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Journal articles on the topic 'Rational Strain, Metabolic Engineering'

Author: Grafiati

Published: 6 September 2023

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Tsouka, Sophia, Meric Ataman, Tuure Hameri, Ljubisa Miskovic, and Vassily Hatzimanikatis. "Constraint-based metabolic control analysis for rational strain engineering." Metabolic Engineering 66 (July 2021): 191–203. http://dx.doi.org/10.1016/j.ymben.2021.03.003.

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Freedman, Benjamin G., Parker W. Lee, and Ryan S. Senger. "Engineering the Metabolic Profile of Clostridium cellulolyticum with Genomic DNA Libraries." Fermentation 9, no. 7 (2023): 605. http://dx.doi.org/10.3390/fermentation9070605.

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Clostridium cellulolyticum H10 (ATCC 35319) has the ability to ferment cellulosic substrates into ethanol and weak acids. The growth and alcohol production rates of the wild-type organism are low and, therefore, targets of metabolic engineering. A genomic DNA expression library was produced by a novel application of degenerate oligonucleotide primed PCR (DOP-PCR) and was serially enriched in C. cellulolyticum grown on cellobiose in effort to produce fast-growing and productive strains. The DNA library produced from DOP-PCR contained gene-sized DNA fragments from the C. cellulolyticum genome and from the metagenome of a stream bank soil sample. The resulting enrichment yielded a conserved phage structural protein fragment (part of Ccel_2823) from the C. cellulolyticum genome that, when overexpressed alone, enabled the organism to increase the ethanol yield by 250% compared to the plasmid control strain. The engineered strain showed a reduced production of lactate and a 250% increased yield of secreted pyruvate. Significant changes in growth rate were not seen in this engineered strain, and it is possible that the enriched protein fragment may be combined with the existing rational metabolic engineering strategies to yield further high-performing cellulolytic strains.

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Burgardt, Arthur, Ludovic Pelosi, Mahmoud Hajj Chehade, Volker F. Wendisch, and Fabien Pierrel. "Rational Engineering of Non-Ubiquinone Containing Corynebacterium glutamicum for Enhanced Coenzyme Q10 Production." Metabolites 12, no. 5 (2022): 428. http://dx.doi.org/10.3390/metabo12050428.

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Coenzyme Q10 (CoQ10) is a lipid-soluble compound with important physiological functions and is sought after in the food and cosmetic industries owing to its antioxidant properties. In our previous proof of concept, we engineered for CoQ10 biosynthesis the industrially relevant Corynebacterium glutamicum, which does not naturally synthesize any CoQ. Here, liquid chromatography–mass spectrometry (LC–MS) analysis identified two metabolic bottlenecks in the CoQ10 production, i.e., low conversion of the intermediate 10-prenylphenol (10P-Ph) to CoQ10 and the accumulation of isoprenologs with prenyl chain lengths of not only 10, but also 8 to 11 isopentenyl units. To overcome these limitations, the strain was engineered for expression of the Ubi complex accessory factors UbiJ and UbiK from Escherichia coli to increase flux towards CoQ10, and by replacement of the native polyprenyl diphosphate synthase IspB with a decaprenyl diphosphate synthase (DdsA) to select for prenyl chains with 10 isopentenyl units. The best strain UBI6-Rs showed a seven-fold increased CoQ10 content and eight-fold increased CoQ10 titer compared to the initial strain UBI4-Pd, while the abundance of CoQ8, CoQ9, and CoQ11 was significantly reduced. This study demonstrates the application of the recent insight into CoQ biosynthesis to improve metabolic engineering of a heterologous CoQ10 production strain.

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Zhu, Linghuan, Sha Xu, Youran Li, and Guiyang Shi. "Improvement of 2-phenylethanol production in Saccharomyces cerevisiae by evolutionary and rational metabolic engineering." PLOS ONE 16, no. 10 (2021): e0258180. http://dx.doi.org/10.1371/journal.pone.0258180.

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2-Phenylethanol (2-PE) is a valuable aromatic compound with favorable flavors and good properties, resulting in its widespread application in the cosmetic, food and medical industries. In this study, a mutant strain, AD032, was first obtained by adaptive evolution under 2-PE stress. Then, a fusion protein from the Ehrlich pathway, composed of tyrB from Escherichia coli, kdcA from Lactococcus lactis and ADH2 from Saccharomyces cerevisiae, was constructed and expressed. As a result, 3.14 g/L 2-PE was achieved using L-phenylalanine as a precursor. To further increase 2-PE production, L-glutamate oxidase from Streptomyces overexpression was applied for the first time in our research to improve the supply of α-ketoglutarate in the transamination of 2-PE synthesis. Furthermore, we found that the disruption of the pyruvate decarboxylase encoding gene PDC5 caused an increase in 2-PE production, which has not yet been reported. Finally, assembly of the efficient metabolic modules and process optimization resulted in the strain RM27, which reached 4.02 g/L 2-PE production from 6.7 g/L L-phenylalanine without in situ product recovery. The strain RM27 produced 2-PE (0.8 mol/mol) with L-phenylalanine as a precursor, which was considerably high, and displayed manufacturing potential regarding food safety and process simplification aspects. This study suggests that innovative strategies regarding metabolic modularization provide improved prospects for 2-PE production in food exploitation.

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Nevoigt, Elke. "Progress in Metabolic Engineering of Saccharomyces cerevisiae." Microbiology and Molecular Biology Reviews 72, no. 3 (2008): 379–412. http://dx.doi.org/10.1128/mmbr.00025-07.

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SUMMARY The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

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Natarajan, Aravind, Thapakorn Jaroentomeechai, Mingji Li, Cameron J. Glasscock, and Matthew P. DeLisa. "Metabolic engineering of glycoprotein biosynthesis in bacteria." Emerging Topics in Life Sciences 2, no. 3 (2018): 419–32. http://dx.doi.org/10.1042/etls20180004.

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The demonstration more than a decade ago that glycoproteins could be produced in Escherichia coli cells equipped with the N-linked protein glycosylation machinery from Campylobacter jejuni opened the door to using simple bacteria for the expression and engineering of complex glycoproteins. Since that time, metabolic engineering has played an increasingly important role in developing and optimizing microbial cell glyco-factories for the production of diverse glycoproteins and other glycoconjugates. It is becoming clear that future progress in creating efficient glycoprotein expression platforms in bacteria will depend on the adoption of advanced strain engineering strategies such as rational design and assembly of orthogonal glycosylation pathways, genome-wide identification of metabolic engineering targets, and evolutionary engineering of pathway performance. Here, we highlight recent advances in the deployment of metabolic engineering tools and strategies to develop microbial cell glyco-factories for the production of high-value glycoprotein targets with applications in research and medicine.

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Tafur Rangel, Albert E., Abel García Oviedo, Freddy Cabrera Mojica, Jorge M. Gómez, and Andrés Fernando Gónzalez Barrios. "Development of an integrating systems metabolic engineering and bioprocess modeling approach for rational strain improvement." Biochemical Engineering Journal 178 (January 2022): 108268. http://dx.doi.org/10.1016/j.bej.2021.108268.

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Zhang, Xiaomei, Zhenhang Sun, Jinyu Bian, et al. "Rational Metabolic Engineering Combined with Biosensor-Mediated Adaptive Laboratory Evolution for l-Cysteine Overproduction from Glycerol in Escherichia coli." Fermentation 8, no. 7 (2022): 299. http://dx.doi.org/10.3390/fermentation8070299.

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l-Cysteine is an important sulfur-containing amino acid with numerous applications in the pharmaceutical and cosmetic industries. The microbial production of l-cysteine has received substantial attention, and the supply of the precursor l-serine is important in l-cysteine biosynthesis. In this study, to achieve l-cysteine overproduction, we first increased l-serine production by deleting genes involved in the pathway of l-serine degradation to glycine (serine hydroxymethyl transferase, SHMT, encoded by glyA genes) in strain 4W (with l-serine titer of 1.1 g/L), thus resulting in strain 4WG with l-serine titer of 2.01 g/L. Second, the serine-biosensor based on the transcriptional regulator NCgl0581 of C. glutamicum was constructed in E. coli, and the validity and sensitivity of the biosensor were demonstrated in E. coli. Then 4WG was further evolved through adaptive laboratory evolution (ALE) combined with serine-biosensor, thus yielding the strain 4WGX with 4.13 g/L l-serine production. Moreover, the whole genome of the evolved strain 4WGX was sequenced, and ten non-synonymous mutations were found in the genome of strain 4WGX compared with strain 4W. Finally, 4WGX was used as the starting strain, and deletion of the l-cysteine desulfhydrases (encoded by tnaA), overexpression of serine acetyltransferase (encoded by cysE) and the key enzyme of transport pathway (encoded by ydeD) were performed in strain 4WGX. The recombinant strain 4WGX-∆tnaA-cysE-ydeD can produce 313.4 mg/L of l-cysteine using glycerol as the carbon source. This work provides an efficient method for the biosynthesis of value-added commodity products associated with glycerol conversion.

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Iacometti, Camillo, Katharina Marx, Maria Hönick, et al. "Activating Silent Glycolysis Bypasses in Escherichia coli." BioDesign Research 2022 (May 12, 2022): 1–17. http://dx.doi.org/10.34133/2022/9859643.

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All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal and the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal seemed to immediately operate in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary to restore growth of the strain. Furthermore, we engineered the “serine shunt” which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection, we performed an adaptive laboratory evolution study using an enolase deletion strain. Our experiments suggest that the evolved mutants use the serine shunt. Our study reveals the flexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism.

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Jeong, Sun-Wook, Jun-Ho Kim, Ji-Woong Kim, Chae Yeon Kim, Su Young Kim, and Yong Jun Choi. "Metabolic Engineering of Extremophilic Bacterium Deinococcus radiodurans for the Production of the Novel Carotenoid Deinoxanthin." Microorganisms 9, no. 1 (2020): 44. http://dx.doi.org/10.3390/microorganisms9010044.

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Deinoxanthin, a xanthophyll derived from Deinococcus species, is a unique organic compound that provides greater antioxidant effects compared to other carotenoids due to its superior scavenging activity against singlet oxygen and hydrogen peroxide. Therefore, it has attracted significant attention as a next-generation organic compound that has great potential as a natural ingredient in a food supplements. Although the microbial identification of deinoxanthin has been identified, mass production has not yet been achieved. Here, we report, for the first time, the development of an engineered extremophilic microorganism, Deinococcus radiodurans strain R1, that is capable of producing deinoxanthin through rational metabolic engineering and process optimization. The genes crtB and dxs were first introduced into the genome to reinforce the metabolic flux towards deinoxanthin. The optimal temperature was then identified through a comparative analysis of the mRNA expression of the two genes, while the carbon source was further optimized to increase deinoxanthin production. The final engineered D. radiodurans strain R1 was able to produce 394 ± 17.6 mg/L (102 ± 11.1 mg/g DCW) of deinoxanthin with a yield of 40.4 ± 1.2 mg/g sucrose and a productivity of 8.4 ± 0.2 mg/L/h from 10 g/L of sucrose. The final engineered strain and the strategies developed in the present study can act as the foundation for the industrial application of extremophilic microorganisms.

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Fuchino, Katsuya, Uldis Kalnenieks, Reinis Rutkis, Mara Grube, and Per Bruheim. "Metabolic Profiling of Glucose-Fed Metabolically Active Resting Zymomonas mobilis Strains." Metabolites 10, no. 3 (2020): 81. http://dx.doi.org/10.3390/metabo10030081.

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Zymomonas mobilis is the most efficient bacterial ethanol producer and its physiology is potentially applicable to industrial-scale bioethanol production. However, compared to other industrially important microorganisms, the Z. mobilis metabolome and adaptation to various nutritional and genetic perturbations have been poorly characterized. For rational metabolic engineering, it is essential to understand how central metabolism and intracellular redox balance are maintained in Z. mobilis under various conditions. In this study, we applied quantitative mass spectrometry-based metabolomics to explore how glucose-fed non-growing Z. mobilis Zm6 cells metabolically adapt to change of oxygen availability. Mutants partially impaired in ethanol synthesis (Zm6 adhB) or oxidative stress response (Zm6 cat) were also examined. Distinct patterns of adaptation of central metabolite pools due to the change in cultivation condition and between the mutants and Zm6 reference strain were observed. Decreased NADH/NAD ratio under aerobic incubation corresponded to higher concentrations of the phosphorylated glycolytic intermediates, in accordance with predictions of the kinetic model of Entner–Doudoroff pathway. The effects on the metabolite pools of aerobic to anaerobic transition were similar in the mutants, yet less pronounced. The present data on metabolic plasticity of non-growing Z. mobilis cells will facilitate the further metabolic engineering of the respective strains and their application as biocatalysts.

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Arora, Neha, Hong-Wei Yen, and George P. Philippidis. "Harnessing the Power of Mutagenesis and Adaptive Laboratory Evolution for High Lipid Production by Oleaginous Microalgae and Yeasts." Sustainability 12, no. 12 (2020): 5125. http://dx.doi.org/10.3390/su12125125.

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Oleaginous microalgae and yeasts represent promising candidates for large-scale production of lipids, which can be utilized for production of drop-in biofuels, nutraceuticals, pigments, and cosmetics. However, low lipid productivity and costly downstream processing continue to hamper the commercial deployment of oleaginous microorganisms. Strain improvement can play an essential role in the development of such industrial microorganisms by increasing lipid production and hence reducing production costs. The main means of strain improvement are random mutagenesis, adaptive laboratory evolution (ALE), and rational genetic engineering. Among these, random mutagenesis and ALE are straight forward, low-cost, and do not require thorough knowledge of the microorganism’s genetic composition. This paper reviews available mutagenesis and ALE techniques and screening methods to effectively select for oleaginous microalgae and yeasts with enhanced lipid yield and understand the alterations caused to metabolic pathways, which could subsequently serve as the basis for further targeted genetic engineering.

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Wang, Chenyang, Qinyu Li, Peng Zhou, Xiaojia Chen, Jiping Shi, and Zhijun Zhao. "Bioprocess Engineering, Transcriptome, and Intermediate Metabolite Analysis of L-Serine High-Yielding Escherichia coli W3110." Microorganisms 10, no. 10 (2022): 1927. http://dx.doi.org/10.3390/microorganisms10101927.

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L-serine is widely used in the food, cosmetic, and pharmaceutical industries. However, the complicated metabolic network and regulatory mechanism of L-serine production lead to the suboptimal productivity of the direct fermentation of L-serine and limits its large-scale industrial production. In this study, a high-yield L-serine production Escherichia coli strain was constructed by a series of defined genetic modification methodologies. First, L-serine-mediated feedback inhibition was removed and L-serine biosynthetic pathway genes (serAfr, serC, and serB) associated with phosphoglycerate kinase (pgk) were overexpressed. Second, the L-serine conversion pathway was further examined by introducing a glyA mutation (K229G) and deleting other degrading enzymes based on the deletion of initial sdaA. Finally, the L-serine transport system was rationally engineered to reduce uptake and accelerate L-serine export. The optimally engineered strain produced 35 g/L L-serine with a productivity of 0.98 g/L/h and a yield of 0.42 g/g glucose in a 5-L fermenter, the highest productivity and yield of L-serine from glucose reported to date. Furthermore, transcriptome and intermediate metabolite of the high-yield L-serine production Escherichia coli strain were analyzed. The results demonstrated the regulatory mechanism of L-serine production is delicate, and that combined metabolic and bioprocess engineering strategies for L-serine producing strains can improve the productivity and yield.

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Xu, Feng, Xiang Ke, Ming Hong, et al. "Exploring the metabolic fate of propanol in industrial erythromycin-producing strain via 13C labeling experiments and enhancement of erythromycin production by rational metabolic engineering of Saccharopolyspora erythraea." Biochemical and Biophysical Research Communications 542 (February 2021): 73–79. http://dx.doi.org/10.1016/j.bbrc.2021.01.024.

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Wang, Xuan, Xianhao Xu, Jiaheng Liu, et al. "Metabolic Engineering of Saccharomyces cerevisiae for Efficient Retinol Synthesis." Journal of Fungi 9, no. 5 (2023): 512. http://dx.doi.org/10.3390/jof9050512.

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Retinol, the main active form of vitamin A, plays a role in maintaining vision, immune function, growth, and development. It also inhibits tumor growth and alleviates anemia. Here, we developed a Saccharomyces cerevisiae strain capable of high retinol production. Firstly, the de novo synthesis pathway of retinol was constructed in S. cerevisiae to realize the production of retinol. Second, through modular optimization of the metabolic network of retinol, the retinol titer was increased from 3.6 to 153.6 mg/L. Then, we used transporter engineering to regulate and promote the accumulation of the intracellular precursor retinal to improve retinol production. Subsequently, we screened and semi-rationally designed the key enzyme retinol dehydrogenase to further increase the retinol titer to 387.4 mg/L. Lastly, we performed two-phase extraction fermentation using olive oil to obtain a final shaking flask retinol titer of 1.2 g/L, the highest titer reported at the shake flask level. This study laid the foundation for the industrial production of retinol.

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Xu, Jian, Li Zhou та Zhemin Zhou. "Enhancement of β-Alanine Biosynthesis in Escherichia coli Based on Multivariate Modular Metabolic Engineering". Biology 10, № 10 (2021): 1017. http://dx.doi.org/10.3390/biology10101017.

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β-alanine is widely used as an intermediate in industrial production. However, the low production of microbial cell factories limits its further application. Here, to improve the biosynthesis production of β-alanine in Escherichia coli, multivariate modular metabolic engineering was recruited to manipulate the β-alanine biosynthesis pathway through keeping the balance of metabolic flux among the whole metabolic network. The β-alanine biosynthesis pathway was separated into three modules: the β-alanine biosynthesis module, TCA module, and glycolysis module. Global regulation was performed throughout the entire β-alanine biosynthesis pathway rationally and systematically by optimizing metabolic flux, overcoming metabolic bottlenecks and weakening branch pathways. As a result, metabolic flux was channeled in the direction of β-alanine biosynthesis without huge metabolic burden, and 37.9 g/L β-alanine was generated by engineered Escherichia coli strain B0016-07 in fed-batch fermentation. This study was meaningful to the synthetic biology of β-alanine industrial production.

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Lee, Sang Jun, Dong-Yup Lee, Tae Yong Kim, Byung Hun Kim, Jinwon Lee, and Sang Yup Lee. "Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation." Applied and Environmental Microbiology 71, no. 12 (2005): 7880–87. http://dx.doi.org/10.1128/aem.71.12.7880-7887.2005.

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ABSTRACT Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.

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Wang, Qingzhao, Mark S. Ou, Y. Kim, L. O. Ingram, and K. T. Shanmugam. "Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase." Applied and Environmental Microbiology 76, no. 7 (2010): 2107–14. http://dx.doi.org/10.1128/aem.02545-09.

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ABSTRACT During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y ATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y ATP suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.

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Dwijayanti, Ari, Marko Storch, Guy-Bart Stan, and Geoff S. Baldwin. "A modular RNA interference system for multiplexed gene regulation." Nucleic Acids Research 50, no. 3 (2022): 1783–93. http://dx.doi.org/10.1093/nar/gkab1301.

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Abstract The rational design and realisation of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional regulation of gene expression in Escherichia coli. The regulatory function of mARi was characterised in a range of relevant genetic contexts, demonstrating its independence from other genetic control elements and the gene of interest, and providing new insight into the design rules of RNA based regulation in E. coli, while a range of cellular contexts also demonstrated it to be independent of growth-phase and strain type. Importantly, the extensibility and orthogonality of mARi enables the simultaneous post-transcriptional regulation of multi-gene systems as both single-gene cassettes and poly-cistronic operons. To facilitate adoption, mARi was designed to be directly integrated into the modular BASIC DNA assembly framework. We anticipate that mARi-based genetic control within an extensible DNA assembly framework will facilitate metabolic engineering, layered genetic control, and advanced genetic circuit applications.

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Sheremetieva, M. E., K. E. Anufriev, T. M. Khlebodarova, N. A. Kolchanov, and A. S. Yanenko. "Rational metabolic engineering of <i>Corynebacterium glutamicum</i> to create a producer of L-valine." Vavilov Journal of Genetics and Breeding 26, no. 8 (2023): 743–57. http://dx.doi.org/10.18699/vjgb-22-90.

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L-Valine is one of the nine amino acids that cannot be synthesized de novo by higher organisms and must come from food. This amino acid not only serves as a building block for proteins, but also regulates protein and energy metabolism and participates in neurotransmission. L-Valine is used in the food and pharmaceutical industries, medicine and cosmetics, but primarily as an animal feed additive. Adding L-valine to feed, alone or mixed with other essential amino acids, allows for feeds with lower crude protein content, increases the quality and quantity of pig meat and broiler chicken meat, as well as improves reproductive functions of farm animals. Despite the fact that the market for L-valine is constantly growing, this amino acid is not yet produced in our country. In modern conditions, the creation of strains-producers and organization of L-valine production are especially relevant for Russia. One of the basic microorganisms most commonly used for the creation of amino acid producers, along with Escherichia coli, is the soil bacterium Corynebacterium glutamicum. This review is devoted to the analysis of the main strategies for the development of L- valine producers based on C. glutamicum. Various aspects of L-valine biosynthesis in C. glutamicum are reviewed: process biochemistry, stoichiometry and regulation, enzymes and their corresponding genes, export and import systems, and the relationship of L-valine biosynthesis with central cell metabolism. Key genetic elements for the creation of C. glutamicum-based strains-producers are identified. The use of metabolic engineering to enhance L-valine biosynthesis reactions and to reduce the formation of byproducts is described. The prospects for improving strains in terms of their productivity and technological characteristics are shown. The information presented in the review can be used in the production of producers of other amino acids with a branched side chain, namely L-leucine and L-isoleucine, as well as D-pantothenate.

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Pyne, Michael E., Stanislav Sokolenko, Xuejia Liu, et al. "Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum." Applied and Environmental Microbiology 82, no. 17 (2016): 5375–88. http://dx.doi.org/10.1128/aem.01354-16.

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ABSTRACTCrude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry,Clostridium pasteurianumhas gained prominence as a result of its unique capacity to convert waste glycerol inton-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product ofC. pasteurianumglycerol fermentation. Here, we report rational metabolic engineering ofC. pasteurianumfor enhancedn-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in thedhaTdisruption mutant (dhaT::Ll.LtrB). Metabolite characterization of thedhaT::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowestC. pasteurianum1,3-PDO titer reported to date (0.58 g liter−1). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively ton-butanol (8.6 g liter−1), yielding a highn-butanol selectivity of 0.83 gn-butanol g−1of solvents compared to 0.51 gn-butanol g−1of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis ofdhaT::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation byC. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol.IMPORTANCEOrganisms from the genusClostridiumare perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability ofC. pasteurianumto sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involvingC. pasteurianum. To our knowledge, theC. pasteurianum dhaTdisruption mutant derived in this study is the only organism that produces both 1,2- and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production ofn-butanol from waste glycerol.

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Choi, Bo Hyun, Hyun Joon Kang, Sun Chang Kim, and Pyung Cheon Lee. "Organelle Engineering in Yeast: Enhanced Production of Protopanaxadiol through Manipulation of Peroxisome Proliferation in Saccharomyces cerevisiae." Microorganisms 10, no. 3 (2022): 650. http://dx.doi.org/10.3390/microorganisms10030650.

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Isoprenoids, which are natural compounds with diverse structures, possess several biological activities that are beneficial to humans. A major consideration in isoprenoid production in microbial hosts is that the accumulation of biosynthesized isoprenoid within intracellular membranes may impede balanced cell growth, which may consequently reduce the desired yield of the target isoprenoid. As a strategy to overcome this suggested limitation, we selected peroxisome membranes as depots for the additional storage of biosynthesized isoprenoids to facilitate increased isoprenoid production in Saccharomyces cerevisiae. To maximize the peroxisome membrane storage capacity of S.cerevisiae, the copy number and size of peroxisomes were increased through genetic engineering of the expression of three peroxisome biogenesis-related peroxins (Pex11p, Pex34p, and Atg36p). The genetically enlarged and high copied peroxisomes in S.cerevisiae were stably maintained under a bioreactor fermentation condition. The peroxisome-engineered S.cerevisiae strains were then utilized as host strains for metabolic engineering of heterologous protopanaxadiol pathway. The yields of protopanaxadiol from the engineered peroxisome strains were ca 78% higher than those of the parent strain, which strongly supports the rationale for harnessing the storage capacity of the peroxisome membrane to accommodate the biosynthesized compounds. Consequently, this study presents in-depth knowledge on peroxisome biogenesis engineering in S.cerevisiae and could serve as basic information for improvement in ginsenosides production and as a potential platform to be utilized for other isoprenoids.

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Paramasivan, Kalaivani, Aneesha Abdulla, Nabarupa Gupta, and Sarma Mutturi. "In silico target-based strain engineering of Saccharomyces cerevisiae for terpene precursor improvement." Integrative Biology 14, no. 2 (2022): 25–36. http://dx.doi.org/10.1093/intbio/zyac003.

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Abstract Systems-based metabolic engineering enables cells to enhance product formation by predicting gene knockout and overexpression targets using modeling tools. FOCuS, a novel metaheuristic tool, was used to predict flux improvement targets in terpenoid pathway using the genome-scale model of Saccharomyces cerevisiae, iMM904. Some of the key knockout target predicted includes LYS1, GAP1, AAT1, AAT2, TH17, KGD-m, MET14, PDC1 and ACO1. It was also observed that the knockout reactions belonged either to fatty acid biosynthesis, amino acid synthesis pathways or nucleotide biosynthesis pathways. Similarly, overexpression targets such as PFK1, FBA1, ZWF1, TDH1, PYC1, ALD6, TPI1, PDX1 and ENO1 were established using three different existing gene amplification algorithms. Most of the overexpression targets belonged to glycolytic and pentose phosphate pathways. Each of these targets had plausible role for improving flux toward sterol pathway and were seemingly not artifacts. Moreover, an in vitro study as validation was carried with overexpression of ALD6 and TPI1. It was found that there was an increase in squalene synthesis by 2.23- and 4.24- folds, respectively, when compared with control. In general, the rationale for predicting these in silico targets was attributed to either increasing the acetyl-CoA precursor pool or regeneration of NADPH, which increase the sterol pathway flux.

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Pyne, Michael, Murray Moo-Young, Duane Chung, and C. Chou. "Antisense-RNA-Mediated Gene Downregulation in Clostridium pasteurianum." Fermentation 1, no. 1 (2015): 113–26. http://dx.doi.org/10.3390/fermentation1010113.

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Clostridium pasteurianum is receiving growing attention for its unique metabolic properties, particularly its ability to convert waste glycerol and glycerol-rich byproducts into butanol, a prospective biofuel. Genetic tool development and whole genome sequencing have recently been investigated to advance the genetic tractability of this potential industrial host. Nevertheless, methodologies for tuning gene expression through plasmid-borne expression and chromosomal gene downregulation are still absent. Here we demonstrate plasmid-borne heterologous gene expression and gene knockdown using antisense RNA in C. pasteurianum. We first employed a common thermophilic β-galactosidase (lacZ) gene reporter system from Thermoanaerobacterium thermosulfurogenes to characterize two promoters involved in the central fermentative metabolism of C. pasteurianum. Due to a higher level of constitutive lacZ expression compared to the ferredoxin gene (fdx) promoter, the thiolase (thl) promoter was selected to drive expression of asRNA. Expression of a lacZ asRNA resulted in 52%–58% downregulation of β-galactosidase activity compared to the control strain throughout the duration of culture growth. Subsequent implementation of our asRNA approach for downregulation of the native hydrogenase I gene (hydA) in C. pasteurianum resulted in altered end product distribution, characterized by an increase in production of reduced metabolites, particularly butyrate (40% increase) and ethanol (25% increase). Knockdown of hydA was also accompanied by increased acetate formation and lower levels of 1,3-propanediol, signifying a dramatic shift in cellular metabolism in response to inhibition of the hydrogenase enzyme. The methodologies described herein for plasmid-based heterologous gene expression and antisense-RNA-mediated gene knockdown should promote rational metabolic engineering of C. pasteurianum for enhanced production of butanol as a prospective biofuel.

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Deeba, Farha, Kukkala Kiran Kumar, Girish H. Rajacharya, and Naseem A. Gaur. "Metabolomic Profiling Revealed Diversion of Cytidinediphosphate-Diacylglycerol and Glycerol Pathway towards Denovo Triacylglycerol Synthesis in Rhodosporidium toruloides." Journal of Fungi 7, no. 11 (2021): 967. http://dx.doi.org/10.3390/jof7110967.

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Oleaginous yeast Rhodosporidium toruloides has great biotechnological potential and scientific interest, yet the molecular rationale of its cellular behavior to carbon and nitrogen ratios with concurrent lipid agglomeration remains elusive. Here, metabolomics adaptations of the R. toruloides in response to varying glucose and nitrogen concentrations have been investigated. In preliminary screening we found that 5% glucose (w/v) was optimal for further analysis in Rhodosporidium toruloides 3641. Hereafter, the effect of complementation to increase lipid agglomeration was evaluated with different nitrogen sources and their concentration. The results obtained illustrated that the biomass (13 g/L) and lipid (9.1 g/L) production were maximum on 5% (w/v) glucose and 0.12% (NH4)2SO4. Furthermore, to shed lights on lipid accumulation induced by nitrogen-limitation, we performed metabolomic analysis of the oleaginous yeast R. toruloides 3641. Significant changes were observed in metabolite concentrations by qualitative metabolomics through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), which were mapped onto the governing metabolic pathways. Notable finding in this strain concerns glycerol and CDP-DAG metabolism wherein reduced production of glycerol and phospholipids induced a bypass leading to enhanced de-novo triacylglyceride synthesis. Collectively, our findings help in understanding the central carbon metabolism of R. toruloides which may assist in developing rationale metabolic models and engineering efforts in this organism.

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Neves, Rui P. P., Bruno Araújo, Maria J. Ramos, and Pedro A. Fernandes. "Feedback Inhibition of DszC, a Crucial Enzyme for Crude Oil Biodessulfurization." Catalysts 13, no. 4 (2023): 736. http://dx.doi.org/10.3390/catal13040736.

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The Rhodococcus erythropolis (strain IGTS8) bacterium has a tremendous industrial interest as it can remove sulfur from crude oil through its four-enzyme (DszA-D) 4S metabolic pathway. DszC is one of the rate-limiting enzymes of the pathway and the one that most suffers from feedback inhibition. We have combined molecular docking and molecular dynamics simulations to identify binding sites through which two products of the 4S pathway, 2-hydroxybiphenyl and 2′-hydroxybiphenyl-2-sulfinate, induce DszC feedback inhibition. We have identified four potential binding sites: two adjacent binding sites close to the 280–295 lid loop proposed to contribute to DszC oligomerization and proper binding of the flavin mononucleotide cofactor, and two other close to the active site of DszC and the substrate binding site. By considering (i) the occupancy of the binding sites and (ii) the similar inhibitor poses, we propose that the mechanism of feedback inhibition of DszC occurs through disturbance of the DszC oligomerization and consequent binding of the flavin mononucleotide due to the weakening of the interactions between the 280–295 lid loop, and both the 131–142 loop and the C-terminal tail. Nevertheless, inhibitor binding close to the active site or the substrate binding sites also compromises critical interactions within the active site of DszC. The disclosed molecular details provide valuable insight for future rational enzyme engineering protocols to develop DszC mutants more resistant against the observed feedback inhibition mechanism.

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Huang, Mingtao, Yunpeng Bai, Staffan L. Sjostrom, et al. "Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast." Proceedings of the National Academy of Sciences 112, no. 34 (2015): E4689—E4696. http://dx.doi.org/10.1073/pnas.1506460112.

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There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

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Pan, Guohui, Zhengren Xu, Zhikai Guo, et al. "Discovery of the leinamycin family of natural products by mining actinobacterial genomes." Proceedings of the National Academy of Sciences 114, no. 52 (2017): E11131—E11140. http://dx.doi.org/10.1073/pnas.1716245115.

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Nature’s ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF–SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF–SH didomains. Further analysis of the representative genomes from each of the clades identified 28 lnm-type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature’s rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature’s biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.

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Darbani, Behrooz. "Genome Evolutionary Dynamics Meets Functional Genomics: A Case Story on the Identification of SLC25A44." International Journal of Molecular Sciences 22, no. 11 (2021): 5669. http://dx.doi.org/10.3390/ijms22115669.

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Gene clusters are becoming promising tools for gene identification. The study reveals the purposive genomic distribution of genes toward higher inheritance rates of intact metabolic pathways/phenotypes and, thereby, higher fitness. The co-localization of co-expressed, co-interacting, and functionally related genes was found as genome-wide trends in humans, mouse, golden eagle, rice fish, Drosophila, peanut, and Arabidopsis. As anticipated, the analyses verified the co-segregation of co-localized events. A negative correlation was notable between the likelihood of co-localization events and the inter-loci distances. The evolution of genomic blocks was also found convergent and uniform along the chromosomal arms. Calling a genomic block responsible for adjacent metabolic reactions is therefore recommended for identification of candidate genes and interpretation of cellular functions. As a case story, a function in the metabolism of energy and secondary metabolites was proposed for Slc25A44, based on its genomic local information. Slc25A44 was further characterized as an essential housekeeping gene which has been under evolutionary purifying pressure and belongs to the phylogenetic ETC-clade of SLC25s. Pathway enrichment mapped the Slc25A44s to the energy metabolism. The expression of peanut and human Slc25A44s in oocytes and Saccharomyces cerevisiae strains confirmed the transport of common precursors for secondary metabolites and ubiquinone. These results suggest that SLC25A44 is a mitochondrion-ER-nucleus zone transporter with biotechnological applications. Finally, a conserved three-amino acid signature on the cytosolic face of transport cavity was found important for rational engineering of SLC25s.

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Wiedemann, Beate, and Eckhard Boles. "Codon-Optimized Bacterial Genes Improve l-Arabinose Fermentation in Recombinant Saccharomyces cerevisiae." Applied and Environmental Microbiology 74, no. 7 (2008): 2043–50. http://dx.doi.org/10.1128/aem.02395-07.

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ABSTRACT Bioethanol produced by microbial fermentations of plant biomass hydrolysates consisting of hexose and pentose mixtures is an excellent alternative to fossil transportation fuels. However, the yeast Saccharomyces cerevisiae, commonly used in bioethanol production, can utilize pentose sugars like l-arabinose or d-xylose only after heterologous expression of corresponding metabolic pathways from other organisms. Here we report the improvement of a bacterial l-arabinose utilization pathway consisting of l-arabinose isomerase from Bacillus subtilis and l-ribulokinase and l-ribulose-5-P 4-epimerase from Escherichia coli after expression of the corresponding genes in S. cerevisiae. l-Arabinose isomerase from B. subtilis turned out to be the limiting step for growth on l-arabinose as the sole carbon source. The corresponding enzyme could be effectively replaced by the enzyme from Bacillus licheniformis, leading to a considerably decreased lag phase. Subsequently, the codon usage of all the genes involved in the l-arabinose pathway was adapted to that of the highly expressed genes encoding glycolytic enzymes in S. cerevisiae. Yeast transformants expressing the codon-optimized genes showed strongly improved l-arabinose conversion rates. With this rational approach, the ethanol production rate from l-arabinose could be increased more than 2.5-fold from 0.014 g ethanol h−1 (g dry weight)−1 to 0.036 g ethanol h−1 (g dry weight)−1 and the ethanol yield could be increased from 0.24 g ethanol (g consumed l-arabinose)−1 to 0.39 g ethanol (g consumed l-arabinose)−1. These improvements make up a new starting point for the construction of more-efficient industrial l-arabinose-fermenting yeast strains by evolutionary engineering.

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Carlson, Ross, David Fell, and Friedrich Srienc. "Metabolic pathway analysis of a recombinant yeast for rational strain development." Biotechnology and Bioengineering 79, no. 2 (2002): 121–34. http://dx.doi.org/10.1002/bit.10305.

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Wu, Sijia, Wenjuan Chen, Sujuan Lu, Hailing Zhang, and Lianghong Yin. "Metabolic Engineering of Shikimic Acid Biosynthesis Pathway for the Production of Shikimic Acid and Its Branched Products in Microorganisms: Advances and Prospects." Molecules 27, no. 15 (2022): 4779. http://dx.doi.org/10.3390/molecules27154779.

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The shikimate pathway is a necessary pathway for the synthesis of aromatic compounds. The intermediate products of the shikimate pathway and its branching pathway have promising properties in many fields, especially in the pharmaceutical industry. Many important compounds, such as shikimic acid, quinic acid, chlorogenic acid, gallic acid, pyrogallol, catechol and so on, can be synthesized by the shikimate pathway. Among them, shikimic acid is the key raw material for the synthesis of GS4104 (Tamiflu®), an inhibitor of neuraminidase against avian influenza virus. Quininic acid is an important intermediate for synthesis of a variety of raw chemical materials and drugs. Gallic acid and catechol receive widespread attention as pharmaceutical intermediates. It is one of the hotspots to accumulate many kinds of target products by rationally modifying the shikimate pathway and its branches in recombinant strains by means of metabolic engineering. This review considers the effects of classical metabolic engineering methods, such as central carbon metabolism (CCM) pathway modification, key enzyme gene modification, blocking the downstream pathway on the shikimate pathway, as well as several expansion pathways and metabolic engineering strategies of the shikimate pathway, and expounds the synthetic biology in recent years in the application of the shikimate pathway and the future development direction.

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Michael, Drew G., Ezekiel J. Maier, Holly Brown, et al. "Model-based transcriptome engineering promotes a fermentative transcriptional state in yeast." Proceedings of the National Academy of Sciences 113, no. 47 (2016): E7428—E7437. http://dx.doi.org/10.1073/pnas.1603577113.

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The ability to rationally manipulate the transcriptional states of cells would be of great use in medicine and bioengineering. We have developed an algorithm, NetSurgeon, which uses genome-wide gene-regulatory networks to identify interventions that force a cell toward a desired expression state. We first validated NetSurgeon extensively on existing datasets. Next, we used NetSurgeon to select transcription factor deletions aimed at improving ethanol production in Saccharomyces cerevisiae cultures that are catabolizing xylose. We reasoned that interventions that move the transcriptional state of cells using xylose toward that of cells producing large amounts of ethanol from glucose might improve xylose fermentation. Some of the interventions selected by NetSurgeon successfully promoted a fermentative transcriptional state in the absence of glucose, resulting in strains with a 2.7-fold increase in xylose import rates, a 4-fold improvement in xylose integration into central carbon metabolism, or a 1.3-fold increase in ethanol production rate. We conclude by presenting an integrated model of transcriptional regulation and metabolic flux that will enable future efforts aimed at improving xylose fermentation to prioritize functional regulators of central carbon metabolism.

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Chen, Zhen, Rajesh Reddy Bommareddy, Doinita Frank, Sugima Rappert, and An-Ping Zeng. "Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum." Applied and Environmental Microbiology 80, no. 4 (2013): 1388–93. http://dx.doi.org/10.1128/aem.03535-13.

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ABSTRACTAllosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition ofCorynebacterium glutamicumPEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into theppcgene, encoding PEPC of the lysine-producing strainC. glutamicumLC298, resulted in ∼37% improved lysine production.In vitroenzyme assays and13C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.

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Lee, Sang Yup. "Metabolic Engineering and Synthetic Biology in Strain Development." ACS Synthetic Biology 1, no. 11 (2012): 491–92. http://dx.doi.org/10.1021/sb300109d.

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Long, Matthew R., Wai Kit Ong, and Jennifer L. Reed. "Computational methods in metabolic engineering for strain design." Current Opinion in Biotechnology 34 (August 2015): 135–41. http://dx.doi.org/10.1016/j.copbio.2014.12.019.

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Bonk, Brian M., Yekaterina Tarasova, Michael A. Hicks, Bruce Tidor, and Kristala L. J. Prather. "Rational design of thiolase substrate specificity for metabolic engineering applications." Biotechnology and Bioengineering 115, no. 9 (2018): 2167–82. http://dx.doi.org/10.1002/bit.26737.

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Woodruff, Lauren B. A., Brian L. May, Joseph R. Warner, and Ryan T. Gill. "Towards a metabolic engineering strain “commons”: AnEscherichia coliplatform strain for ethanol production." Biotechnology and Bioengineering 110, no. 5 (2013): 1520–26. http://dx.doi.org/10.1002/bit.24840.

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Desai, Ruchir P., and Eleftherios T. Papoutsakis. "Antisense RNA Strategies for Metabolic Engineering of Clostridium acetobutylicum." Applied and Environmental Microbiology 65, no. 3 (1999): 936–45. http://dx.doi.org/10.1128/aem.65.3.936-945.1999.

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ABSTRACT We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by ∼100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes.

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Hendry, John I., Anindita Bandyopadhyay, Shyam Srinivasan, Himadri B. Pakrasi, and Costas D. Maranas. "Metabolic model guided strain design of cyanobacteria." Current Opinion in Biotechnology 64 (August 2020): 17–23. http://dx.doi.org/10.1016/j.copbio.2019.08.011.

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Mukhopadhyay, N. K., K. N. Ishihara, S. Ranganathan, and K. Chattopadhyay. "Rational approximant structures and phason strain in icosahedral quasicrystalline phases." Acta Metallurgica et Materialia 39, no. 6 (1991): 1151–59. http://dx.doi.org/10.1016/0956-7151(91)90203-d.

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Libourel, Igor G. L., and Yair Shachar-Hill. "Metabolic Flux Analysis in Plants: From Intelligent Design to Rational Engineering." Annual Review of Plant Biology 59, no. 1 (2008): 625–50. http://dx.doi.org/10.1146/annurev.arplant.58.032806.103822.

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Stafford, Daniel E., and Gregory Stephanopoulos. "Metabolic engineering as an integrating platform for strain development." Current Opinion in Microbiology 4, no. 3 (2001): 336–40. http://dx.doi.org/10.1016/s1369-5274(00)00214-9.

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Biggs, Bradley Walters, Brecht De Paepe, Christine Nicole S. Santos, Marjan De Mey, and Parayil Kumaran Ajikumar. "Multivariate modular metabolic engineering for pathway and strain optimization." Current Opinion in Biotechnology 29 (October 2014): 156–62. http://dx.doi.org/10.1016/j.copbio.2014.05.005.

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Kawaguchi, Hideo, Alain A. Vertï¿½s, Shohei Okino, Masayuki Inui, and Hideaki Yukawa. "Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum." Applied and Environmental Microbiology 72, no. 5 (2006): 3418–28. http://dx.doi.org/10.1128/aem.72.5.3418-3428.2006.

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ABSTRACT The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertï¿½s, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

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Kroukamp, Heinrich, Riaan den Haan, John‐Henry van Zyl, and Willem Heber van Zyl. "Rational strain engineering interventions to enhance cellulase secretion by Saccharomyces cerevisiae." Biofuels, Bioproducts and Biorefining 12, no. 1 (2017): 108–24. http://dx.doi.org/10.1002/bbb.1824.

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Tenhaef, Niklas, Robert Stella, Julia Frunzke, and Stephan Noack. "Automated Rational Strain Construction Based on High-Throughput Conjugation." ACS Synthetic Biology 10, no. 3 (2021): 589–99. http://dx.doi.org/10.1021/acssynbio.0c00599.

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Yi, Xiunan, and Hal S. Alper. "Considering Strain Variation and Non-Type Strains for Yeast Metabolic Engineering Applications." Life 12, no. 4 (2022): 510. http://dx.doi.org/10.3390/life12040510.

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A variety of yeast species have been considered ideal hosts for metabolic engineering to produce value-added chemicals, including the model organism Saccharomyces cerevisiae, as well as non-conventional yeasts including Yarrowia lipolytica, Kluyveromyces marxianus, and Pichia pastoris. However, the metabolic capacity of these microbes is not simply dictated or implied by genus or species alone. Within the same species, yeast strains can display distinct variations in their phenotypes and metabolism, which affect the performance of introduced pathways and the production of interesting compounds. Moreover, it is unclear how this metabolic potential corresponds to function upon rewiring these organisms. These reports thus point out a new consideration for successful metabolic engineering, specifically: what are the best strains to utilize and how does one achieve effective metabolic engineering? Understanding such questions will accelerate the host selection and optimization process for generating yeast cell factories. In this review, we survey recent advances in studying yeast strain variations and utilizing non-type strains in pathway production and metabolic engineering applications. Additionally, we highlight the importance of employing portable methods for metabolic rewiring to best access this metabolic diversity. Finally, we conclude by highlighting the importance of considering strain diversity in metabolic engineering applications.

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Tilloy, Valentin, Anne Ortiz-Julien, and Sylvie Dequin. "Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions." Applied and Environmental Microbiology 80, no. 8 (2014): 2623–32. http://dx.doi.org/10.1128/aem.03710-13.

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ABSTRACTThere is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine's sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation ofSaccharomyces cerevisiaewine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.

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Wei, Zeng, Xianai Shi, Rong Lian, Weibin Wang, Wenrong Hong, and Shaobin Guo. "Exclusive Production of Gentamicin C1a from Micromonospora purpurea by Metabolic Engineering." Antibiotics 8, no. 4 (2019): 267. http://dx.doi.org/10.3390/antibiotics8040267.

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Gentamicin C1a is an important precursor to the synthesis of etimicin, a potent antibiotic. Wild type Micromonospora purpurea Gb1008 produces gentamicin C1a, besides four other gentamicin C components: C1, C2, C2a, and C2b. While the previously reported engineered strain M. purpurea GK1101 can produce relatively high titers of C1a by blocking the genK pathway, a small amount of undesirable C2b is still being synthesized in cells. Gene genL (orf6255) is reported to be responsible for converting C1a to C2b and C2 to C1 in Micromonospora echinospora ATCC15835. In this work, we identify the genL that is also responsible for the same methylation in Micromonospora purpurea. Based on M. purpurea GK1101, we construct a new strain with genL inactivated and show that no C2b is produced in this strain. Therefore, we successfully engineer a strain of M. purpurea that solely produces gentamicin C1a. This strain can potentially be used in the industrial production of C1a for the synthesis of etimicin.

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