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The MEP and MVA pathways. In the MVA pathway, a series of enzymes catalyze two molecules of acetyl-CoA into IPP, which is then isomerized by IDI to its isomer—DMAPP. In the MEP pathway, glyceraldehyde 3-phosphate and pyruvate are converted to IPP and DMAPP. At the same time, IDI as an isomerase, functions to balance the amount of IPP and DMAPP. Under the catalysis by the downstream enzymes, DMAPP enters the synthesis pathway of the target product
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Lycopene is a terpenoid pigment that has diverse applications in the food and medicine industries. A prospective approach for lycopene production is by metabolic engineering in microbial hosts, such as Escherichia coli. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) is one of the rate-limiting enzymes in the lycopene biosynthetic...
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Pathway engineering is a useful technology for producing desired compounds on a large scale by modifying the biosynthetic pathways of host organisms using genetic engineering. We focused on acetoacetate esters as novel low-cost substrates and established an efficient terpenoid production system using pathway-engineered recombinant Escherichia coli....
Carotenoid pigments are important components of the human diet. Carrots are rich source of dietary pro-vitamin A, antioxidants and minerals. Since carotenoid play, essential biological roles in carrot and the genes coding for the carotenoid pathway enzymes are evolutionarily conserved.Compounds for carotenoid biosynthesis pathway derived from the i...
Red yeasts, mainly included in the genera Rhodotorula, Rhodosporidiobolus, and Sporobolomyces, are renowned biocatalysts for the production of a wide range of secondary metabolites of commercial interest, among which lipids, carotenoids, and other isoprenoids. The production of all these compounds is tightly interrelated as they share acetyl-CoA an...
Citations
... Protein engineering could involve using more active homologous enzymes from alternative organisms which can improve product yield [91][92][93][94]. Alternatively, targeted mutagenesis of key enzymes could also improve activity towards substrates [95]. Extracellular and intracellular time-series metabolomics data obtained from cell cultures of wild-type E. coli engineered to overproduce limonene (EcoCTs03) used for model development. ...
Limonene is a valuable monoterpene utilised in biofuel, pharmaceutical, material science, food and beverage industries. Recently, microorganisms, such as engineered Escherichia coli, have emerged as potential alternatives for limonene production in the laboratory. However, there are general issues such as low enzyme abundance or activities, and regulatory feedback/forward inhibition resulting in reduced yields. To better understand the underlying metabolism, both mechanistically and dynamically, here we adopted a systems biology approach for cell cultures of engineered E. coli strain K-12 MG1655 producing limonene. Firstly, we generated time-series intra- and extra-cellular targeted metabolomics data and, secondly, developed a dynamic model based on enzyme dynamics to interpret the data. The initial wild-type model tracked native metabolic networks such as the glycolytic, tricarboxylic acid cycle, mixed fermentation, and deoxyxylulose 5-phosphate pathway, as well as the engineered mevalonate pathway. After several iterations of model fitting with experimental profiles, which also included 13C tracer studies, we utilised the finalised model to perform in silico knockouts (KOs) of all enzymes in the model to identify crucial bottleneck(s) for optimal limonene yields. The simulations indicated that the suppression of ALDH/ADH (aldehyde dehydrogenase/alcohol dehydrogenase) and LDH (lactate dehydrogenase), and the enhancement of HK (hexokinase) would increase limonene yields. These results were experimentally confirmed where ALDH-ADH and LDH KOs produced 8- to 11-fold enhancements, while HK overexpression resulted in an 8-fold increase in limonene yield. We believe that our systems biology approach can be extended to study other microbes for re-engineering strains for optimal target compound production.
... To enhance the activity of S. cerevisiae IDI, authors employed a directed evolution strategy involving error-prone polymerase chain reaction (PCR). Subsequent fermentation experiments demonstrated that the mutant IDI exhibited a 1.8-fold increase in lycopene production [70]. ...
... When researchers overexpressed tHMGR from X. dendrorhous, a truncated form of HMGR that spans the membrane, in S. cerevisiae, there was a 2.2-fold enhancement in β-carotene production [72]. In Y. lipolytica, overexpression of the bottleneck genes HMG1 and GGS1 led to a remarkable 10.8-fold improvement in lycopene yield [73]. ments demonstrated that the mutant IDI exhibited a 1.8-fold increase in lycopene produc-tion [70]. ...
... Overexpressed AMP deaminase-encoding gene AMPD approximately 3-fold increase in lycopene content [57] E. coli Deletion of central carbon metabolic gene zwf 130% enhancement in lycopene production [62] E. coli Directed evolution of isopentenyl diphosphate isomerase (IDI) 2.1-fold increase in lycopene yield [70] S. cerevisia Overexpressed the fatty acid desaturase gene OLE1; deletion of the Seipin gene FLD1 25% increase in lycopene yield [77] Y. lipolytica Overexpressed the bottleneck genes HMG1 and GGS1 increased the lycopene content 10.8-fold [73] S. cerevisia ...
Carotenoids are naturally occurring pigments that are abundant in the natural world. Due to their excellent antioxidant attributes, carotenoids are widely utilized in various industries, including the food, pharmaceutical, cosmetic industries, and others. Plants, algae, and microorganisms are presently the main sources for acquiring natural carotenoids. However, due to the swift progress in metabolic engineering and synthetic biology, along with the continuous and thorough investigation of carotenoid biosynthetic pathways, recombinant strains have emerged as promising candidates to produce carotenoids. The identification and manipulation of gene targets that influence the accumulation of the desired products is a crucial challenge in the construction and metabolic regulation of recombinant strains. In this review, we provide an overview of the carotenoid biosynthetic pathway, followed by a summary of the methodologies employed in the discovery of gene targets associated with carotenoid production. Furthermore, we focus on discussing the gene targets that have shown potential to enhance carotenoid production. To facilitate future research, we categorize these gene targets based on their capacity to attain elevated levels of carotenoid production.
... The Cpd I-H 2 O 2 complex was found to form a Cpd II complex by transferring a hydrogen atom from hydrogen peroxide to ferrous ions, during which H 2 O 2 may diffuse into the active center through the protein channel, and thus the increased volume of the protein channel and the active center has a positive effect on enhancing protein activity. Elevated protein-substrate affinity can effectively increase the catalytic activity of the enzyme [65]. The molecular docking results in this study showed that the GpCAT1 protein has higher affinity for H 2 O 2 , so it is hypothesized that the significant elevation of CAT enzyme activity in the leaf tissues of G. przewalskii under NaCl stress is related to its mutation site. ...
The antioxidant enzyme system is the main defense system responsible for maintaining cellular reactive oxygen species (ROS) homeostasis and normal plant growth and development after saline stress. In this study, we identified and characterized the members of the SOD, APX and CAT gene families of the antioxidant enzyme system in Gymnocarpos przewalskii, using plant physiology and molecular biology methods, and analyzed the pattern of enzyme activity in response to NaCl stress. It was found that seven, six and two genes of SOD, APX and CAT gene families, respectively, were expressed in the leaf tissue of G. przewalskii, in which most of the genes were significantly upregulated under NaCl stress, and the enzymatic activities were in accordance with the gene expression. Three positive selection sites in the GpCAT1 gene can increase the hydrophilicity of the GpCAT1 protein, increase the volume of the active site and increase the affinity for H2O2, thus improving the catalytic efficiency of GpCAT1. The results of the present study provide new insights for further investigations of the evolution and function of the SOD, APX and CAT gene families in G. przewalskii and their essential roles under salt stress, and the findings will be useful for revealing the molecular mechanism of salt tolerance and breeding of salt-tolerant plants.
... As reported, the rationally designed GGPPS with multisite mutations from Nicotiana tabacum exhibited significantly higher efficiency for GGPP synthesis than the wild-type gene, increasing carotenoid levels greatly [29]. A directed evolution method was applied to improve the enzyme activity, half-life and substrate affinity of IDI from Saccharomyces cerevisiae [56]. Additionally, multienzyme assembly using docking domains to increase cascade catalytic efficiency was also a potential strategy for enhancing carotenoid production [23]. ...
Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo β-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of β-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in β-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, β-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.
... It requires knowledge about the three-dimensional structure of the enzyme and the chemical mechanism of the reaction. This approach often fails, although successes have been achieved (Wu et al., 2017;Chen et al., 2018). Combinatorial methods include directed evolution which does not require extensive knowledge about the enzyme. ...
... Adopting this screening strategy, an improved variant form of IDI bearing triple-mutation (L141H/ Y195F/W256C) with a catalytic activity of 2.53-fold higher than the wild-type was selected after a directed evolution and site-saturation mutagenesis process. The final strain expressing the mutated enzyme produced more than 1.2 g/L of lycopene, a 2.8-fold increment as compared to the wild-type (Chen et al. 2018a). Wang et al. also developed a novel high-throughput screening method based on DMAPP toxicity to screen for enhanced isoprene synthases (ISPS). ...
Terpenoids form the most diversified class of natural products, which have gained application in the pharmaceutical, food, transportation, and fine and bulk chemical industries. Extraction from naturally occurring sources does not meet industrial demands, whereas chemical synthesis is often associated with poor enantio-selectivity, harsh working conditions, and environmental pollutions. Microbial cell factories come as a suitable replacement. However, designing efficient microbial platforms for isoprenoid synthesis is often a challenging task. This has to do with the cytotoxic effects of pathway intermediates and some end products, instability of expressed pathways, as well as high enzyme promiscuity. Also, the low enzymatic activity of some terpene synthases and prenyltransferases, and the lack of an efficient throughput system to screen improved high-performing strains are bottlenecks in strain development. Metabolic engineering and synthetic biology seek to overcome these issues through the provision of effective synthetic tools. This review sought to provide an in-depth description of novel strategies for improving cell factory performance. We focused on improving transcriptional and translational efficiencies through static and dynamic regulatory elements, enzyme engineering and high-throughput screening strategies, cellular function enhancement through chromosomal integration, metabolite tolerance, and modularization of pathways.
Graphical Abstract
... Sitesaturation mutagenesis can also be performed on the basis of target sites selected by random mutagenesis. 59 Nevertheless, terpene synthases, especially sesquiterpene synthases, have low sequence similarity and there are few published crystal structures, resulting in limited support for homology modeling to obtain reliable target-site information. This is still a bottleneck that needs to be resolved. ...
Terpenes are a large class of secondary metabolites with diverse structures and functions that are commonly used as valuable raw materials in food, cosmetics, and medicine. With the development of metabolic engineering and emerging synthetic biology tools, these important terpene compounds can be sustainably produced using different microbial chassis. Currently, yeasts such as Saccharomyces cerevisiae and Yarrowia lipolytica have received extensive attention as potential hosts for the production of terpenes due to their clear genetic background and endogenous mevalonate pathway. In this review, we summarize the natural terpene biosynthesis pathways and various engineering strategies, including enzyme engineering, pathway engineering, and cellular engineering, to further improve the terpene productivity and strain stability in these two widely used yeasts. In addition, the future prospects of yeast-based terpene production are discussed in light of the current progress, challenges, and trends in this field. Finally, guidelines for future studies are also emphasized.
... Beyond these strategies, the reconstruction of alternative pathways for DMAPP production has also appeared as an attractive approach to produce high levels of this compound. This strategy has been widely applied in E. coli by introducing a heterologous MVA pathway from another microorganism, namely from S. cerevisiae, to increase IPP and DMAPP (Cao et al., 2019;Liu et al., 2018b). For example, Cao et al. (2019) constructed a heterologous MVA pathway in E. coli. ...
Prenylflavonoids are flavonoid-derived compounds characterized by the presence of a lipophilic prenylated side-chain in the flavonoid skeleton. These compounds are present in several food supplements and food products, and have a wide variety of recognized biological activities, namely estrogenic, antioxidant, anti-inflammatory and anticancer. Since these compounds are present in nature in very low amounts, their extraction from plants is not enough to fulfill the current demand, besides being an inefficient and environmentally unfriendly process. For these reasons, the use of microorganisms as microbial cell factories represents an interesting alternative to produce prenylflavonoids in a faster and cheaper way. Saccharomyces cerevisiae has been used as chassis to produce prenylflavonoids. Moreover, Escherichia coli can also emerge as an alternative chassis to produce these compounds. However, there is still a long way before prenylflavonoids can be produced by heterologous organisms with relevant yields and titers. In this review, we highlight the biosynthetic pathways involved in the production of prenylflavonoids. Additionally, we review the advances, challenges and strategies on the heterologous production of these compounds in a competitive way.
... In a previous study, enzyme mutants with high activity were obtained through directed evolution of the rate-limiting enzymes mevalonate kinase (MK) and IDI in the MVA pathway. 81,82 Moreover, the sequential error-prone polymerase chain reaction (PCR) was selected for screening random mutants of FS with high catalytic activity. 62 In this study, more than 500 FS mutant strains were obtained by measuring the level of intracellular inorganic pyrophosphate (PPi). ...
... In another study, alkaline medium with pH ranging from 7 to 8 enhanced farnesol accumulation by S. cerevisiae [100]. It was also reported that the optimum pH for the wild type S. cerevisiae, and mutant strain in isopentenyl diphosphate isomerase activity (one of the important rate-limiting enzymes in terpenoid production), was 7.5 [101]. Alkaline pH might enhance the synthesis and solubility of terpenoids in the cultivation medium, but growth and biomass accumulation might be low, not allowing high cell density cultivation as desirable in industrial terpenoids production. ...
Terpenoids, also known as isoprenoids, are a broad and diverse class of plant natural products with significant industrial and pharmaceutical importance. Many of these natural products have antitumor, anti-inflammatory, antibacterial, antiviral, and antimalarial effects, support transdermal absorption, prevent and treat cardiovascular diseases, and have hypoglycemic activities. Production of these compounds are generally carried out through extraction from their natural sources or chemical synthesis. However, these processes are generally unsustainable, produce low yield, and result in wasting of substantial resources, most of them limited. Microbial production of terpenoids provides a sustainable and environment-friendly alternative. In recent years, the yeast Saccharomyces cerevisiae has become a suitable cell factory for industrial terpenoid biosynthesis due to developments in omics studies (genomics, transcriptomics, metabolomics, proteomics), and mathematical modeling. Besides that, fermentation development has a significant importance on achieving high titer, yield, and productivity (TYP) of these compounds. Up to now, there have been many studies and reviews reporting metabolic strategies for terpene biosynthesis. However, fermentation strategies have not been yet comprehensively discussed in the literature. This review summarizes recent studies of recombinant production of pharmaceutically important terpenoids by engineered yeast, S. cerevisiae, with special focus on fermentation strategies to increase TYP in order to meet industrial demands to feed the pharmaceutical market. Factors affecting recombinant terpenoids production are reviewed (strain design and fermentation parameters) and types of fermentation process (batch, fed-batch, and continuous) are discussed.
