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Pigments are widely used in the food, cosmetic, textile, pharmaceutical, and materials industries. Demand for natural pigments has been increasing due to concerns regarding potential health problems and environmental pollution from synthetic pigments. Microbial production of natural pigments is a promising alternative to chemical synthesis or extraction from natural sources. Here, we discuss yeasts as promising chassis for producing natural pigments with their advantageous traits such as genetic amenability, safety, rapid growth, metabolic diversity, and tolerance. Metabolic engineering strategies and optimizing strategies in downstream process to enhance production of natural pigments are thoroughly reviewed. We discuss the challenges, including expanding the range of natural pigments and improving their feasibility of industrial scale-up, as well as the potential strategies for future development.

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Pyrimidine nucleosides, as intermediate materials of significant commercial value, find extensive applications in the pharmaceutical industry. However, the current production of pyrimidine nucleosides largely relies on chemical synthesis, creating environmental problems that do not align with sustainable development goals. Recent progress in systemic metabolic engineering and synthetic biology has enabled the synthesis of natural products like pyrimidine nucleosides through microbial fermentation, offering a more sustainable alternative. Nevertheless, the intricate and tightly regulated biosynthetic pathways involved in the microbial production of pyrimidine nucleosides pose a formidable challenge. This study focuses on metabolic engineering and synthetic biology strategies aimed at enhancing pyrimidine nucleoside production. These strategies include gene modification, transcriptional regulation, metabolic flux analysis, cofactor balance optimization, and transporter engineering. Finally, this research highlights the challenges involved in the further development of pyrimidine nucleoside-producing strains and offers potential solutions in order to provide theoretical guidance for future research endeavors in this field.

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Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

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Ergothioneine (EGT) is a naturally occurring derivative of histidine with diverse applications in the medicine, cosmetic, and food industries. Nevertheless, its sustainable biosynthesis faces hurdles due to the limited biosynthetic pathways, complex metabolic network of precursors, and high cost associated with fermentation. Herein, efforts were made to address these limitations first by reconstructing a novel EGT biosynthetic pathway from Methylobacterium aquaticum in Escherichia coli and optimizing it through plasmid copy number. Subsequently, the supply of precursor amino acids was promoted by engineering the global regulator, recruiting mutant resistant to feedback inhibition, and blocking competitive pathways. These metabolic modifications resulted in a significant improvement in EGT production, increasing from 35 to 130 mg/L, representing a remarkable increase of 271.4%. Furthermore, an economical medium was developed by replacing yeast extract with corn steep liquor, a byproduct of wet milling of corn. Finally, the production of EGT reached 595 mg/L with a productivity of 8.2 mg/L/h by exploiting fed-batch fermentation in a 10 L bioreactor. This study paves the way for exploring and modulating a de novo biosynthetic pathway for efficient and low-cost fermentative production of EGT.

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Vanillin is one of the world's most extensively used flavoring agents with high application value. However, the yield of vanillin biosynthesis remains limited due to the low efficiency of substrate uptake and the inhibitory effect on cell growth caused by vanillin. Here, we screened high-efficiency ferulic acid importer TodX and vanillin exporters PP_0178 and PP_0179 by overexpressing genes encoding candidate transporters in a vanillin-producing engineered Escherichia coli strain VA and further constructed an autoregulatory bidirectional transport system by coexpressing TodX and PP_0178/PP_0179 with a vanillin self-inducible promoter ADH7. Compared with strain VA, strain VA-TodX-PP_0179 can efficiently transport ferulic acid across the cell membrane and convert it to vanillin, which significantly increases the substrate utilization rate efficiency (14.86%) and vanillin titer (51.07%). This study demonstrated that the autoregulatory bidirectional transport system significantly enhances the substrate uptake efficiency while alleviating the vanillin toxicity issue, providing a promising viable route for vanillin biosynthesis.

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Aromatic amino acids (AAA) and derived compounds have enormous commercial value with extensive applications in the food, chemical and pharmaceutical fields. Microbial production of AAA and derived compounds is a promising prospect for its environmental friendliness and sustainability. However, low yield and production efficiency remain major challenges for realizing industrial production. With the advancement of synthetic biology, microbial production of AAA and derived compounds has been significantly facilitated. In this review, a comprehensive overview on the current progresses, challenges and corresponding solutions for AAA and derived compounds biosynthesis is provided. The most cutting-edge developments of synthetic biology technology in AAA and derived compounds biosynthesis, including CRISPR-based system, genetically encoded biosensors and synthetic genetic circuits, were highlighted. Finally, future prospects of modern strategies conducive to the biosynthesis of AAA and derived compounds are discussed. This review offers guidance on constructing microbial cell factory for aromatic compound using synthetic biology technology.

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BACKGROUND: Chelerythrine is an important alkaloid used in agriculture and medicine. However, its structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from plants. Here, we reconstructed and optimized the complete biosynthesis pathway for chelerythrine from (S)-reticuline in Saccharomyces cerevisiae using genetic reprogramming. RESULTS: The first-generation strain Z4 capable of producing chelerythrine was obtained via heterologous expression of seven plant-derived enzymes (McoBBE, TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, and PsCPR) in S. cerevisiae W303-1 A. When this strain was cultured in the synthetic complete (SC) medium supplemented with 100 µM of (S)-reticuline for 10 days, it produced up to 0.34 µg/L chelerythrine. Furthermore, efficient metabolic engineering was performed by integrating multiple-copy rate-limiting genes (TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR, INO2, and AtATR1), tailoring the heme and NADPH engineering, and engineering product trafficking by heterologous expression of MtABCG10 to enhance the metabolic flux of chelerythrine biosynthesis, leading to a nearly 900-fold increase in chelerythrine production. Combined with the cultivation process, chelerythrine was obtained at a titer of 12.61 mg per liter in a 0.5 L bioreactor, which is over 37,000-fold higher than that of the first-generation recombinant strain. CONCLUSIONS: This is the first heterologous reconstruction of the plant-derived pathway to produce chelerythrine in a yeast cell factory. Applying a combinatorial engineering strategy has significantly improved the chelerythrine yield in yeast and is a promising approach for synthesizing functional products using a microbial cell factory. This achievement underscores the potential of metabolic engineering and synthetic biology in revolutionizing natural product biosynthesis.

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High-energy-density liquid fuels (HED fuels) are essential for volume-limited aerospace vehicles and could serve as energetic additives for conventional fuels. Terpene-derived HED biofuel is an important research field for green fuel synthesis. The direct extraction of terpenes from natural plants is environmentally unfriendly and costly. Designing efficient synthetic pathways in microorganisms to achieve high yields of terpenes shows great potential for the application of terpene-derived fuels. This review provides an overview of the current research progress of terpene-derived HED fuels, surveying terpene fuel properties and the current status of biosynthesis. Additionally, we systematically summarize the engineering strategies for biosynthesizing terpenes, including mining and engineering terpene synthases, optimizing metabolic pathways and cell-level optimization, such as the subcellular localization of terpene synthesis and adaptive evolution. This article will be helpful in providing insight into better developing terpene-derived HED fuels.

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Synthetic cell factory offers substantial advantages in economically efficient production of biofuels, chemicals, and pharmaceutical compounds. However, to create a high-performance synthetic cell factory, precise regulation of cellular material and energy flux is essential. In this context, protein components including enzymes, transcription factor-based biosensors and transporters play pivotal roles. Protein engineering aims to create novel protein variants with desired properties by modifying or designing protein sequences. This review focuses on summarizing the latest advancements of protein engineering in optimizing various aspects of synthetic cell factory, including: enhancing enzyme activity to eliminate production bottlenecks, altering enzyme selectivity to steer metabolic pathways towards desired products, modifying enzyme promiscuity to explore innovative routes, and improving the efficiency of transporters. Furthermore, the utilization of protein engineering to modify protein-based biosensors accelerates evolutionary process and optimizes the regulation of metabolic pathways. The remaining challenges and future opportunities in this field are also discussed.

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Product secretion from an engineered cell can be advantageous for microbial cell factories. Extensive work on nucleotide manufacturing, one of the most successful microbial fermentation processes, has enabled Corynebacterium stationis to transport nucleotides outside the cell by random mutagenesis; however, the underlying mechanism has not been elucidated, hindering its applications in transporter engineering. Herein, we report the nucleotide-exporting major facilitator superfamily (MFS) transporter from the C. stationis genome and its hyperactive mutation at the G64 residue. Structural estimation and molecular dynamics simulations suggested that the activity of this transporter improved via two mechanisms: (1) enhancing interactions between transmembrane helices through the conserved "RxxQG" motif along with substrate binding and (2) trapping substrate-interacting residue for easier release from the cavity. Our results provide novel insights into how MFS transporters change their conformation from inward- to outward-facing states upon substrate binding to facilitate efflux and can contribute to the development of rational design approaches for efflux improvements in microbial cell factories. KEYPOINTS: • An MFS transporter from C. stationis genome and its mutation at residue G64 were assessed • It enhanced the transporter activity by strengthening transmembrane helix interactions and trapped substrate-interacting residues • Our results contribute to rational design approach development for efflux improvement.

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Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid-a crucial precursor to artemisinin-is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing.

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Branched-chain amino acids (BCAAs) such as L-valine, L-leucine, and L-isoleucine are widely used in food and feed. To comply with sustainable development goals, commercial production of BCAAs has been completely replaced with microbial fermentation. However, the efficient production of BCAAs by microorganisms remains a serious challenge due to their staggered metabolic networks and cell growth. To overcome these difficulties, systemic metabolic engineering has emerged as an effective and feasible strategy for the biosynthesis of BCAA. This review firstly summarizes the research advances in the microbial synthesis of BCAAs and representative engineering strategies. Second, systematic methods, such as high-throughput screening, adaptive laboratory evolution, and omics analysis, can be used to analyses the synthesis of BCAAs at the whole-cell level and further improve the titer of target chemicals. Finally, new tools and engineering strategies that may increase the production output and development direction of the microbial production of BCAAs are discussed.

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Methylated natural products are widely spread in nature. S-Adenosyl-l-methionine (SAM) is the secondary abundant cofactor and the primary methyl donor, which confer natural products with structural and functional diversification. The increasing demand for SAM-dependent natural products (SdNPs) has motivated the development of microbial cell factories (MCFs) for sustainable and efficient SdNP production. Insufficient and unsustainable SAM availability hinders the improvement of SdNP MCF performance. From the perspective of developing MCF, this review summarized recent understanding of de novo SAM biosynthesis and its regulatory mechanism. SAM is just the methyl mediator but not the original methyl source. Effective and sustainable methyl source supply is critical for efficient SdNP production. We compared and discussed the innate and relatively less explored alternative methyl sources and identified the one involving cheap one-carbon compound as more promising. The SAM biosynthesis is synergistically regulated on multilevels and is tightly connected with ATP and NAD(P)H pools. We also covered the recent advancement of metabolic engineering in improving intracellular SAM availability and SdNP production. Dynamic regulation is a promising strategy to achieve accurate and dynamic fine-tuning of intracellular SAM pool size. Finally, we discussed the design and engineering constraints underlying construction of SAM-responsive genetic circuits and envisioned their future applications in developing SdNP MCFs.

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The industrial bacterium Bacillus licheniformis has long been used as a microbial factory for the production of enzymes due to its ability to secrete copious amounts of native extracellular proteins and its generally regarded as safe (GRAS) status. However, most attempts to use B. licheniformis to produce heterologous and cytoplasmic enzymes primarily via the general secretory (Sec) pathway have had limited success. The twin-arginine transport (Tat) pathway offers a promising alternative for the extracellular export of Sec-incompatible proteins because it transports full, correctly folded proteins. However, compared to the Sec pathway, the yields of the Tat pathway have historically been too low for commercial use. To improve the export efficiency of the Tat pathway, we identified the optimal Tat-dependent signal peptides and increased the abundance of the Tat translocases, the signal peptidase (SPase), and the intracellular chaperones. These strategic modifications significantly improved the Tat-dependent secretion of the cytoplasmic enzyme arginase into the culture medium using B. licheniformis. The extracellular enzymatic activity of arginase showed a 5.2-fold increase after these modifications. Moreover, compared to the start strain B. licheniformis 0F3, the production of extracellular GFP was improved by 3.8 times using the strategic modified strain B. licheniformis 0F13, and the extracellular enzymatic activity of SOX had a 1.3-fold increase using the strain B. licheniformis 0F14. This Tat-based production chassis has the potential for enhanced production of Sec-incompatible enzymes, therefore expanding the capability of B. licheniformis as an efficient cellular factory for the production of high-value proteins. KEY POINTS: • Systematic genetic modification of Tat-pathway in B. licheniformis. • Significant enhancement of the secretion capacity of Tat pathway for delivery the cytoplasmic enzyme arginase. • A new platform for efficient extracellular production of Sec-incompatible enzymes.

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Microbial cell factories (MCFs) convert low-cost carbon sources into valuable compounds. The CRISPR/Cas9 system has revolutionized MCF construction as a remarkable genome editing tool with unprecedented programmability. Recently, the CRISPR toolbox has been significantly expanded through the exploration of new CRISPR systems, the engineering of Cas effectors, and the incorporation of other effectors, enabling multi-level regulation and gene editing free of double-strand breaks. This expanded CRISPR toolbox powerfully promotes MCF construction by facilitating pathway construction, enzyme engineering, flux redistribution, and metabolic burden control. In this article, we summarize different CRISPR tool designs and their applications in MCF construction for gene editing, transcriptional regulation, and enzyme modulation. Finally, we also discuss future perspectives for the development and application of the CRISPR toolbox.

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With the development of metabolic engineering and synthetic biology, microbial cell factories (MCFs) have provided an efficient and sustainable method to synthesize a series of chemicals from renewable feedstocks. However, the efficiency of MCFs is usually limited by the inappropriate status of protein. Thus, engineering status of protein is essential to achieve efficient bioproduction with high titer, yield and productivity. In this review, we summarize the engineering strategies for metabolic protein status, including protein engineering for boosting microbial catalytic efficiency, protein modification for regulating microbial metabolic capacity, and protein assembly for enhancing microbial synthetic capacity. Finally, we highlight future challenges and prospects of improving microbial cell factories by engineering status of protein.

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Porphyrins, phycobilins, and their proteins have abundant π-electrons and strongly absorb visible light, some of which bind a metal ion in the center. Because of the structural and optical properties, they not only play critical roles as an essential component in natural systems but also have attracted much attention as a high value specialty chemical in various fields, including renewable energy, cosmetics, medicines, and foods. However, their commercial application seems to be still limited because the market price of porphyrins and phycobilins is generally expensive to apply them easily. Furthermore, their petroleum-based chemical synthesis is energy-intensive and emits a pollutant. Recently, to replace petroleum-based production, many studies on the bioproduction of metalloporphyrins, including Zn-porphyrin, Co-porphyrin, and heme, porphyrin derivatives including chlorophyll, biliverdin, and phycobilins, and their proteins including hemoproteins, phycobiliproteins, and phytochromes from renewable carbon sources using microbial cell factories have been reported. This review outlines recent advances in the bioproduction of porphyrins, phycobilins, and their proteins using microbial cell factories developed by various microbial biotechnology techniques, provides well-organized information on metabolic regulations of the porphyrin metabolism, and then critically discusses challenges and future perspectives. Through these, it is expected to be able to achieve possible solutions and insights and to develop an outstanding platform to be applied to the industry in future research.

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Hydroxylated steroids are value-added products with diverse biological activities mediated by cytochrome P450 enzymes, however, few has been thoroughly characterized in fungi. This study introduces a rapid identification strategy for filamentous fungi P450 enzymes through transcriptome and bioinformatics analysis. Five novel enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1 and CYP68N3) were identified and characterized in Saccharomyces cerevisiae or Aspergillus oryzae. Molecular docking and dynamics simulations were employed to elucidate hydroxylation preferences of CYP68J5 (11α, 7α bihydroxylase) and CYP68N1 (11α hydroxylase). Additionally, redox partners (cytochrome P450 reductase and cytochrome b5) and ABC transporter were co-expressed with CYP68N1 to enhance 11α-OH-androstenedione (11α-OH-4AD) production. The engineered cell factory, co-expressing CPR1 and CYP68N1, achieved a significant increase of 11α-OH-4AD production, reaching 0.845 g·L-1, which increased by 14 times compared to the original strain. This study provides a comprehensive approach for identifying and implementing novel cytochrome P450 enzymes, paving the way for sustainable production of steroidal products.

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BACKGROUND: Microbes have been used as cell factories to synthesize various chemical compounds. Recent advances in synthetic biological technologies have accelerated the increase in the number and capacity of microbial cell factories; the variety and number of synthetic compounds produced via these cell factories have also grown substantially. However, no database is available that provides detailed information on the microbial cell factories and the synthesized compounds. RESULTS: In this study, we established MCF2Chem, a manually curated knowledge base on the production of biosynthetic compounds using microbial cell factories. It contains 8888 items of production records related to 1231 compounds that were synthesizable by 590 microbial cell factories, including the production data of compounds (titer, yield, productivity, and content), strain culture information (culture medium, carbon source/precursor/substrate), fermentation information (mode, vessel, scale, and condition), and other information (e.g., strain modification method). The database contains statistical analyses data of compounds and microbial species. The data statistics of MCF2Chem showed that bacteria accounted for 60% of the species and that "fatty acids", "terpenoids", and "shikimates and phenylpropanoids" accounted for the top three chemical products. Escherichia coli, Saccharomyces cerevisiae, Yarrowia lipolytica, and Corynebacterium glutamicum synthesized 78% of these chemical compounds. Furthermore, we constructed a system to recommend microbial cell factories suitable for synthesizing target compounds and vice versa by combining MCF2Chem data, additional strain- and compound-related data, the phylogenetic relationships between strains, and compound similarities. CONCLUSIONS: MCF2Chem provides a user-friendly interface for querying, browsing, and visualizing detailed statistical information on microbial cell factories and their synthesizable compounds. It is publicly available at https://mcf.lifesynther.com . This database may serve as a useful resource for synthetic biologists.

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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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