Peptide-based medications hold immense potential in addressing a wide range of human disorders and discomforts. However, their widespread utilization encounters two major challenges: preservation and production efficiency. Cyclotides, a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), exhibit unique characteristics, such as a cyclic backbone and cystine knot, enhancing their stability and contributing to a wide range of pharmacological properties exhibited by these compounds. Cyclotides are efficient in the biomedical (e.g., antitumor, antidiabetic, antimicrobial, antiviral) and agrochemical fields by exhibiting activity against pests and plant diseases. Furthermore, their structural attributes make them suitable as molecular scaffolds for grafting and drug delivery. Notably, the mutated variant of kalata B1 cyclotide ([T20K] kalata B1) has recently entered phase 1 of human clinical trials for multiple sclerosis, building upon the success observed in animal trials. To enable large-scale production of cyclotides, it is crucial to further explore their remarkable structural and bioactive properties. This necessitates extensive research focused on enhancing the efficiency of the processes required for their production. This study provides a comprehensive review of the biological synthesis methods of cyclotides, with particular emphasis on various expression systems, namely bacteria, plants, yeast, and cell-free systems. By investigating these expression systems, it becomes possible to design production systems that are adaptable, economically viable, and efficient for generating active and pure cyclotides at an industrial scale. The advantages of biological synthesis over chemical synthesis are thoroughly explored, highlighting the potential of these expression systems in meeting the demands of large-scale cyclotide production.

The biomolecular condensates (BCs) formed by proteins through phase separation provide the necessary space and raw materials for the orderly progression of cellular activities, and on this basis, various membraneless organelles (MLOs) are formed. The occurrence of eukaryotic phase separation is driven by multivalent interactions from intrinsically disordered regions (IDRs) and/or specific protein/nucleic acid binding domains and is regulated by various environmental factors. In plant and animal cells, the MLOs involved in gene expression regulation, stress response, and mitotic control display similar functions and mechanisms. In contrast, the phase separation related to reproductive development and immune regulation differs significantly between the two kingdoms owing to their distinct cell structures and nutritional patterns. In addition, animals and plants each exhibit unique protein phase separation activities, such as neural regulation and light signal response. By comparing the similarities and differences in the formation mechanism and functional regulation of known protein phase separation, we elucidated its importance in the evolution, differentiation, and environmental adaptation of both animals and plants. The significance of studying protein phase separation for enhancing biological quality of life has been further emphasized.

Vibrational spectroscopy is a nondestructive analysis technique that depends on the periodic variations in dipole moments and polarizabilities resulting from the molecular vibrations of molecules/atoms. These methods have important advantages over conventional analytical techniques, including (a) their simplicity in terms of implementation and operation, (b) their adaptability to on-line and on-farm applications, (c) making measurement in a few minutes, and (d) the absence of dangerous solvents throughout sample preparation or measurement. Food safety is a concept that requires the assurance that food is free from any physical, chemical, or biological hazards at all stages, from farm to fork. Continuous monitoring should be provided in order to guarantee the safety of the food. Regarding their advantages, vibrational spectroscopic methods, such as Fourier-transform infrared (FTIR), near-infrared (NIR), and Raman spectroscopy, are considered reliable and rapid techniques to track food safety- and food authenticity-related issues throughout the food chain. Furthermore, coupling spectral data with chemometric approaches also enables the discrimination of samples with different kinds of food safety-related hazards. This review deals with the recent application of vibrational spectroscopic techniques to monitor various hazards related to various foods, including crops, fruits, vegetables, milk, dairy products, meat, seafood, and poultry, throughout harvesting, transportation, processing, distribution, and storage.

have been widely used in industrial compound production for their incomplete oxidation ability. However, they are often subjected to a wide variety of severe environmental stresses, such as extreme pH, high temperature, osmotic pressure, and organic solvents, which greatly repress microbial growth viability and productivity. As typical biocatalysis chassis cells with a high tolerance to external environmental stresses, it is extremely important to construct highly tolerant chassis cells and understand the tolerance mechanisms of and how different stresses interact with the cell: membranes, phospholipid bilayers, transporters, and chaperone proteins. In this review, we discuss and summarize the mechanisms of environmental stress tolerance in , and the promising strategies that can be used to further construct tolerant strains are prospected.

Resveratrol is an antioxidant abundant in plants like grapes and peanuts and has garnered significant attention for its potential therapeutic applications. This review explores its chemical attributes, stability, and solubility, influencing its diverse applications and bioavailability. Resveratrol's multifaceted therapeutic roles encompass: antioxidant, cardioprotective, anti-inflammatory, neuroprotective, anti-aging, and anticancer properties. While traditionally studied in preclinical settings, a surge in clinical trials underscores resveratrol's promise for human health. Over 250 recent clinical trials investigate its effects alone and in combination with other compounds. Commercially utilized in food, cosmetics, supplements, and pharmaceuticals, the resveratrol market is expanding, driven by microbial fermentation. Microbes offer advantages over plant extraction and chemical synthesis, providing cost-effective, pure, and sustainable production. Microbial biosynthesis can be attained from carbon sources, such as glucose or xylose, among others, which can be obtained from renewable resources or agro-industrial wastes. While has been the most used host, non-conventional yeasts like and bacteria like have also demonstrated potential. Genetic modifications such as increasing acetyl-CoA/malonyl-CoA pools, boosting the shikimate pathway, or multi-copy expression of pathway genes, allied to the optimization of fermentation strategies have been promising in increasing titers. Microbial biosynthesis of resveratrol aligns with the shift toward sustainable and renewable bio-based compounds, exemplifying a circular bioeconomy. Concluding, microbial fermentation presents a promising avenue for efficient resveratrol production, driven by genetic engineering, pathway optimization, and fermentation strategies. These advances hold the key to unlocking the potential of resveratrol for diverse therapeutic applications, contributing to a greener and sustainable future.

Agricultural byproducts generally contain abundant bioactive compounds (e.g., cellulose/hemicellulose, phenolic compounds (PCs), and dietary fibers (DFs)), but most of them are neglected and underutilized. Owing to the complicated and rigid structures of agricultural byproducts, a considerable amount of bioactive compounds are entrapped in the polymer matrix, impeding their further development and utilization. In recent years, the prominent performance of cellulolytic fungi to grow and degrade agricultural byproducts has been applied to achieve efficient biotransformation of byproducts to high-value compounds, which is a green and sustainable strategy for the reutilization of agricultural byproducts. This review comprehensively summarizes recent progress in the value-added biotransformation of agricultural byproducts by cellulolytic fungi, including (1) direct utilization of agricultural byproducts for biochemicals and bioethanol production via a consolidated bioprocessing, (2) recovery and biotransformation of bounded PCs from agricultural byproducts for higher bioactive properties, as well as (3) modification and conversion of insoluble DF from agricultural byproducts to produce functional soluble DF. The functional enzymes, potential mechanisms, and metabolic pathways involved are emphasized. Moreover, promising advantages and current bottlenecks using cellulolytic fungi have also been elucidated, shedding further perspectives for sustainable and efficient reutilization of agricultural byproducts by cellulolytic fungi.

Millets, often overlooked as food crops, have regained potential as promising stable food sources of bioactive compounds to regulate blood sugar levels in the diabetic populace. This comprehensive review delves into various millet varieties, processing methods, and extraction techniques aimed at isolating bioactive compounds. The review elucidates the inhibitory effects of millet-derived bioactive compounds on key enzymes involved in carbohydrate metabolism, such as α-amylase and α-glucosidase. It further explores the relationship between the antibacterial activity of phenols, flavonoids, and anthocyanins in millets and their role in amylase inhibition. In particular, phenols, flavonoids, and proteins found in millets play pivotal roles in inhibiting enzymes responsible for glucose digestion and absorption. However, processing methods can either enhance or reduce the bioactive compounds, thereby influencing enzyme inhibition capacity. Studies underscore the presence of phenolic compounds with notable inhibitory activity in: foxtail, finger, barnyard, and pearl millet varieties. Furthermore, extraction techniques, such as Soxhlet and ultrasonic-assisted extraction, emerge as efficient methods for isolating bioactive compounds, thus enhancing their therapeutic efficacy. This review highlights the challenges in preserving the inhibitory activity of millets during processing and optimizing processing methods to ensure better retention of bioactive compounds. It also emphasizes the utilization of millet as a natural dietary supplement or functional food to manage diabetes and promote overall well-being.

The development and commercialization of bio-based and biodegradable polyhydroxyalkanoates (PHAs) biopolymers could be crucial for the transition toward a sustainable circular economy. However, despite potential traditional and novel applications in the packaging, textiles, agriculture, automotive, electronics, and biomedical industries, the commercialization of PHAs is limited by their current market competitiveness. This review provides the first critical assessment of the current pure culture pilot-scale PHA literature, which could be crucial in translating promising laboratory-scale developments into industrial-scale commercial PHA production. It will also complement reviews of mixed microbial cultures currently dominating pilot-scale PHA literature. Pure culture fermentations could provide advantages, such as ease of characterizing microbial producers' behavior, higher PHA productivities, and better alignment with existing PHA commercialization and industrial biotechnology approaches. Key aspects, including producer organisms, fermentation volumes and schemes, control schemes, optimization, and properties of the polymers produced, are discussed in-depth, to elucidate important trends, achievements, and knowledge gaps. Furthermore, specific ways for future pilot-scale studies to help address current PHA commercialization challenges are also identified. The insights, and recommendations provided will be extremely beneficial for the future development of PHA production, at both pilot and commercial scales, whilst also being beneficial to the production of other microbial polymers and industrial biotechnology as a whole.

Astaxanthin (AXT), a natural carotenoid, has strong antioxidant and anti-ageing effects and can reduce ultraviolet light-induced damage to cells and DNA, stimulate the immune system, and improve cardiovascular disease prognosis. Despite its wide applications in the: nutraceutical, cosmetic, aquaculture, and pharmaceutical industries, AXT industrial production and application are hindered by natural source scarcity, low production efficiency, and high requirements. This review compares the qualitative differences of AXT derived from different natural sources, evaluates the upstream procedures for AXT expression in different chassis organisms, and investigates synthetic biology- and cell factory-based strategies for the industrial production of natural AXT. Synthetic biology is a promising novel strategy for reprogramming plants or microorganisms to produce AXT. Additionally, genetic engineering using cell factories extends beyond terrestrial applications, as it may contribute to the long-term sustainability of human health during space exploration and migration endeavors. This review provides a theoretical basis for the efficient and accurate genetic engineering of AXT from the microalga , providing a valuable reference for future research on the biomanufacturing of AXT and other biological metabolites.

Statins are the most prescribed drug for regulating the high cholesterol level in the blood, which can lead to severe complications, such as cardiovascular diseases and other health complications. A wide range of analytical techniques have been employed for the quantification of statins from various origins, including fermentation derived (lovastatin, pravastatin, and compactin), semi-synthetic (simvastatin), and synthetic (atorvastatin, rosuvastatin, and fluvastatin) routes. The presence of more than one structural form and structural analogue generated in the biosynthesis pathway, as well as reaction intermediates and macromolecules in the clinical sample, complicates the quantification of statins. Furthermore, significant concentrations of statins in environmental samples pose serious health and ecology hazards, and estimating statins in those diluted samples is extremely difficult. On the other hand, the: cost, accurate estimation of the desired one from other structural forms, sample complexity, time, limits of detection and quantification, were major criteria distinguishing the usability of each technique. As a result, the current manuscript focuses on analytical techniques such as molecular spectroscopy (normal and derivatives UV-Visible spectrophotometer), chromatography (TLC, HP-TLC, HPLC, GC, swing column, micellar, and supercritical fluid), mass spectroscopy (HPLC-MS/MS and GC-MS/MS), sequential flow injection, capillary electrophoresis, and cyclic voltammetry, as well as their: optimal operating conditions, limits of detection and quantification, advancements, and limitations. Furthermore, various online and offline sample preparations (precipitation, solid phase extraction, liquid-liquid extraction, and micellar extraction) have been highlighted as an essential pretreatment technique to avoid the interference caused by structural analogues and other macromolecules. The greener and more sustainable concepts used in analytical approaches for the quantification statins are also highlighted with current advancements.

Geraniol, an acyclic monoterpene alcohol, has significant potential applications in various fields, including: food, cosmetics, biofuels, and pharmaceuticals. However, the current sources of geraniol mainly include plant tissue extraction or chemical synthesis, which are unsustainable and suffer severely from high energy consumption and severe environmental problems. The process of microbial production of geraniol has recently undergone vigorous development. Particularly, the sustainable construction of recombinant (13.2 g/L) and (5.5 g/L) laid a solid foundation for the microbial production of geraniol. In this review, recent advances in the development of geraniol-producing strains, including: metabolic pathway construction, key enzyme improvement, genetic modification strategies, and cytotoxicity alleviation, are critically summarized. Furthermore, the key challenges in scaling up geraniol production and future perspectives for the development of robust geraniol-producing strains are suggested. This review provides theoretical guidance for the industrial production of geraniol using microbial cell factories.

Acetyl-CoA is an intermediate metabolite in cellular central metabolism. It's a precursor for various valuable commercial products, including: terpenoids, fatty acids, and polyketides. With the advancement of metabolic and synthetic biology tools, microbial cell factories have been constructed for the efficient synthesis of acetyl-CoA and derivatives, with the and as two prominent chassis. This review summarized the recent developments in the biosynthetic pathways and metabolic engineering approaches for acetyl-CoA and its derivatives synthesis in these two yeasts. First, the metabolic routes involved in the biosynthesis of acetyl-CoA and derived products were outlined. Then, the advancements in metabolic engineering strategies for channeling acetyl-CoA toward the desired products were summarized, with particular emphasis on: enhancing metabolic flux in different organelles, refining precursor CoA synthesis, optimizing substrate utilization, and modifying protein acetylation level. Finally, future developments in advancing the metabolic engineering strategies for acetyl-CoA and related derivatives synthesis, including: reducing CO emissions, dynamically regulating metabolic pathways, and exploring the regulatory functions between acetyl-CoA levels and protein acetylation, are highlighted. This review provided new insights into regulating acetyl-CoA synthesis to create more effective microbial cell factories for bio-manufacturing.

Cyanobacteria, the only oxygenic photoautotrophs among prokaryotes, are developing as both carbon building blocks and energetic self-supported chassis for the generation of various bioproducts. However, one of the challenges to optimize it as a more sustainable platform is how to release intracellular bioproducts for an easier downstream biorefinery process. To date, the major method used for cyanobacterial cell lysis is based on mechanical force, which is energy-intensive and economically unsustainable. Phage-mediated bacterial cell lysis is species-specific and highly efficient and can be conducted under mild conditions; therefore, it has been intensively studied as a bacterial cell lysis weapon. In contrast to heterotrophic bacteria, biological cell lysis studies in cyanobacteria are lagging behind. In this study, we reviewed cyanobacterial cell envelope features that could affect cell strength and elicited a thorough presentation of the necessary phage lysin components for efficient cell lysis. We then summarized all bioengineering manipulated pipelines for lysin component optimization and further revealed the challenges for each intent-oriented application in cyanobacterial cell lysis. In addition to applied biotechnology usage, the significance of phage-mediated cyanobacterial cell lysis could also advance sophisticated biochemical studies and promote biocontrol of toxic cyanobacteria blooms.

The skin aging process is a complex interaction of genetic, epigenetic, and environmental factors, such as chemical pollution and UV radiation. There is growing evidence that biosurfactants, especially those of microbial origin, have distinct age-supportive effects through different mechanisms, such as stimulation of fibroblast growth, high antioxidant capacities, and favorable anti-inflammatory properties. With a growing financial contribution of more than 15 m€per year, microbial surfactants (MSs) display unique biological effects on the skin including improved cell mobility, better nutrient access, and facilitated cellular growth under harsh conditions. Their biodegradable nature, unusual surface activity, good safety profile and tolerance to high temperature and pH variations widen their potential spectrum in biomedical and pharmaceutical applications. MSs typically have lower critical micelle concentration (CMC) levels than chemical surfactants enhancing their effectiveness. As natural surfactants, MSs are considered possible "green" alternatives to synthetic surfactants with better biodegradability, sustainability, and beneficial functional properties. This review therefore aims to explore the potential impacts of MSs as anti-aging ingredients.

Compounds containing chiral C-N bonds play a vital role in the composition of biologically active natural products and small pharmaceutical molecules. Therefore, the development of efficient and convenient methods for synthesizing compounds containing chiral C-N bonds is a crucial area of research. Nicotinamide-dependent oxidoreductases (NDOs) emerge as promising biocatalysts for asymmetric synthesis of chiral C-N bonds due to their mild reaction conditions, exceptional stereoselectivity, high atom economy, and environmentally friendly nature. This review aims to present the structural characteristics and catalytic mechanisms of various NDOs, including imine reductases/ketimine reductases, reductive aminases, EneIRED, and amino acid dehydrogenases. Additionally, the review highlights protein engineering strategies employed to modify the stereoselectivity, substrate specificity, and cofactor preference of NDOs. Furthermore, the applications of NDOs in synthesizing essential medicinal chemicals, such as noncanonical amino acids and chiral amine compounds, are extensively examined. Finally, the review outlines future perspectives by addressing challenges and discussing the potential of utilizing NDOs to establish efficient biosynthesis platforms for C-N bond synthesis. In conclusion, NDOs provide an economical, efficient, and environmentally friendly toolbox for asymmetric synthesis of C-N bonds, thus contributing significantly to the field of pharmaceutical chemical development.

CRISPR-based diagnostics (CRISPR/Dx) have revolutionized the field of molecular diagnostics. It enables home self-test, field-deployable, and point-of-care testing (POCT). Despite the great potential of CRISPR/Dx in diagnoses of biologically complex diseases, preamplification of the template often is required for the sensitive detection of low-abundance nucleic acids. Various amplification-free CRISPR/Dx systems were recently developed to enhance signal detection at sufficient sensitivity. Broadly, these amplification-free CRISPR/Dx systems are classified into five groups depending on the signal enhancement strategies employed: CRISPR/Cas12a and/or CRISPR/Cas13a are integrated with: (1) other catalytic enzymes (Cas14a, Csm6, Argonaute, duplex-specific nuclease, nanozyme, or T7 exonuclease), (2) rational-designed oligonucleotides (multivalent aptamer, tetrahedral DNA framework, RNA G-quadruplexes, DNA roller machine, switchable-caged guide RNA, hybrid locked RNA/DNA probe, hybridized cascade probe, or "U" rich stem-loop RNA), (3) nanomaterials (nanophotonic structure, gold nanoparticle, micromotor, or microbeads), (4) electrochemical and piezoelectric plate biosensors (SERS nanoprobes, graphene field-effect transistor, redox probe, or primer exchange reaction), or (5) cutting-edge detection technology platforms (digital bioanalysis, droplet microfluidic, smartphone camera, or single nanoparticle counting). Herein, we critically discuss the advances, pitfalls and future perspectives for these amplification-free CRISPR/Dx systems in nucleic acids detection. The continued refinement of these CRISPR/Dx systems will pave the road for rapid, cost-effective, ultrasensitive, and ultraspecific on-site detection without resorting to target amplification, with the ultimate goal of establishing CRISPR/Dx as the paragon of diagnostics.

Buckwheat ( spp.) is a typical pseudocereal, valued for its extensive nutraceutical potential as well as its centuries-old cultivation. Tartary buckwheat and common buckwheat have been used globally and become well-known nutritious foods due to their high quantities of: proteins, flavonoids, and minerals. Moreover, its increasing demand makes it critical to improve nutraceutical, traits and yield. In this review, bioactive compounds accumulated in buckwheat were comprehensively evaluated according to their chemical structure, properties, and physiological function. Biosynthetic pathways of flavonoids, phenolic acids, and fagopyrin were methodically summarized, with the regulation of flavonoid biosynthesis. Although there are classic synthesis pathways presented in the previous research, the metabolic flow of how these certain compounds are being synthesized in buckwheat still remains uncovered. The functional genes involved in the biosynthesis of flavonols, stress response, and plant development were identified based on multi-omics research. Furthermore, it delves into the applications of multi-omics in improving buckwheat's agronomic traits, including: yield, nutritional content, stress resilience, and bioactive compounds biosynthesis. While pangenomics combined with other omics to mine elite genes, the regulatory network and mechanism of specific agronomic traits and biosynthetic of bioactive components, and developing a more efficient genetic transformation system for genetic engineering require further investigation for the execution of breeding designs aimed at enhancing desirable traits in buckwheat. This critical review will provide a comprehensive understanding of multi-omics for nutraceutical enhancement and traits improvement in buckwheat.

The ornithine-urea cycle (OUC) in fungal cells has biotechnological importance and many physiological functions and is closely related to the acetyl glutamate cycle (AGC). Fumarate can be released from argininosuccinate under the catalysis of argininosuccinate lyase in OUC which is regulated by the Ca signaling pathway and over 93.9 ± 0.8 g/L fumarate can be yielded by the engineered strain of var. i in the presence of CaCO. Furthermore, 2.1 ± 0.02 mg of L-ornithine (L-Orn)/mg of the protein also can be synthesized OUC by the engineered strains of . Fumarate can be transformed into many drugs and amino acids and L-Orn can be converted into siderophores (1.7 g/L), putrescine (33.4 g/L) and L-piperazic acid (L-Piz) (3.0 g/L), by different recombinant strains of . All the fumarate, L-Orn, siderophore, putrescine and L-Piz have many applications. As the yeast-like fungi and the promising chassis, spp, have many advantages over any other fungal strains. Further genetic manipulation and bioengineering will enhance the biosynthesis of fumarate and L-Orn and their derivates.

Developing proteins with increased chemical space by expanding the amino acids alphabet has been an emerging technique to compete for the obstacle encountered by their need in various applications. 3,4-Dihydroxyphenylalanine (L-DOPA) catecholic unnatural amino acid is abundantly present in mussels foot proteins through post-translational modification of tyrosine to give a strong adhesion toward wet rocks. L-DOPA forms: bidentate coordination, H-bonding, metal-ligand complexes, long-ranged electrostatic, and van der Waals interactions a pair of donor hydroxyl groups. Incorporating catechol in proteins through genetic code expansion paved the way for developing: protein-based bio-sensor, implant coating, bio-conjugation, adhesive bio-materials, biocatalyst, metal interaction and nano-biotechnological applications. The increased chemical spaces boost the protein properties by offering a new chemically active interaction ability to the protein. Here, we review the technique employed to develop a genetically expanded organism with catechol to provide novel properties and functionalities; and we highlight the importance of L-DOPA incorporated proteins in biomedical and industrial fields.

With antibiotic resistance on the rise, there is an urgent need for new antibacterial drugs and products to treat or prevent infection. Many such products in current use, for example human and veterinary antibiotics and antimicrobial food preservatives, were discovered and developed from nature. Natural selection acts on all living organisms and the presence of bacterial competitors or pathogens in an environment can favor the evolution of antibacterial adaptations. In this review, we ask if vultures, blow flies and other carrion users might be a good starting point for antibacterial discovery based on the selection pressure they are under from bacterial disease. Dietary details are catalogued for over 600 of these species, bacterial pathogens associated with the diets are described, and an overview of the antibacterial defenses contributing to disease protection is given. Biotechnological applications for these defenses are then discussed, together with challenges facing developers and possible solutions. Examples include use of (a) the antimicrobial peptide (AMP) gene to improve crop resistance to bacterial disease, (b) peptide antibiotics such as serrawettin W2 as antibacterial drug leads, (c) lectins for targeted drug delivery, (d) bioconversion-generated chitin as an antibacterial biomaterial, (e) bacteriocins as antibacterial food preservatives and (f) mutualistic microbiota bacteria as alternatives to antibiotics in animal feed. We show that carrion users encounter a diverse range of bacterial pathogens through their diets and interactions, have evolved many antibacterial defenses, and are a promising source of genes, molecules, and microbes for medical, agricultural, and food industry product development.

Microbes have been extensively utilized for their sustainable and scalable properties in synthesizing desired bio-products. However, insufficient knowledge about intracellular metabolism has impeded further microbial applications. The genome-scale metabolic models (GEMs) play a pivotal role in facilitating a global understanding of cellular metabolic mechanisms. These models enable rational modification by exploring metabolic pathways and predicting potential targets in microorganisms, enabling precise cell regulation without experimental costs. Nonetheless, simplified GEM only considers genome information and network stoichiometry while neglecting other important bio-information, such as enzyme functions, thermodynamic properties, and kinetic parameters. Consequently, uncertainties persist particularly when predicting microbial behaviors in complex and fluctuant systems. The advent of the omics era with its massive quantification of genes, proteins, and metabolites under various conditions has led to the flourishing of multi-constrained models and updated algorithms with improved predicting power and broadened dimension. Meanwhile, machine learning (ML) has demonstrated exceptional analytical and predictive capacities when applied to training sets of biological big data. Incorporating the discriminant strength of ML with GEM facilitates mechanistic modeling efficiency and improves predictive accuracy. This paper provides an overview of research innovations in the GEM, including multi-constrained modeling, analytical approaches, and the latest applications of ML, which may contribute comprehensive knowledge toward genetic refinement, strain development, and yield enhancement for a broad range of biomolecules.