Improving lycopene production in Escherichia coli by engineering metabolic control

Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.     

Rational design of thiolase substrate specificity for metabolic engineering applications

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3‐hydroxyacid (3HA) pathway, also known as the reverse β‐oxidation or coenzyme‐A‐dependent chain‐elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model‐guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3‐hydroxy‐hexanoic acid and 3‐hydroxybutyric acid. We identify thiolase mutants that result in nearly 10‐fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence–structure–function relationship for this important class of enzymes.     

Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: Pathway stoichiometry,free-energy conservation and redox-cofactor balancing

Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of this yeast require acetyl coenzyme A (acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic acetyl-CoA consumes 2 ATP equivalents in the acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for acetyl-CoA synthesis. Moreover, this analysis showed that combination of different acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories.     

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

Bioconversion of xylose—the second most abundant sugar in nature—into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.     

In vivo interpretation of model predicted inhibition in acrylate pathway engineered Lactococcus lactis

To maximize the productivity of engineered metabolic pathway, in silico model is an established means to provide features of enzyme reaction dynamics. In our previous study, Escherichia coli engineered with acrylate pathway yielded low propionic acid titer. To understand the bottleneck behind this low productivity, a kinetic model was developed that incorporates the enzymatic reactions of the acrylate pathway. The resulting model was capable of simulating the fluxes reported under in vitro studies with good agreement, suggesting repression of propionyl-CoA transferase (Pct) by carboxylate metabolites as the main limiting factor for propionate production. Furthermore, the predicted flux control coefficients of the pathway enzymes under steady state conditions revealed that the control of flux is shared between Pct and lactoyl-CoA dehydratase. Increase in lactate concentration showed gradual decrease in flux control coefficients of Pct that in turn confirmed the control exerted by the carboxylate substrate. To interpret these in silico predictions under in vivo system, an organized study was conducted with a lactic acid bacteria strain engineered with acrylate pathway. Analysis reported a decreased product formation rate on attainment of inhibitory titer by suspected metabolites and supported the model.     

谷氨酸棒杆菌代谢工程高效生产L-缬氨酸北大核心CSCD

L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。     

Deriving metabolic engineering strategies from genome-scale modeling with flux ratio constraints

Optimized production of bio-based fuels and chemicals from microbial cell factories is a central goal of systems metabolic engineering. To achieve this goal, a new computational method of using flux balance analysis with flux ratios (FBrAtio) was further developed in this research and applied to five case studies to evaluate and design metabolic engineering strategies. The approach was implemented using publicly available genome-scale metabolic flux models. Synthetic pathways were added to these models along with flux ratio constraints by FBrAtio to achieve increased (i) cellulose production from Arabidopsis thaliana; (ii) isobutanol production from Saccharomyces cerevisiae; (iii) acetone production from Synechocystis sp. PCC6803; (iv) H₂ production from Escherichia coli MG1655; and (v) isopropanol, butanol, and ethanol (IBE) production from engineered Clostridium acetobutylicum. The FBrAtio approach was applied to each case to simulate a metabolic engineering strategy already implemented experimentally, and flux ratios were continually adjusted to find (i) the end-limit of increased production using the existing strategy, (ii) new potential strategies to increase production, and (iii) the impact of these metabolic engineering strategies on product yield and culture growth. The FBrAtio approach has the potential to design “fine-tuned” metabolic engineering strategies in silico that can be implemented directly with available genomic tools.     

Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression

Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.     

Metabolite damage and repair in metabolic engineering design

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways – particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile ‘plug and play’ set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.     

Protein design in systems metabolic engineering for industrial strain development

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering.     

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

We applied isotopically nonstationary ¹³C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway (mdh and me) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how ¹³C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.     

酿酒酵母生产羧酸的代谢工程策略

羧酸广泛地应用于食品、医药和化工等行业,具有广阔的市场前景.作为真核模式微生物,酿酒酵母作为代谢工程平台用来生产有机酸具有明显优势.本文论述了酿酒酵母生产重要羧酸的策略:首先构建一条能够和糖酵解途径相连接的高效的重要羧酸积累途径,进而探讨如何将碳代谢流由乙醇转向目的产物,在此基础上研究有机酸的转运及涉及到的能量问题.最后,对当前研究存在的问题进行了分析,并对未来研究方向进行了展望.     

Expanding the metabolic engineering toolbox with directed evolution

Cellular systems can be engineered into factories that produce high-value chemicals from renewable feedstock. Such an approach requires an expanded toolbox for metabolic engineering. Recently, protein engineering and directed evolution strategies have started to play a growing and critical role within metabolic engineering. This review focuses on the various ways in which directed evolution can be applied in conjunction with metabolic engineering to improve product yields. Specifically, we discuss the application of directed evolution on both catalytic and non-catalytic traits of enzymes, on regulatory elements, and on whole genomes in a metabolic engineering context. We demonstrate how the goals of metabolic pathway engineering can be achieved in part through evolving cellular parts as opposed to traditional approaches that rely on gene overexpression and deletion. Finally, we discuss the current limitations in screening technology that hinder the full implementation of a metabolic pathway-directed evolution approach.     

Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration

NADPH is an essential cofactor for the biosynthesis of several high-value chemicals, including isoprenoids, fatty acid-based fuels, and biopolymers. Tunable control over all potentially rate-limiting steps, including the NADPH regeneration rate, is crucial to maximizing production titers. We have rationally engineered a synthetic version of the Entner–Doudoroff pathway from Zymomonas mobilis that increased the NADPH regeneration rate in Escherichia coli MG1655 by 25-fold. To do this, we combined systematic design rules, biophysical models, and computational optimization to design synthetic bacterial operons expressing the 5-enzyme pathway, while eliminating undesired genetic elements for maximum expression control. NADPH regeneration rates from genome-integrated pathways were estimated using a NADPH-binding fluorescent reporter and by the productivity of a NADPH-dependent terpenoid biosynthesis pathway. We designed and constructed improved pathway variants by employing the RBS Library Calculator to efficiently search the 5-dimensional enzyme expression space and by performing 40 cycles of MAGE for site-directed genome mutagenesis. 624 pathway variants were screened using a NADPH-dependent blue fluorescent protein, and 22 were further characterized to determine the relationship between enzyme expression levels and NADPH regeneration rates. The best variant exhibited 25-fold higher normalized mBFP levels when compared to wild-type strain. Combining the synthetic Entner–Doudoroff pathway with an optimized terpenoid pathway further increased the terpenoid titer by 97%.     

Metabolic engineering of Escherichia coli for high production of 1,5-pentanediol via a cadaverine-derived pathway

1,5-Pentanediol (1,5-PDO) is a high value-added chemical which is widely used as a monomer in the polymer industry. There are no natural organisms that could directly produce 1,5-PDO from renewable carbon sources. In this study, we report metabolic engineering of Escherichia coli for high-level production of 1,5-PDO from glucose via a cadaverine-derived pathway. In the newly proposed pathway, cadaverine can be converted to 1,5-PDO via 5-hydroxyvalerate (5-HV) by introducing only one heterologous enzyme in E. coli. Different endogenous genes of E. coli were screened and heterologous carboxylic acid reductase genes were tested to build a functional pathway. Compared to the previously reported pathways, the engineered cadaverine-based pathway has a higher theoretical yield (0.70 mol/mol glucose) and higher catalytic efficiency. By further combining strategies of pathway engineering and process engineering, we constructed an engineered E. coli strain that could produce 2.62 g/L 1,5-PDO in shake-flask and 9.25 g/L 1,5-PDO with a yield of 0.28 mol/mol glucose in fed-batch fermentation. The proposed new pathway and engineering strategies reported here should be useful for developing biological routes to produce 1,5-PDO for real application.     

Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review

Pichia pastoris has been recognized as one of the most industrially important hosts for heterologous protein production. Despite its high protein productivity, the optimization of P. pastoris cultivation is still imperative due to strain- and product-specific challenges such as promoter strength, methanol utilization type and oxygen demand. To address the issues, strategies involving genetic and process engineering have been employed. Optimization of codon usage and gene dosage, as well as engineering of promoters, protein secretion pathways and methanol metabolic pathways have proved beneficial to innate protein expression levels. Large-scale production of proteins via high cell density fermentation additionally relies on the optimization of process parameters including methanol feed rate, induction temperature and specific growth rate. Recent progress related to the enhanced production of proteins in P. pastoris via various genetic engineering and cultivation strategies are reviewed. Insight into the regulation of the P. pastoris alcohol oxidase 1 (AOX1) promoter and the development of methanol-free systems are highlighted. Novel cultivation strategies such as mixed substrate feeding are discussed. Recent advances regarding substrate and product monitoring techniques are also summarized. Application of P. pastoris to the production of biodiesel and other value-added products via metabolic engineering are also reviewed. P. pastoris is becoming an indispensable platform through the use of these combined engineering strategies.     

Engineered affinity proteins—Generation and applications

The use of combinatorial protein engineering to design proteins with novel binding specificities and desired properties has evolved into a powerful technology, resulting in the recent advances in protein library selection strategies and the emerge of a variety of new engineered affinity proteins. The need for different protein library selection methods is due to that each target protein pose different challenges in terms of its availability and inherent properties. At present, alternative engineered affinity proteins are starting to complement and even challenge the classical immunoglobulins in different applications in biotechnology and potentially also for in vivo use as imaging agents or as biotherapeutics. This review article covers the generation and use of affinity proteins generated through combinatorial protein engineering. The most commonly used selection techniques for isolation of desired variants from large protein libraries are described. Different antibody derivatives, as well as a variety of the most validated engineered protein scaffolds, are discussed. In addition, we provide an overview of some of the major present and future applications for these engineered affinity proteins in biotechnology and medicine.