| 1. |
- Yu, Tao, 1988, et al.
(author)
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Metabolic reconfiguration enables synthetic reductive metabolism in yeast
- 2022
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In: Nature Metabolism. - : Springer Science and Business Media LLC. - 2522-5812. ; 4:11, s. 1551-1559
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Journal article (peer-reviewed)abstract
- Cell proliferation requires the integration of catabolic processes to provide energy, redox power and biosynthetic precursors. Here we show how the combination of rational design, metabolic rewiring and recombinant expression enables the establishment of a decarboxylation cycle in the yeast cytoplasm. This metabolic cycle can support growth by supplying energy and increased provision of NADPH or NADH in the cytosol, which can support the production of highly reduced chemicals such as glycerol, succinate and free fatty acids. With this approach, free fatty acid yield reached 40% of theoretical yield, which is the highest yield reported for Saccharomyces cerevisiae to our knowledge. This study reports the implementation of a synthetic decarboxylation cycle in the yeast cytosol, and its application in achieving high yields of valuable chemicals in cell factories. Our study also shows that, despite extensive regulation of catabolism in yeast, it is possible to rewire the energy metabolism, illustrating the power of biodesign.
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| 2. |
- Liu, Quanli, 1988, et al.
(author)
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Current state of aromatics production using yeast: achievements and challenges
- 2020
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In: Current Opinion in Biotechnology. - : Elsevier BV. - 0958-1669 .- 1879-0429. ; 65, s. 65-74
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Research review (peer-reviewed)abstract
- Aromatics find a range of applications in the chemical, food, cosmetic and pharmaceutical industries. While production of aromatics on the current market heavily relies on petroleum-derived chemical processes or direct extraction from plants, there is an increasing demand for establishing new renewable and sustainable sources of aromatics. To this end, microbial cell factories-mediated bioproduction using abundant feedstocks comprises a highly promising alternative to aromatics production. In this review, we provide the recent development of de novo biosynthesis of aromatics derived from the shikimate pathway in yeasts, including the model Saccharomyces cerevisiae as well as other non-conventional species. Moreover, we discuss how evolved metabolic engineering tools and strategies contribute to the construction and optimization of aromatics cell factories.
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| 3. |
- Liu, Quanli, 1988, et al.
(author)
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De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories
- 2021
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 12:1
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Journal article (peer-reviewed)abstract
- Isoflavonoids comprise a class of plant natural products with great nutraceutical, pharmaceutical and agricultural significance. Their low abundance in nature and structural complexity however hampers access to these phytochemicals through traditional crop-based manufacturing or chemical synthesis. Microbial bioproduction therefore represents an attractive alternative. Here, we engineer the metabolism of Saccharomyces cerevisiae to become a platform for efficient production of daidzein, a core chemical scaffold for isoflavonoid biosynthesis, and demonstrate its application towards producing bioactive glucosides from glucose, following the screening-reconstruction-application engineering framework. First, we rebuild daidzein biosynthesis in yeast and its production is then improved by 94-fold through screening biosynthetic enzymes, identifying rate-limiting steps, implementing dynamic control, engineering substrate trafficking and fine-tuning competing metabolic processes. The optimized strain produces up to 85.4 mg L−1 of daidzein and introducing plant glycosyltransferases in this strain results in production of bioactive puerarin (72.8 mg L−1) and daidzin (73.2 mg L−1). Our work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added isoflavonoids and the multi-phased framework may be extended to engineer pathways of complex natural products in other microbial hosts.
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| 4. |
- Liu, Yi, 1986, et al.
(author)
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Engineering yeast phospholipid metabolism for de novo oleoylethanolamide production
- 2020
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In: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 16:2, s. 197-205
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Journal article (peer-reviewed)abstract
- Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l−1 and a yield on glucose of 405.8 µg g−1. Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.
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| 5. |
- Pereira, Rui, 1986, et al.
(author)
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Metabolic Engineering of Yeast
- 2021
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In: Metabolic Engineering: Concepts and Applications: Volume 13a and 13b. - : Wiley. ; 13, s. 689-733
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Book chapter (other academic/artistic)abstract
- This chapter focuses on a few examples that can serve as illustrations of how powerful yeast metabolic engineering stands today. Yeast, especially S. cerevisiae, plays an essential role in bioethanol production. Rapid ethanol production by yeast cells makes the fermentation process less susceptible to contamination. Higher alcohols are attractive due to some advantages compared with bioethanol, such as higher energy density, better blending into gasoline, higher octane value, lower hygroscopicity, and less corrosivity. The ethanol production process in the industry is mainly achieved through simultaneous saccharification and fermentation. Production of insulin, by volume the largest pharmaceutical protein produced, has paved the way for a wide use of S. cerevisiae for production of recombinant proteins. Virus like particles are proteins of virus capsid, which are produced by recombinant DNA technology and are important for the development of viral vaccines as they can self-assemble and display similar immunogenic properties as native viruses.
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| 6. |
- Wang, Guokun, 1988, et al.
(author)
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RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
- 2019
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:19, s. 9324-9332
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Journal article (peer-reviewed)abstract
- The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.
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| 7. |
- Li, Xiaowei, 1986, et al.
(author)
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Metabolic network remodelling enhances yeast’s fitness on xylose using aerobic glycolysis
- 2021
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In: Nature Catalysis. - : Springer Science and Business Media LLC. - 2520-1158. ; 4:9, s. 783-796
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Journal article (peer-reviewed)abstract
- The reprogramming of metabolism in response to switching the carbon source from glucose to non-preferred carbon sources is well-studied for yeast. However, understanding how metabolic networks respond to utilize a non-natural carbon source such as xylose is limited due to the incomplete knowledge of cellular response mechanisms. Here we applied a combination of metabolic engineering, systems biology and adaptive laboratory evolution to gain insights into how yeast can perform a global rewiring of cellular processes to efficiently accompany metabolic transitions. Through metabolic engineering, we substantially enhanced the cell growth on xylose after the growth on glucose. Transcriptome analysis of the engineered strains demonstrated that multiple pathways were involved in the cellular reprogramming. Through genome resequencing of the evolved strains and reverse engineering, we further identified that SWI/SNF chromatin remodelling, osmotic response and aldehyde reductase were responsible for the improved growth. Combined, our analysis showed that glycerol-3-phosphate and xylitol serve as two key metabolites that affect cellular adaptation to growth on xylose. [Figure not available: see fulltext.].
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| 8. |
- Li, Yuanzi, et al.
(author)
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De Novo Biosynthesis of Caffeic Acid from Glucose by Engineered Saccharomyces cerevisiae
- 2020
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In: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 9:4, s. 756-765
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Journal article (peer-reviewed)abstract
- Caffeic acid is a plant phenolic compound possessing extensive pharmacological activities. Here, we identified that p-coumaric acid 3-hydroxylase from Arabidopsis thaliana was capable of hydroxylating p-coumaric acid to form caffeic acid in Saccharomyces cerevisiae. Then, we introduced a combined caffeic acid biosynthetic pathway into S. cerevisiae and obtained 0.183 mg L-1 caffeic acid from glucose. Next we improved the tyrosine biosynthesis in S. cerevisiae by blocking the pathway flux to aromatic alcohols and eliminating the tyrosine-induced feedback inhibition resulting in caffeic acid production of 2.780 mg L-1. Finally, the medium was optimized, and the highest caffeic acid production obtained was 11.432 mg L-1 in YPD medium containing 4% glucose. This study opens a route to produce caffeic acid from glucose in S. cerevisiae and establishes a platform for the biosynthesis of caffeic acid derived metabolites.
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| 9. |
- Li, Yuanzi, et al.
(author)
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Optimization of the l-tyrosine metabolic pathway in Saccharomyces cerevisiae by analyzing p-coumaric acid production
- 2020
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In: 3 Biotech. - : Springer Science and Business Media LLC. - 2190-572X .- 2190-5738. ; 10:6
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Journal article (peer-reviewed)abstract
- In this study, we applied a series of genetic modifications to wild-type S. cerevisiae strain BY4741 to address the bottlenecks in the l-tyrosine pathway. A tyrosine ammonia-lyase (TAL) gene from Rhodobacter capsulatus, which can catalyze conversion of l-tyrosine into p-coumaric acid, was overexpressed to facilitate the analysis of l-tyrosine and test the strain's capability to synthesize heterologous derivatives. First, we enhanced the supply of precursors by overexpressing transaldolase gene TAL1, enolase II gene ENO2, and pentafunctional enzyme gene ARO1 resulting in a 1.55-fold increase in p-coumaric acid production. Second, feedback inhibition of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase was relieved by overexpressing the mutated feedback-resistant ARO4(K229L) and ARO7(G141S), and a 3.61-fold improvement of p-coumaric acid production was obtained. Finally, formation of byproducts was decreased by deleting pyruvate decarboxylase gene PDC5 and phenylpyruvate decarboxylase gene ARO10, and p-coumaric acid production was increased 2.52-fold. The best producer-when TAL1, ENO2, ARO1, ARO4(K229L), ARO7(G141S), and TAL were overexpressed, and PDC5 and ARO10 were deleted-increased p-coumaric acid production by 14.08-fold (from 1.4 to 19.71 mg L-1). Our study provided a valuable insight into the optimization of l-tyrosine metabolic pathway.
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| 10. |
- Liu, Quanli, 1988, et al.
(author)
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Modular Pathway Rewiring of Yeast for Amino Acid Production
- 2018
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In: Methods in Enzymology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 608, s. 417-439
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Book chapter (other academic/artistic)abstract
- Amino acids find various applications in biotechnology in view of their importance in the food, feed, pharmaceutical, and personal care industries as nutrients, additives, and drugs, respectively. For the large-scale production of amino acids, microbial cell factories are widely used and the development of amino acid-producing strains has mainly focused on prokaryotes Corynebacterium glutamicum and Escherichia coli. However, the eukaryote Saccharomyces cerevisiae is becoming an even more appealing microbial host for production of amino acids and derivatives because of its superior molecular and physiological features, such as amenable to genetic engineering and high tolerance to harsh conditions. To transform S. cerevisiae into an industrial amino acid production platform, the highly coordinated and multiple layers regulation in its amino acid metabolism should be relieved and reconstituted to optimize the metabolic flux toward synthesis of target products. This chapter describes principles, strategies, and applications of modular pathway rewiring in yeast using the engineering of L-ornithine metabolism as a paradigm. Additionally, detailed protocols for in vitro module construction and CRISPR/Cas-mediated pathway assembly are provided.
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| 11. |
- Liu, Quanli, 1988, et al.
(author)
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Rewiring carbon metabolism in yeast for high level production of aromatic chemicals
- 2019
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 10:1
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Journal article (peer-reviewed)abstract
- The production of bioactive plant compounds using microbial hosts is considered a safe, cost-competitive and scalable approach to their production. However, microbial production of some compounds like aromatic amino acid (AAA)-derived chemicals, remains an outstanding metabolic engineering challenge. Here we present the construction of a Saccharomyces cerevisiae platform strain able to produce high levels of p-coumaric acid, an AAA-derived precursor for many commercially valuable chemicals. This is achieved through engineering the AAA biosynthesis pathway, introducing a phosphoketalose-based pathway to divert glycolytic flux towards erythrose 4-phosphate formation, and optimizing carbon distribution between glycolysis and the AAA biosynthesis pathway by replacing the promoters of several important genes at key nodes between these two pathways. This results in a maximum p-coumaric acid titer of 12.5 g L−1 and a maximum yield on glucose of 154.9 mg g−1.
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| 12. |
- Mao, Jiwei, 1990, et al.
(author)
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Fine-tuning of p-coumaric acid synthesis to increase (2S)-naringenin production in yeast
- 2023
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In: Metabolic Engineering. - 1096-7176 .- 1096-7184. ; 79, s. 192-202
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Journal article (peer-reviewed)abstract
- (2S)-Naringenin is a key precursor for biosynthesis of various high-value flavonoids and possesses a variety of nutritional and pharmaceutical properties on human health. Systematic optimization approaches have been employed to improve (2S)-naringenin production in different microbial hosts. However, very few studies have focused on the spatiotemporal distribution of (2S)-naringenin and the related pathway intermediate p-coumaric acid, which is an important factor for efficient production. Here, we first optimized the (2S)-naringenin biosynthetic pathway by alleviating the bottleneck downstream of p-coumaric acid and increasing malonyl-CoA supply, which improved (2S)-naringenin production but significant accumulation of p-coumaric acid still existed extracellularly. We thus established a dual dynamic control system through combining a malonyl-CoA biosensor regulator and an RNAi strategy, to autonomously control the synthesis of p-coumaric acid with the supply of malonyl-CoA. Furthermore, screening potential transporters led to identification of Pdr12 for improved (2S)-naringenin production and reduced accumulation of p-coumaric acid. Finally, a titer of 2.05 g/L (2S)-naringenin with negligible accumulation of p-coumaric acid was achieved in a fed batch fermentation. Our work highlights the importance of systematic control of pathway intermediates for efficient microbial production of plant natural products.
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| 13. |
- Wu, Yuzhen, et al.
(author)
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Comparative transcriptome analysis of genomic region deletion strain with enhanced l-tyrosine production in Saccharomyces cerevisiae
- 2020
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In: Biotechnology Letters. - : Springer Science and Business Media LLC. - 1573-6776 .- 0141-5492. ; 42:3, s. 453-460
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Journal article (peer-reviewed)abstract
- Objective To determine the effect of large genomic region deletion in a Saccharomyces cerevisiae strain on tyrosine yield and to identify new genetic modification targets through transcriptome analysis. Results TAL was used to produce p-coumaric acid (p-CA) from tyrosine to quantity tyrosine yield. S. cerevisiae mutant strain NK14 with deletion of a 23.8 kb genomic region was identified to have p-CA production of 10.3 mg L- 1, while the wild-type strain BY4741 had p-CA production of 1.06 mg L- 1. Analysis of growth patterns and stress tolerance showed that the deletion did not affect the growth phenotype of NK14. Transcriptome analysis suggested that, compared to BY4741, genes related to glycolysis (ENO2, TKL1) and the tyrosine pathway (ARO1, ARO2, ARO4, ARO7, TYR1) were upregulated in NK14 at different levels. Besides genes related to the tyrosine biosynthetic pathway, amino acid transporters (AVT6, VBA5, THI72) and transcription factor (ARO80) also showed changes in transcription levels. Conclusions We developed a strain with improved tyrosine yield and identified new genetic modification candidates for tyrosine production.
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| 14. |
- Yu, Tao, 1986, et al.
(author)
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Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals
- 2017
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 8, s. Article number: 15587-
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Journal article (peer-reviewed)abstract
- Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae . VLCFAs can be selectively modified towards the fatty alcohol docosanol (C22H46O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l(-1) in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.
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| 15. |
- Yu, Tao, 1986, et al.
(author)
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Reprogramming Yeast Metabolism from Alcoholic Fermentation to Lipogenesis
- 2018
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In: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 174:6, s. 1549-1572
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Journal article (peer-reviewed)abstract
- Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism's natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.
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| 16. |
- Yu, Tao, 1986, et al.
(author)
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Strategies and challenges for metabolic rewiring
- 2019
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In: Current Opinion in Systems Biology. - : Elsevier BV. - 2452-3100. ; 15, s. 30-38
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Research review (peer-reviewed)abstract
- Metabolic engineering is often centred on rewiring cellular metabolism to improve the production of chemicals. Turning cells into efficient factories is challenging because of ubiquitous and tightly regulated metabolic interactions. Tools and strategies from different disciplines, including systems biology, synthetic biology and evolutionary engineering, have been integrated into metabolic engineering to overcome challenges with rewiring metabolism for cell factory development. In this review, we summarise the recent development of tools and strategies for dynamic pathway regulation, compartmentalisation, modular and evolutionary engineering. In addition, we describe how systems biology tools benefit metabolic rewiring for advancing the development of cell factories.
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