RESUMEN
Flavonoids, a significant group of natural polyphenolic compounds, possess a broad spectrum of pharmacological effects. Recent advances in the systematic metabolic engineering of yeast cell factories (YCFs) provide new opportunities for enhanced flavonoid production. Herein, we outline the latest research progress on typical flavonoid products in YCFs. Advanced engineering strategies involved in flavonoid biosynthesis are discussed in detail, including enhancing precursor supply, cofactor engineering, optimizing core pathways, eliminating competitive pathways, relieving transport limitations, and dynamic regulation. Additionally, we highlight the existing problems in the biosynthesis of flavonoid glucosides in yeast, such as endogenous degradation of flavonoid glycosides, substrate promiscuity of UDP-glycosyltransferases, and an insufficient supply of UDP-sugars, with summaries on the corresponding solutions. Discussions also cover other typical postmodifications like prenylation and methylation, and the recent biosynthesis of complex flavonoid compounds in yeast. Finally, a series of advanced technologies are envisioned, i.e., semirational enzyme engineering, ML/DL algorithn, and systems biology, with the aspiration of achieving large-scale industrial production of flavonoid compounds in the future.
Asunto(s)
Flavonoides , Ingeniería Metabólica , Saccharomyces cerevisiae , Flavonoides/biosíntesis , Flavonoides/metabolismo , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
(R)-3-Hydroxybutyric acid (R-3HB) is an important chiral chemical with extensive applications in the agricultural, food, and chemical industries. The synthesis of R-3HB by microbial fermentation is of interest due to its remarkable stereoselectivity and economy. However, the low production of R-3HB failed to meet the needs of large-scale industrial production. In this study, an engineered strain for the efficient biosynthesis of R-3HB was constructed through a three-pronged approach encompassing biosynthetic pathway optimization, engineering of NADPH regenerators, and central metabolism regulation. The engineered strain Q5081 produced 75.7 g/L R-3HB, with a productivity of 1.26 g/L/h and a yield of 0.34 g/g glucose in fed-batch fermentation, showing the highest reported titer and productivity of R-3HB to date. We also performed transcriptome sequencing and annotation to illustrate the mechanism underlying the enhanced R-3HB production. The systematic metabolic engineering by a three-pronged approach demonstrated the feasibility of improving the biosynthesis, and the engineered strain Q5081 has the potential for widespread applications in the industrial production of R-3HB.
Asunto(s)
Ácido 3-Hidroxibutírico , Escherichia coli , Fermentación , Ingeniería Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/biosíntesis , Ácido 3-Hidroxibutírico/química , Vías BiosintéticasRESUMEN
Industrial production of bioactive compounds from actinobacteria, such as erythromycin and its derivatives, faces challenges in achieving optimal yields. To this end, the Design-Build-Test-Learn (DBTL) framework, a systematic metabolic engineering approach, was employed to enhance erythromycin production in Saccharopolyspora erythraea (S. erythraea) E3 strain. A genetically modified strain, S. erythraea E3-CymRP21-dcas9-sucC (S. erythraea CS), was developed by suppressing the sucC gene using an inducible promoter and dcas9 protein. The strain exhibited improved erythromycin synthesis, attributed to enhanced precursor synthesis and increased NADPH availability. Transcriptomic and metabolomic analyses revealed altered central carbon metabolism, amino acid metabolism, energy metabolism, and co-factor/vitamin metabolism in CS. Augmented amino acid metabolism led to nitrogen depletion, potentially causing cellular autolysis during later fermentation stages. By refining the fermentation process through ammonium sulfate supplementation, erythromycin yield reached 1125.66 mg L-1, a 43.5% increase. The results demonstrate the power of the DBTL methodology in optimizing erythromycin production, shedding light on its potential for revolutionizing antibiotic manufacturing in response to the global challenge of antibiotic resistance.
Asunto(s)
Eritromicina , Fermentación , Ingeniería Metabólica , Saccharopolyspora , Eritromicina/biosíntesis , Ingeniería Metabólica/métodos , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Antibacterianos/biosíntesis , Antibacterianos/metabolismoRESUMEN
Vitamin B5, also called d-pantothenic acid, is an essential vitamin in the human body and is widely used in pharmaceuticals, nutritional supplements, food, and cosmetics. However, few studies have investigated the microbial production of d-pantothenic acid, especially in Saccharomyces cerevisiae. By employing a systematic optimization strategy, we screened seven key genes in d-pantothenic acid biosynthesis from diverse species, including bacteria, yeast, fungi, algae, plants, animals, etc., and constructed an efficient heterologous d-pantothenic acid pathway in S. cerevisiae. By adjusting the copy number of the pathway modules, knocking out the endogenous bypass gene, balancing NADPH utilization, and regulating the GAL inducible system, a high-yield d-pantothenic acid-producing strain, DPA171, which can regulate gene expression using glucose, was constructed. By optimizing fed-batch fermentation, DPA171 produced 4.1 g/L d-pantothenic acid, which is the highest titer in S. cerevisiae to date. This study provides guidance for the development of vitamin B5 microbial cell factories.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Pantoténico/genética , Ácido Pantoténico/metabolismo , Ingeniería Metabólica , Proteínas de Saccharomyces cerevisiae/metabolismo , FermentaciónRESUMEN
Fungal azaphilones have attracted considerable interest as they exhibit great potential in food and pharmacological industries. However, there is a severe bottleneck in the low production in wild strains and the ability to genetically engineer azaphilone-producing fungi. Using Monascus azaphilones (MAs) as an example, we demonstrate a systematic metabolic engineering strategy for improving the production of MAs. In this study, Monascus purpureus HJ11 was systematically engineered through a combination of promoter engineering, gene knockout, rate-limiting enzyme overexpression, repression of the competing pathway, enzyme engineering, and metabolic rebalance. The maximum yield and titer of MAs successfully increased to 906 mg/g dry cell weight (DCW) and 14.6 g/L, respectively, 2.6 and 3.7 times higher than those reported in the literature. Our successful model not only offers a practical and efficient way to improve the azaphilone production but also sheds light on the potential of systematic metabolic engineering in nonmodel fungi as a chassis for the production of high-value chemicals.
Asunto(s)
Monascus , Benzopiranos , Ingeniería Metabólica , Monascus/genética , Pigmentos BiológicosRESUMEN
D-glucaric acid (GA) has been identified as among promising biotechnological alternatives to oil-based chemicals. GA and its derivatives are widely used in food additives, dietary supplements, drugs, detergents, corrosion inhibitors and biodegradable materials. The increasing availability of a GA market is improving the cost-effectiveness and efficiency of various biosynthetic pathways. In this study, an engineered Escherichia coli strain GA10 was constructed by systematic metabolic engineering. This involved redirecting metabolic flux into the GA biosynthetic pathways, blocking the conversion pathways of d-glucuronic acid (GlcA) and GA into by-products, introducing an in situ NAD+ regeneration system and fine-tuning the activity of the key enzyme, myo-inositol oxygenase (Miox). Subsequently, the culture medium was optimized to achieve the best performance of the GA10 strain. GA was produced at 5.35â¯g/L (extracellular and intracellular), with a maximized yield of â¼0.46â¯mol/mol on d-glucose and glycerol, by batch fermentation. This work demonstrates efficient biosynthetic pathways of GA in E. coli by metabolic engineering and should accelerate the application of GA biosynthetic pathways in industrial processes.
Asunto(s)
Escherichia coli/metabolismo , Ácido Glucárico/metabolismo , Ingeniería Metabólica , Vías Biosintéticas , Biotecnología , Escherichia coli/enzimología , Inositol-Oxigenasa/metabolismoRESUMEN
Lycopene is widely used in foods, cosmetics, nutritional supplements, and pharmaceuticals. Microbial production of lycopene has been intensively studied. However, there are few systematic engineering studies on Saccharomyces cerevisiae aimed at achieving high-yield lycopene production. In the current study, by employing a systematic optimization strategy, we screened the key lycopene biosynthetic genes, crtE, crtB, and crtI, from diverse organisms. By adjusting the copy number of these three key genes, knocking out endogenous bypass genes, increasing the supply of the precursor acetyl-CoA, balancing NADPH utilization, and regulating the GAL-inducible system, we constructed a high-yield lycopene-producing strain BS106, which can produce 310 mg/L lycopene in shake-flask fermentation, with gene expression controlled by glucose. In optimized two-stage fed-batch fermentation, BS106 produced 3.28 g/L lycopene in a 7 L fermenter, which is the highest concentration achieved in S. cerevisiae to date. It will decrease the consumption of tomatoes for lycopene extraction and increase the market supply of lycopene.
Asunto(s)
Licopeno/metabolismo , Ingeniería Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vías Biosintéticas , Fermentación , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Saccharomyces cerevisiae is an efficient host for natural-compound production and preferentially employed in academic studies and bioindustries. However, S. cerevisiae exhibits limited production capacity for lipophilic natural products, especially compounds that accumulate intracellularly, such as polyketides and carotenoids, with some engineered compounds displaying cytotoxicity. In this study, we used a nature-inspired strategy to establish an effective platform to improve lipid oil-triacylglycerol (TAG) metabolism and enable increased lycopene accumulation. Through systematic traditional engineering methods, we achieved relatively high-level production at 56.2â¯mg lycopene/g cell dry weight (cdw). To focus on TAG metabolism in order to increase lycopene accumulation, we overexpressed key genes associated with fatty acid synthesis and TAG production, followed by modulation of TAG fatty acyl composition by overexpressing a fatty acid desaturase (OLE1) and deletion of Seipin (FLD1), which regulates lipid-droplet size. Results showed that the engineered strain produced 70.5â¯mg lycopene/g cdw, a 25% increase relative to the original high-yield strain, with lycopene production reaching 2.37 g/L and 73.3 mg/g cdw in fed-batch fermentation and representing the highest lycopene yield in S. cerevisiae reported to date. These findings offer an effective strategy for extended systematic metabolic engineering through lipid engineering.
Asunto(s)
Metabolismo de los Lípidos/genética , Licopeno/metabolismo , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/biosíntesis , Fermentación , Eliminación de Gen , Técnicas de Inactivación de Genes , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Redes y Vías Metabólicas/genética , NADP/metabolismo , Plásmidos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Triglicéridos/metabolismoRESUMEN
The primary aims and challenges associated with microbial fermentation include achieving faster cell growth, higher productivity, and more robust production processes. Genome-scale biological models, predicting the formation of an interaction among genetic materials, enzymes, and metabolites, constitute a systematic and comprehensive platform to analyze and optimize the microbial growth and production of biological products. Genome-scale biological models can help optimize microbial growth-associated traits by simulating biomass formation, predicting growth rates, and identifying the requirements for cell growth. With regard to microbial product biosynthesis, genome-scale biological models can be used to design product biosynthetic pathways, accelerate production efficiency, and reduce metabolic side effects, leading to improved production performance. The present review discusses the development of microbial genome-scale biological models since their emergence and emphasizes their pertinent application in improving industrial microbial fermentation of biological products.