Recent Progress In Metabolic Engineering Research Articles

Advances in the biosynthesis and metabolic engineering of rare ginsenosides

Rare ginsenosides are the deglycosylated secondary metabolic derivatives of major ginsenosides, and they are more readily absorbed into the bloodstream and function as active substances. The traditional preparation methods hindered the potential application of these effective components. The continuous elucidation of ginsenoside biosynthesis pathways has rendered the production of rare ginsenosides using synthetic biology techniques effective for their large-scale production. Previously, only the progress in the biosynthesis and biotechnological production of major ginsenosides was highlighted. In this review, we summarized the recent advances in the identification of key enzymes involved in the biosynthetic pathways of rare ginsenosides, especially the glycosyltransferases (GTs). Then the construction of microbial chassis for the production of rare ginsenosides, mainly in Saccharomyces cerevisiae, was presented. In the future, discovery of more GTs and improving their catalytic efficiencies are essential for the metabolic engineering of rare ginsenosides. This review will give more clues and be helpful for the characterization of the biosynthesis and metabolic engineering of rare ginsenosides. KEY POINTS: • The key enzymes involved in the biosynthetic pathways of rare ginsenosides are summarized. • The recent progress in metabolic engineering of rare ginsenosides is presented. • The discovery of glycosyltransferases is essential for the microbial production of rare ginsenosides in the future.

Advances in metabolic engineering of cyanobacteria for production of biofuels

Current trends in fossil fuel use, having a direct impact on the global carbon cycle, could potentially be mitigated by adopting the use of biofuels with sustainable features. While bioethanol is currently being used as a major biofuel, especially for transportation worldwide, it is hygroscopic and contains less energy. Therefore, the prospect of biofuels has now shifted to higher alcohols, alkanes, and other molecules that have similar properties to existing gasoline and diesel fuels. Microalgae and cyanobacteria are aquatic photosynthetic microorganisms that can convert CO2 and water into carbon-rich lipids, a substrate for biodiesel production, more efficiently than the agricultural oleaginous crops, making them a superior option to traditional oil-seed crops. Cyanobacteria are gram-negative photosynthetic bacteria that have instrumental roles in global biological carbon sequestration, oxygen evolution, and nitrogen fixation. Furthermore, cyanobacteria have established genetic backgrounds, flexibility in genetic manipulation, and tolerance to the introduction of foreign genes. Metabolic engineering strategies in these photosynthetic hosts have resulted in the conception of cell factories capable of one-step synthesis of biofuel molecules from CO2, light, and water. Two freshwater cyanobacteria species, Synechocystis sp. PCC 6803 (Synechocystis 6803) and Synechococcus elongatus sp. PCC 7492 (Synechococcus 7492), and marine Synechococcus sp. PCC 7002 (Synechococcus 7002) are among the highly studied cyanobacteria for the production of biofuels. Here, we reviewed the recent progress in metabolic engineering and synthetic biology approaches in cyanobacteria for the synthesis of various biofuel molecules, ranging from alcohols to biodiesels and terpenes or terpenoids.

Recent Advances in Heterologous Synthesis Paving Way for Future Green-Modular Bioindustries: A Review With Special Reference to Isoflavonoids.

Isoflavonoids are well-known plant secondary metabolites that have gained importance in recent time due to their multiple nutraceutical and pharmaceutical applications. In plants, isoflavonoids play a role in plant defense and can confer the host plant a competitive advantage to survive and flourish under environmental challenges. In animals, isoflavonoids have been found to interact with multiple signaling pathways and have demonstrated estrogenic, antioxidant and anti-oncologic activities in vivo. The activity of isoflavonoids in the estrogen pathways is such that the class has also been collectively called phytoestrogens. Over 2,400 isoflavonoids, predominantly from legumes, have been identified so far. The biosynthetic pathways of several key isoflavonoids have been established, and the genes and regulatory components involved in the biosynthesis have been characterized. The biosynthesis and accumulation of isoflavonoids in plants are regulated by multiple complex environmental and genetic factors and interactions. Due to this complexity of secondary metabolism regulation, the export and engineering of isoflavonoid biosynthetic pathways into non-endogenous plants are difficult, and instead, the microorganisms Saccharomyces cerevisiae and Escherichia coli have been adapted and engineered for heterologous isoflavonoid synthesis. However, the current ex-planta production approaches have been limited due to slow enzyme kinetics and traditionally laborious genetic engineering methods and require further optimization and development to address the required titers, reaction rates and yield for commercial application. With recent progress in metabolic engineering and the availability of advanced synthetic biology tools, it is envisaged that highly efficient heterologous hosts will soon be engineered to fulfill the growing market demand.

Recent progress in metabolic engineering of Corynebacterium glutamicum for the production of C4, C5, and C6 chemicals

Recent progress in metabolic engineering of Corynebacterium glutamicum for the production of C4, C5, and C6 chemicals

Recent Progress on Chemical Production From Non-food Renewable Feedstocks Using Corynebacterium glutamicum.

Due to the non-renewable nature of fossil fuels, microbial fermentation is considered a sustainable approach for chemical production using glucose, xylose, menthol, and other complex carbon sources represented by lignocellulosic biomass. Among these, xylose, methanol, arabinose, glycerol, and other alternative feedstocks have been identified as superior non-food sustainable carbon substrates that can be effectively developed for microbe-based bioproduction. Corynebacterium glutamicum is a model gram-positive bacterium that has been extensively engineered to produce amino acids and other chemicals. Recently, in order to reduce production costs and avoid competition for human food, C. glutamicum has also been engineered to broaden its substrate spectrum. Strengthening endogenous metabolic pathways or assembling heterologous ones enables C. glutamicum to rapidly catabolize a multitude of carbon sources. This review summarizes recent progress in metabolic engineering of C. glutamicum toward a broad substrate spectrum and diverse chemical production. In particularly, utilization of lignocellulosic biomass-derived complex hybrid carbon source represents the futural direction for non-food renewable feedstocks was discussed.

Recent progress in metabolic engineering of microbial formate assimilation.

Formate can be efficiently produced via electrochemical or photochemical catalytic conversion of CO2, and it can be directly used as an organic carbon source by microorganisms. In theory, formate can be used as the sole carbon source for the microbial production of high-value-added chemicals. Consequently, the construction of efficient formate-assimilation pathways in microorganisms is essential for the utilization of cheap, renewable one-carbon compounds. This paper summarizes new methods of formate synthesis, as well as the natural formate utilization pathways of microorganisms with their advantages and disadvantages. Furthermore, it reviews recent progress in the design of utilization pathways for formate in microbial cells through metabolic engineering and synthetic biology. Besides, we also use the pathway-prediction algorithm comb-FBA to rationally design completely new one-carbon compounds utilization pathways. The pathway with the highest efficiency, named GAA, was corroborated by the in vitro experiments showing a carbon molar yield up to 88%. Finally, it discusses the main problems and challenges presently existing in the pathway design and strain improvement for microbial utilization of formate. KEY POINTS: • Natural and artificial design pathways of formate-assimilation was summarized. • Recent progresses in different hosts and approaches of using one-carbon compounds was reviewed. • Metabolic engineering and synthetic biology methods to improve formate utilization were discussed.

A framework for the identification of promising bio‐based chemicals

Recent progress in metabolic engineering and synthetic biology enables the use of microorganisms for the production of chemicals-"bio-based chemicals." However, it is still unclear which chemicals have the highest economic prospect. To this end, we develop a framework for the identification of such promising ones. Specifically, we first develop a genome-scale constraint-based metabolic modeling approach, which is used to identify a candidate pool of 209 chemicals (together with the estimated yield, productivity, and residence time for each) from the intersection of the high-production-volume chemicals and the KEGG and MetaCyc databases. Second, we design three screening criteria based on a chemical's profit margin, market volume, and market size. The total process cost, including the downstream separation cost, is systematically incorporated into the evaluation. Third, given the three aforementioned criteria, we identify 32 products as economically promising if the maximum yields can be achieved, and 22 products if the maximum productivities can be achieved. The breakeven titer that renders zero profit margin for each product is also presented. Comparisons between extracellular and intracellular production, as well as Escherichia coli and Saccharomyces cerevisiae systems are also discussed. The proposed framework provides important guidance for future studies in the production of bio-based chemicals. It is also flexible in that the databases, yield estimations, and criteria can be modified to customize the screening.

Recent advances in metabolic engineering of Corynebacterium glutamicum as a potential platform microorganism for biorefinery

AbstractThe fermentative production of platform chemicals in biorefineries is a sustainable alternative to current petroleum‐refining processes. Industrial microorganisms, such as Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum, have been engineered as microbial cell factories that are able to utilize biomass for the production of value‐added platform chemicals and polymers. Compared to E. coli and S. cerevisiae, C. glutamicum displays weak carbon catabolite repression and can co‐utilize mixed sugars as carbon sources, without any significant growth retardation. Pathways for the utilization of alternative carbon sources, such as d‐xylose and l‐arabinose from lignocellulosic biomass, lactose and galactose from whey, glycerol from biodiesel, and methanol from natural gas refineries, have been evaluated for chemical production. However, the application of C. glutamicum in biorefineries is limited because it does not secrete hydrolases for the efficient utilization of cellulose, xylan, and starch from lignocellulosic and starch biomass. To solve the limitation, C. glutamicum has been engineered for the consolidated bioprocessing of biomass by the heterologous expression of amylolytic and cellulolytic enzymes. Recently, C. glutamicum has been extensively engineered for polyamide monomer production owing to its ability to produce l‐lysine and l‐glutamate. This review summarizes recent advances in the development of C. glutamicum strains that can utilize renewable biomass resources for the production of industrially important chemicals. It highlights recent progress in metabolic engineering for the production of polyamide monomers. © 2018 Society of Chemical Industry and John Wiley & Sons, Ltd

Accessing New Monomers from Biopriviledged Molecules By Coupling Metabolic Engineering and Electrosynthesis

Recent progress in metabolic engineering has generated new microorganisms able to convert carbohydrates and waste lignin into biopriviledged molecules.1 One of these molecules, muconic acid, can be further diversified to adipic acid and terephthalic acid, which are central to the production of Nylon-6,6 and polyethylene terephthalate (PET). Its semi-hydrogenation also leads to new molecules such as trans-3-hexenedioic acid (3HDA), a compound that enables the synthesis of bio-advantaged Nylon-6,6. However, to achieve economically sustainable conversion, it is crucial to streamline the fermentation and downstream catalytic processing steps. This generates new challenges as biogenic impurities inherent to the fermentation broth can deactivate Pt-group metal catalysts. An alternative approach would be to utilize novel conversion schemes using robust base metals that are stable in the presence of biogenic impurities.2 We show that electrosynthesis represents a promising approach for the direct processing of unpurified broths.2,3 The water, salts, and acidic impurities already present in the medium provide a suitable electrolyte, while protons in the solution provide the hydrogen required for the desired transformation. Muconic acid was converted to 3HDA with a 94% yield and 100% faradaic efficiency using lead (Pb) electrodes.4 Under these conditions, 3HDA can be produced at a competitive price of $2.00 kg-1. The reaction mechanism and the origin of the high selectivity to 3HDA will be discussed. 1. B.H. Shanks and P. Keeling, Bioprivileged Molecules: Creating Value from Biomass, Green Chem. 2017, advance article. https://dx.doi.org/10.1039/C7GC00296C2. M. Suastegui, J. E. Matthiesen, J. M. Carraher, N. Hernandez, N. Rodriguez Quiroz, A. Okerlund, E. W. Cochran, Z. Shao, J.-P. Tessonnier, Combining Metabolic Engineering and Electrocatalysis: Application to the Production of Polyamides from Sugar, Angew. Chem. Int. Ed., 2016, 55, 2368-2373. http://dx.doi.org/10.1002/anie.2015096533. J. E. Matthiesen, J. M. Carraher, M. Vasiliu, D. A. Dixon, J.-P. Tessonnier, Electrochemical Conversion of Muconic Acid to Biobased Diacid Monomers, ACS Sustainable Chem. Eng., 2016, 4, 3575-3585. http://dx.doi.org/10.1021/acssuschemeng.6b006794. J. E. Matthiesen, M. Suástegui, Y. Wu, M. Viswanathan, Y. Qu, M. Cao, N. Rodriguez-Quiroz, A. Okerlund, G. Kraus, D. R. Raman, Z. Shao, J.-P. Tessonnier, Electrochemical Conversion of Biologically Produced Muconic Acid: Key Considerations for Scale-up and Corresponding Technoeconomic Analysis, ACS Sustainable Chem. Eng., 2016, 12, 7098-7109. http://dx.doi.org/10.1021/acssuschemeng.6b01981

Recent progress in metabolic engineering for the production of biofuels and biochemicals from renewable sources with particular emphasis on catabolite regulation and its modulation

Recent metabolic engineering practice was briefly reviewed including the case where a mixture of multiple sugars obtained from lignocellulose, etc. was used as a carbon source to produce a variety of biofuels and biochemicals for the realization of green society. In the wild type Escherichia coli, sequential utilization of carbon sources is observed as known as diauxie phenomenon due to carbon catabolite repression (CCR), where much attention has been focused on co-consumption of multiple sugars to improve the productivities of the target metabolites. Although co-consumption of multiple sugars can be attained by modulating phosphotransferase system (PTS) and mgsA, pgi, etc. in E. coli, the glucose uptake rate inherently became lower, and thus the productivity of such metabolite as ethanol may not be improved, where this may be improved by amplifying the non-PTS pathway genes such as galP and glk. It should be noted that the modulation of PTS gene might change the robustness from the systems biology point of view.

Quantitative iTRAQ LC–MS/MS Proteomics Reveals Metabolic Responses to Biofuel Ethanol in Cyanobacterial Synechocystis sp. PCC 6803

Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.

Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria

In amino acid production by coryneform bacteria, study on relationship between change in enzyme activities and production of a target amino acid is important. In glutamate production, Kawahara et al. discovered that the effect of decrease in 2-oxoglutamate dehydrogenase complex (ODHC) on glutamate production is essential (Kawahara et al., Biosci. Biotechnol. Biochem. 61(7) (1997) 1109). Significant reduction of the ODHC activity was observed in the cells under the several glutamate-productive conditions in Corynebacterium glutamicum. Recent progress in metabolic engineering enables us to quantitatively compare the flux redistribution of the different strains after change in enzyme activity precisely. In this paper, relationship between flux redistribution and change in enzyme activities after biotin deletion and addition of detergent (Tween 40) was studied in two coryneform bacteria, C. glutamicum and a newly isolated strain, Corynebacterium efficiens (Fudou et al., Int. J. Syst. Evol. Microbiol. 52(Part 4) 1127), based on metabolic flux analysis (MFA). It was observed that in both species the specific activities of isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) did not significantly change throughout the fermentation, while that of the ODHC significantly decreased after biotin depletion and Tween 40 addition. Flux redistribution clearly occurred after the decrease in ODHC specific activity. The difference in glutamate production between C. glutamicum and C. efficiens was caused by the difference in the degree of decrease in ODHC specific activity. The difference in Michaelis–Menten constants or K m value between ICDH, GDH, and ODHC explained the mechanism of flux redistribution at the branch point of 2-oxoglutarate. It was found that the K m values of ICDH and ODHC were much lower than that of GDH for both strains. It was quantitatively proved that the ODHC plays the most important role in controlling flux distribution at the key branch point of 2-oxoglutarate in both coryneform bacteria. Flux redistribution mechanism was well simulated by a Michaelis–Menten-based model with kinetic parameters. The knowledge of the mechanism of flux redistribution will contribute to improvement of glutamate production in coryneform bacteria.