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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.
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Monoterpenoids are an important subclass of terpenoids that play important roles in the energy, cosmetics, pharmaceuticals, and fragrances fields. With the development of biotechnology, microbial synthesis of monoterpenoids has received great attention. Yeasts such Saccharomyces cerevisiae and Yarrowia lipolytica are emerging as potential hosts for monoterpenoids production because of unique advantages including rapid growth cycles, mature gene editing tools, and clear genetic background. Recently, advancements in metabolic engineering and fermentation engineering have significantly enhanced the accumulation of monoterpenoids in cell factories. First, this review introduces the biosynthetic pathway of monoterpenoids and comprehensively summarizes the latest production strategies, which encompass enhancing precursor flux, modulating the expression of rate-limited enzymes, suppressing competitive pathway flux, mitigating cytotoxicity, optimizing substrate utilization, and refining the fermentation process. Subsequently, this review introduces four representative monoterpenoids. Finally, we outline the future prospects for efficient construction cell factories tailored for the production of monoterpenoids and other terpenoids.
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Ingeniería Metabólica , Monoterpenos , Saccharomyces cerevisiae , Yarrowia , Yarrowia/metabolismo , Yarrowia/genética , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Monoterpenos/metabolismo , Fermentación , Vías Biosintéticas/genética , Terpenos/metabolismo , Edición Génica/métodosRESUMEN
5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid essential for synthesizing tetrapyrrole compounds, including heme, chlorophyll, cytochrome, and vitamin B12. As a plant growth regulator, 5-ALA is extensively used in agriculture to enhance crop yield and quality. The complexity and low yield of chemical synthesis methods have led to significant interest in the microbial synthesis of 5-ALA. Advanced strategies, including the: enhancement of precursor and cofactor supply, compartmentalization of key enzymes, product transporters engineering, by-product formation reduction, and biosensor-based dynamic regulation, have been implemented in bacteria for 5-ALA production, significantly advancing its industrialization. This article offers a comprehensive review of recent developments in 5-ALA production using engineered bacteria and presents new insights to propel the field forward.
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Aspergillus niger is an efficient cell factory for organic acids production, particularly l-malic acid, through genetic manipulation. However, the traditional method of collecting A. niger spores for inoculation is labor-intensive and resource-consuming. In our study, we used the CRISPR-Cas9 system to replace the promoter of brlA, a key gene in Aspergillus conidiation, with a xylose-inducible promoter xylP in l-malic acid-producing A. niger strain RG0095, generating strain brlAxylP. When induced with xylose in submerged liquid culture, brlAxylP exhibited significant upregulation of conidiation-related genes. This induction allowed us to easily collect an abundance of brlAxylP spores (>7.1 × 106/mL) in liquid xylose medium. Significantly, the submerged conidiation approach preserves the substantial potential of A. niger as a foundational cellular platform for the biosynthesis of organic acids, including but not limited to l-malic acid. In summary, our study offers a simplified submerged conidiation strategy to streamline the preparation stage and reduce labor and material costs for industrial organic acid production using Aspergillus species.
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Due to the extreme living conditions, extremophiles have unique characteristics in morphology, structure, physiology, biochemistry, molecular evolution mechanism and so on. Extremophiles have superior growth and synthesis capabilities under harsh conditions compared to conventional microorganisms, allowing for unsterilized fermentation processes and thus better performance in low-cost production. In recent years, due to the development and optimization of molecular biology, synthetic biology and fermentation technology, the identification and screening technology of extremophiles has been greatly improved. In this review, we summarize techniques for the identification and screening of extremophiles and review their applications in industrial biotechnology in recent years. In addition, the facts and perspectives gathered in this review suggest that next-generation industrial biotechnology (NGIBs) based on engineered extremophiles holds the promise of simplifying biofuturing processes, establishing open, non-sterilized continuous fermentation production systems, and utilizing low-cost substrates to make NGIBs attractive and cost-effective bioprocessing technologies for sustainable manufacturing.
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Extremófilos , Extremófilos/genética , Biotecnología/métodos , FermentaciónRESUMEN
Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in F. fujikuroi, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5'-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.
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Enzymes, as biocatalysts, play a cumulatively important role in environmental purification and industrial production of chemicals and pharmaceuticals. However, natural enzymes are limited by their physiological properties in practice, which need to be modified driven by requirements. Screening and isolating certain enzyme variants or ideal industrial strains with high yielding of target product enzymes is one of the main directions of enzyme engineering research. Droplet-based high-throughput screening (DHTS) technology employs massive monodisperse emulsion droplets as microreactors to achieve single strain encapsulation, as well as continuous monitoring for the inside mutant library. It can effectively sort out strains or enzymes with desired characteristics, offering a throughput of 108 events per hour. Much of the early literature focused on screening various engineered strains or designing signalling sorting strategies based on DHTS technology. However, the field of enzyme engineering lacks a comprehensive overview of advanced methods for microfluidic droplets and their cutting-edge developments in generation and manipulation. This review emphasizes the advanced strategies and frontiers of microfluidic droplet generation and manipulation facilitating enzyme engineering development. We also introduce design for various screening signals that cooperate with DHTS and devote to enzyme engineering.
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Técnicas Biosensibles , Ensayos Analíticos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento/métodos , Microfluídica/métodosRESUMEN
Gibberellic acid (GA3), produced industrially by Fusarium fujikuroi, stands as a crucial plant growth regulator extensively employed in the agriculture filed while limited understanding of the global metabolic network hinders researchers from conducting rapid targeted modifications. In this study, a small-molecule compounds-based targeting technology was developed to increase GA3 production. Firstly, various small molecules were used to target key nodes of different pathways and the result displayed that supplement of terbinafine improved significantly GA3 accumulation, which reached to 1.08 g/L. Subsequently, lipid and squalene biosynthesis pathway were identified as the key pathways influencing GA3 biosynthesis by transcriptomic analysis. Thus, the strategies including in vivo metabolic engineering modification and in vitro supplementation of lipid substrates were adopted, both contributed to an enhanced GA3 yield. Finally, the engineered strain demonstrated the ability to achieve a GA3 yield of 3.24 g/L in 5 L bioreactor when utilizing WCO as carbon source and feed.
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Fusarium , Giberelinas , Fermentación , Fusarium/genética , Fusarium/química , Reactores Biológicos , LípidosRESUMEN
ß-Carotene is one kind of the most important carotenoids. The major functions of ß-carotene include the antioxidant and anti-cardiovascular properties, which make it a growing market. Recently, the use of metabolic engineering to construct microbial cell factories to synthesize ß-carotene has become the latest model for its industrial production. Among these cell factories, yeasts including Saccharomyces cerevisiae and Yarrowia lipolytica have attracted the most attention because of the: security, mature genetic manipulation tools, high flux toward carotenoids using the native mevalonate pathway and robustness for large-scale fermentation. In this review, the latest strategies for ß-carotene biosynthesis, including protein engineering, promoters engineering and morphological engineering are summarized in detail. Finally, perspectives for future engineering approaches are proposed to improve ß-carotene production.
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Ingeniería Metabólica , Yarrowia , beta Caroteno/genética , beta Caroteno/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Saccharomyces cerevisiae/genética , Regiones Promotoras GenéticasRESUMEN
Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.
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Sistemas CRISPR-Cas , Yarrowia , Sistemas CRISPR-Cas/genética , Yarrowia/genética , Yarrowia/metabolismo , Ácido Araquidónico/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Ingeniería MetabólicaRESUMEN
Liquid fermentation is the primary method for GA3 production usingFusarium fujikuroi. However, production capacity is limited due to unknown metabolic pathways. To address this, we constructed a genome-scale metabolic model (iCY1235) with 1753 reactions, 1979 metabolites, and 1235 genes to understand the GA3 regulation mechanisms. The model was validated by analyzing growth rates under different glucose uptake rates and identifying essential genes. We used the model to optimize fermentation conditions, including carbon sources and dissolved oxygen. Through the OptForce algorithm, we identified 20 reactions as targets. Overexpressing FFUJ_02053 and FFUJ_14337 resulted in a 37.5 and 75% increase in GA3 titers, respectively. These targets enhance carbon flux toward GA3 production. Our model holds promise for guiding the metabolic engineering of F. fujikuroi to achieve targeted overproduction. In summary, our study utilizes the iCY1235 model to understand GA3 regulation, optimize fermentation conditions, and identify specific targets for enhancing GA3 production through metabolic engineering.
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Fusarium , Giberelinas , Giberelinas/metabolismo , Fermentación , Redes y Vías MetabólicasRESUMEN
Bacillus sp. is one of the most distinctive gram-positive bacteria, able to grow efficiently using cheap carbon sources and secrete a variety of useful substances, which are widely used in food, pharmaceutical, agricultural and environmental industries. At the same time, Bacillus sp. is also recognized as a safe genus with a relatively clear genetic background, which is conducive to the industrial production of target metabolites. In this review, we discuss the reasons why Bacillus sp. has been so extensively studied and summarize its advances in systems and synthetic biology, engineering strategies to improve microbial cell properties, and industrial applications in several metabolic engineering applications. Finally, we present the current challenges and possible solutions to provide a reliable basis for Bacillus sp. as a microbial cell factory.
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Bacillus , Bacillus/genética , Ingeniería Metabólica , Biología Sintética , Alimentos , CarbonoRESUMEN
Special environmental microorganisms are considered to be of great industrial application value because of their special genotypes, physiological functions and metabolites. The research and development of special environmental microorganisms will certainly bring about some innovations in biotechnology processes and change the face of bioengineering. The Special Environmental Microbial Database (DSEMR) is a comprehensive database that provides information on special environmental microbial resources and correlates them with synthetic biological parts. DSEMR aggregates information on specific environmental microbial genomes, physiological properties, culture media, biological parts, and metabolic pathways, and provides online tool analysis data, including 5268 strains from 620 genera, 31 media, and 42,126 biological parts. In short, DSEMR will become an important resource for the study of microorganisms in special environments and actively promote the development of synthetic biology.
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Eicosapentaenoic acid (EPA) belonged to the ω-3 series of polyunsaturated fatty acids and had physiological functions lipid as regulating blood lipid and preventing cardiovascular diseases. Schizochytrium sp. was considered to be a potential industrial fermentation strain of EPA because of its fast growth, high oil content, and simple fatty acid composition. However, Schizochytrium sp. produced EPA with low production efficiency and a long synthesis path. This research aims to improve the yield of EPA in Schizochytrium sp. by ARTP mutagenesis and to reveal the mechanism of high-yield EPA through transcriptome analysis. ARTP mutagenesis screening yielded the mutant M12 that whereas the productivity of EPA increased 108% reaching 0.48 g/L, the total fatty acid concentration was 13.82 g/L with an increase of 13.7%. The transcriptomics revealed 2995 differentially expressed genes were identified between M12 and the wild-type strain and transcripts involved in carbohydrate, amino acid, energy, and lipid metabolism were up-regulated. Among them, the hexokinase (HK) and the phosphofructokinase genes (PFK), which can catalyze pyruvate to acetyl-CoA, were increased 2.23-fold and 1.78-fold. Glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GLDH), which can both generate NADPH, were increased by 1.67-fold and 3.11-fold. Furthermore, in the EPA synthesis module, the expression of 3-oxoacyl-[acyl-carrier protein] reductase(fabG) and carbonyl reductase 4 / 3-oxoacyl-[acyl-carrier protein] reductase beta subunit(CBR4), also up-regulated 1.11-fold and 2.67-fold. These may lead to increases in cell growth. The results provide an important reference for further research on promoting fatty acid and EPA accumulation in Schizochytrium sp.
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Ácido Eicosapentaenoico , Estramenopilos , Ácido Eicosapentaenoico/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácidos Grasos/metabolismo , Mutagénesis , Oxidorreductasas/metabolismo , Proteínas Portadoras/genéticaRESUMEN
Acetoin was widely used in food, medicine, and other industries, because of its unique fragrance. Bacillus amyloliquefaciens was recognized as a safe strain and a promising acetoin producer in fermentation. However, due to the complexity of its metabolic network, it had not been fully utilized. Therefore, a genome-scale metabolic network model (iJYQ746) of B. amyloliquefaciens was constructed in this study, containing 746 genes, 1736 reactions, and 1611 metabolites. The results showed that Mg2+, Mn2+, and Fe2+ have inhibitory effects on acetoin. When the stirring speed was 400 rpm, the maximum titer was 49.8 g L-1. Minimization of metabolic adjustments (MOMA) was used to identify potential metabolic modification targets 2-oxoglutarate aminotransferase (serC, EC 2.6.1.52) and glucose-6-phosphate isomerase (pgi, EC 5.3.1.9). These targets could effectively accumulate acetoin by increasing pyruvate content, and the acetoin synthesis rate was increased by 610% and 10%, respectively. This provides a theoretical basis for metabolic engineering to reasonably transform B. amyloliquefaciens and produce acetoin.
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Acetoína , Ingeniería Metabólica , Acetoína/metabolismo , Fermentación , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genéticaRESUMEN
Patchoulol is an important sesquiterpene alcohol with a strong and lasting odor, which has led to prominent applications in perfumes and cosmetics. In this study, systematic metabolic engineering strategies were adopted to create an efficient yeast cell factory for patchoulol overproduction. First, a baseline strain was constructed by selecting a highly active patchoulol synthase. Subsequently, the mevalonate precursor pool was expanded to boost patchoulol synthesis. Moreover, a method for downregulating squalene synthesis based on Cu2+-repressible promoter was optimized, which significantly improved the patchoulol titer by 100.9% to 124 mg/L. In addition, a protein fusion strategy resulted in a final titer of 235 mg/L in shake flasks. Finally, 2.864 g/L patchoulol could be produced in a 5 L bioreactor, representing a remarkable 1684-fold increase compared to the baseline strain. To our knowledge, this is the highest patchoulol titer reported so far.
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Sesquiterpenos , Yarrowia , Yarrowia/genética , Yarrowia/metabolismo , Sesquiterpenos/metabolismo , Reactores Biológicos , Escualeno/metabolismo , Ingeniería Metabólica/métodosRESUMEN
Spores and pollens refer to the reproductive cells of seed plants and asexually reproducing sporophytes, exhibiting a natural core-shell structure and exquisite surface morphology. They possess extraordinary dimensional homogeneity, porosity, amphiphilicity and adhesion. Their sporopollenin exine layer endows them with chemically stable, UV resistant, and biocompatible properties, which can also be facilely functionalized due to sufficient groups on the surface. The unique characteristics of spores and pollens have facilitated a wide range of applications in drug carriers, biological imaging, food science, microrobotics, environmental purification, flexible electronics, cell scaffolds, 3D printing materials and biological detection. This review showcases the common structural composition and physicochemical properties of spores and pollens, describes the extraction and processing methods, and summarizes the recent research on their applications in various fields. Following these sections, this review analyzes the existing challenges in spores and pollen research and provides a future outlook.
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Polen , Esporas , Polen/químicaRESUMEN
BACKGROUND: α-Humulene is an important biologically active sesquiterpene, whose heterologous production in microorganisms is a promising alternative biotechnological process to plant extraction and chemical synthesis. In addition, the reduction of production expenses is also an extremely critical factor in the sustainable and industrial production of α-humulene. In order to meet the requirements of industrialization, finding renewable substitute feedstocks such as low cost or waste substrates for terpenoids production remains an area of active research. RESULTS: In this study, we investigated the feasibility of peroxisome-engineering strain to utilize waste cooking oil (WCO) for high production of α-humulene while reducing the cost. Subsequently, transcriptome analysis revealed differences in gene expression levels with different carbon sources. The results showed that single or combination regulations of target genes identified by transcriptome were effective to enhance the α-humulene titer. Finally, the engineered strain could produce 5.9 g/L α-humulene in a 5-L bioreactor. CONCLUSION: To the best of our knowledge, this is the first report that converted WCO to α-humulene in peroxisome-engineering strain. These findings provide valuable insights into the high-level production of α-humulene in Y. lipolytica and its utilization in WCO bioconversion.