely to be more complex than those required to introduce high metabolic capabilities. Thus, all steps proposed here might need to be repeated until both desired characteristics are achieved. Specifically, if the engineering attempts of step 4 are unable to achieve the expected results with regard to increasing robustness and fitness, the engineering strategy should restart from step 2 and repeated until desired strains are obtained (Figure 2). Pioneering studies of engineering industrial microbes The past decade has witnessed rapid progress in improving microbial metabolic capability with the development of several novel approaches to engineering microbes, including cofactor engineering to shift metabolite profile [41], systems metabolic engineering for improved amino acid production [10], ribosome engineering for improved antibiotics production [42] and global transcription machinery engineering for improved ethanol productivity [24]. However, only a few engineered strains have been used on an industrial scale successfully. Two of these are further discussed below: an E. coli strain producing 1,3propanediol, and bacteria or yeast strains for the production of ethanol from lignocellulosic materials. As an important chemical and raw material, the biotechnological production of 1,3-propanediol has attracted considerable attention. Much effort has been made to engineer the wild types of 1,3-propanediol-producing strains, such as Klebsiella pneumoniae, Citrobacter freundii and Clostridium butyricum, but the maximum titer of 1,3-propanediol that could be achieved in batch and fedbatch fermentation processes reached only 78 g L-1 [12,43,44]. This was due to the intolerance of E. coli to 1,3-propanediol and an imbalance of the metabolic activities of the glycerol dehydratase and 1,3-propanediol oxidoreductase enzymes that are involved in the 1,3-propanediol production pathway. Escherichia coli has several advantages as a biocatalyst for 1,3-propanediol production, including its ability to ferment a range of sugars, the lack of requirements for complex growth factors, and its proven industrial use (e.g. for production of recombinant proteins), despite the fact that it lacks the necessary pathways of converting sugars to 1,3-propanediol. Through a series of engineering events, including the deletion, overexpression or fine-tuning of 25 targeted genes, DuPont (http:// www.dupont.com/) and Genencor (http://www.genencor. com) successfully obtained an engineered E. coli strain that was able to produce a 1,3-propanediol titer of 130 g L-1 using glucose as feedstock [45]. This success constitutes an elegant bench mark of metabolic engineering. However, 669
Opinion using physiological engineering, the number of the metabolic genes that need to be introduced, disrupted or modified could be reduced significantly. The expected high titer and productivity of 1,3-propandiol might be relatively easily obtained through engineering on transcription levels only. The second example concerns recent advances in engineering strains that are able to produce ethanol from lignocellulosic materials. Here, E. coli [46,47], Z. mobilis [32,36–38,48], which produces ethanol from a specific sugar, and S. cerevisiae [24,49,50], which shows a wide spectrum of sugar substrates, have been used extensively as host strains. To improve ethanol yield, genes involved in either xylose metabolism or ethanol production were successfully expressed, while competing branches were simultaneously eliminated [51]. Subsequently, ethanolinduced adaptation [47,49], or evolution of master regulators (e.g. global transcription machinery engineering [24]) were carried out with the goal to meet the major criteria for cellulosic ethanol producing strains: the tolerance to ethanol and resistance to inhibitors [51,52]. Several isolates showing significant improvements could be obtained and demonstrated great potential for industrial application, although they still exhibited some deficiencies that might limit their application in actual bioprocesses. For E. coli LY01, these included the narrow usable pH range and the high sensitivity to a mixture of inhibitors [47], whereas Z. mobilis AX101 was intolerant to acetic acid [37,38]. Another resulting strain, S. cerevisiae INVSC1, showed a low resistance to low pH and inhibitors [53]. We believe that by implementing the approaches and procedures of physiological engineering as illustrated in Figure 2, further breakthroughs can be anticipated in overcoming microbial physiological limits of these host strains. Alternatively, directly engineering the ethanol production ability into a new robust host strain that is tolerant of inhibitors in hydrolysate and high concentration of ethanol, might be able to meet the demand of cellulosic-based ethanol production. Conclusions and future perspectives Increasing demands on biotechnology to provide solutions for renewable energies, global food security and environmental pollution are the main driving forces for the engineering of novel microbes at a systems level that are able to produce biofuels, bulk-chemicals and other biomaterials [54–56]. Compared with their metabolic capability, the physiological status and the resulting physiological functionalities of microbes, including adaptation and robustness to harsh environments, stress tolerance and fitness throughout the entire production process are becoming increasingly relevant for strains that are to be applied in industrial bioprocesses. To obtain these desired industrial strains, we must define the desired engineering target, obtain strains with various desired characteristics, select the right host strain and engineer desired physiological functionalities into microbes by efficient approaches. The concepts and procedures of engineering physiological functionality into microbes discussed here aim to provide incentives for further strain improvements using engineering approaches. 670
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Apart from the example of lignocellulosic ethanol process described, there is increasing demand from chemical companies to produce other bulk chemicals using biotechnological process. These include 1,3-propanediol, acrylic acid, lactic acid, butanol, 1,4-butanediol, succinic acid, isoprene, isoamyl alcohol, adipic acid and citric acid. The ultimate success of biotechnological routes over conventional petrochemical processes will depend on the one hand on whether biomass-derived sugars can be used as the main carbon source to reduce the raw material cost and, on the other hand, on whether the final product titer can be improved enough to decrease the downstream separation cost. These principles can also be applied to improving the efficiency of biotechnological production of other fermentation products, such as amino acids, organic acids, antibiotics, sugar alcohols, polymers, vitamins and enzymes. Four major obstacles for a wide implementation of physiological engineering currently exist: (i) Engineering targets: as the microbial stress response is a complex system, defining a target for physiological engineering can be difficult even with the assistance of systems biology. Computational tools are thus needed to assist the screening and discovery of potential targets by constructing a virtual cell model and in silico analysis; (ii) Evolution pathway: the presence of extremophiles in extreme environments is a consequence of their longterm evolution and/or adaptation. A comprehensive understanding of the mechanisms underlying the evolution pathway can help to design an artificial evolution pathway to facilitate engineering physiological functionalities; (iii) Genetic instability of the engineered strains: engineered cells can be considered as ‘ill’ rather than ‘normal’ cells with the consequence that microbes might tend to return to the ‘normal’ state owing to the robustness of their metabolic and regulatory networks. The construction of microbes with reduced genome size by a synthetic biology approach might help in this regard, and examples demonstrating this possibility have recently emerged (Table 1); (iv) Population behavior: microbes do not exist alone in nature and microflora composed of single strains or a single species are usually less resistant to environmental perturbations and stresses compared with multi-species population. Knowledge from microbial population behaviors will be helpful in designing an efficient single-species biocatalyst, with the ultimate goal to develop bioprocesses that are based on microbial populations. Taken together, physiological engineering, which expands beyond traditional metabolic engineering paradigms, will become increasingly important in strain improvement approaches. Future insights into microbial physiological functionalities and evolutionary programs are likely to provide increasingly powerful techniques for engineering physiological functionalities into microbes to obtain a highly metabolically active, physiologically robust and fit strain, and therefore ensure their successful applications in biotechnology industries.
Opinion Acknowledgements Funding for our research was provided by the National Natural Science Foundation of China (30870040), National Basic Research Program of China (973 Project, 2007CB707803 and 2007CB714301), National High Technology Research and Development Program of China (863 Project, 2006AA020103 and 2006AA02Z237) and Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-G-005, KSCX2-YW-G007, and KSCX1-YW-11C3). Y.L. is supported by the Hundred Talents Program of the Chinese Academy of Sciences. We thank Linjiang Zhu for helpful discussions.
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