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The engineering of microbial biosynthetic pathways is advancing rapidly because of new molecular genetic approaches, sophisticated analysis of metabolic flux and the rapid sequencing of diverse bacterial genomes. The classical methods of mutagenesis/selection, originally applied in the development of amino-acid-over-producing bacteria are now being complemented by an increasingly rational strategy.
Polyketides are an extensive class of secondary metabolites with diverse molecular structures and biological activities. A plasmid-based multicomponent polyketide synthase expression cassette was constructed using a subset of actinorhodin (act) biosynthetic genes (actI-orf1, actI-orf2, actI-orf3, actIII, actVII, and actIV) from Streptomyces coelicolor which specify the construction of the anthraquinone product aloesaponarin II, a molecule derived from acetyl coenzyme A and 7 malonyl coenzyme A extender units. This system was designed as an indicator pathway in Streptomyces parvulus to quantify polyketide product formation and to examine the functional significance of specific polyketide synthase components, including the act beta-ketoacyl synthase (beta-KS; encoded by actI-orf1 and actI-orf2) and the act cyclase/dehydrase (encoded by actVII-orf4). Site-directed mutagenesis of the putative active site Cys (to a Gln) in the actI-orf1 beta-KS product completely abrogated aloesaponarin II production. Changing the putative acyltransferase active-site Ser (to a Leu) located in the actI-orf1 beta-KS product led to significantly reduced but continued production of aloesaponarin II. Replacement of the expression cassette with one containing a mutant form of actI-orf2 gave no production of aloesaponarin II or any other detectable polyketide products. However, an expression cassette containing a mutant form of actVII-orf4 gave primarily mutactin with low-level production of aloesaponarin II.
During the next decade or so, there will be significant and impressive advances in biomolecular engineering, especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology, DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques, DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific disciplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins, including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment or prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Many more industrial enzymes, which will be engineered to confer desirable properties for the process improvement and manufacturing of high-value biomolecular products at a lower production cost, are also anticipated. New metabolites, including novel antibiotics that are active against resistant strains, will also be produced soon by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here, and many of benefits will be derived from this field of scientific research for years to come if we are willing to put it to good use.
Natural products (NPs) remain a cornerstone of modern drug discovery, offering stereochemical complexity and diverse bioactivities that precisely modulate therapeutic targets, refined through billions of years of evolution. However, their research has long been hindered by inefficient, empirical workflows, high resource consumption, structural complexity, and the "multicomponent, multi-target" nature of their mechanisms. The exponential growth of genomic, metabolomic, and spectral data has overwhelmed conventional analytical methods, exposing critical bottlenecks in handling high-dimensional, heterogeneous datasets that exceed human interpretive capacity. Artificial intelligence (AI) is emerging as a transformative paradigm to address these challenges, integrating multi-omics and chemical data to shift NP research from fragmented empiricism toward mechanism-driven, precision-oriented development. By leveraging deep learning architectures- including graph neural networks, Transformers, and diffusion-based generative models-AI enables systematic decoding of NP biosynthesis, automated structure elucidation, rational target identification, knowledge extraction from vast unstructured scientific literature, and de novo molecular design. This review comprehensively surveys recent advances in AI applications across the full NP discovery and development pipeline, encompassing genome mining, structure-based and ligand-based virtual screening, multimodal structural characterization, lead optimization, and biosynthetic pathway engineering. We further examine the emerging roles of protein-centric, molecule- centric, and multimodal foundation models, as well as large language models, in bridging genotype-to-chemotype gaps and unlocking unstructured scientific knowledge. Finally, we discuss critical challenges including data scarcity, representational limitations for complex stereochemistry, physical plausibility in generative models, and the urgent need for experimental validation, while outlining future directions toward autonomous experimentation, closed-loop optimization, and human-AI collaborative discovery.
Flavonoid glycosides and triterpenoid saponins are bioactive plant metabolites with broad applications in food, medicine, and agriculture. These compounds are typically synthesized through glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGTs). In this study, phylogenetic analysis across multiple species revealed a lineage-specific expansion of the UGT73 family in legumes such as Entada phaseoloides and Glycine max. The genome of the medicinal legume E. phaseoloides was re-annotated using integrated Oxford Nanopore Technologies and Illumina transcriptomic data to identify target genes. Four expanded UGT73 family genes were selected and functionally characterized. UGT73AA6 specifically glycosylates flavonoids, while UGT73CG48 and UGT73CG49 catalyze glycosylation of both flavonoids and pentacyclic triterpenoids. UGT73CG49 exhibits higher catalytic activity for the glucosylation of flavonoids and pentacyclic triterpenes compared to its xylosylation activity. Structural modeling and molecular docking identified key active sites, and site-directed mutagenesis revealed Gly194 as a critical residue enhancing catalytic activity in UGT73CG49. This study provides new insights into the functional evolution and metabolic versatility of the UGT73 family in legumes. The identification and engineering of UGT73 genes from E. phaseoloides lay a foundation for future applications in biosynthetic pathway engineering and the industrial production of high-value glycosides.
Evodia rutaecarpa is a valuable medicinal plant, yet its non-medicinal tissues remain largely underexplored. Here, we integrated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based widely targeted metabolomics and RNA sequencing (RNA-seq) transcriptomics to systematically profile the metabolic and transcriptional landscapes of roots, stems, leaves, and flowers of Evodia rutaecarpa (Juss.) Benth. Our aim was to characterize tissue-specific metabolic differentiation and its underlying transcriptional regulatory mechanisms. Metabolomic analysis, employing principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) with robust model parameters (R2Y > 0.9, Q2 > 0.5), identified 3090 differential metabolite features (variable importance in projection, VIP > 1.0; p < 0.05) across the four tissues, which exhibited distinct tissue-specific clustering patterns. Integrated Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and weighted gene co-expression network analysis (WGCNA) revealed that roots specifically accumulated quinolone alkaloids and flavonoid glycosides, accompanied by the coordinated upregulation of genes involved in flavonoid and phenylpropanoid biosynthetic pathways. In contrast, stems, leaves, and flowers were enriched in indole alkaloids (evodiamine and rutaecarpine) and volatile oil precursors, with concurrent upregulation of genes involved in tryptophan metabolism and indole alkaloid biosynthesis (e.g., tryptophan decarboxylase, TDC; s N-methyltransferase, NMT). Notably, leaves and flowers displayed particularly high accumulation levels of these bioactive alkaloids, suggesting their potential as alternative sources for industrial and pharmaceutical applications. WGCNA further identified multiple transcription factors and structural gene modules tightly correlated with evodiamine accumulation, offering promising candidate regulators for future biosynthetic pathway engineering. Collectively, this multi-omics integration study systematically elucidates the tissue-partitioned secondary metabolism of Evodia rutaecarpa (Juss.) Benth. and provides a solid scientific foundation for full-plant resource utilization, targeted development of non-medicinal tissues, and future metabolic engineering of indole alkaloid production.
Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.
In many economically important plant species, the chemical composition of one or more non-protein compounds determines the value of the plant and may have an important role in protecting the plant from environmental stress, including pests, drought, salt, temperature and light. A number of potential opportunities exist whereby the range or amount of such valuable compounds can be increased by genetic engineering.
The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants. We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli. We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids. One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3, 4,3',4'-tetradehydrolycopene. This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products. One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E. coli. This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry.
Decaprenoxanthin (DPXT) is an unusual bacterial C50 carotenoid that has historically received limited attention despite its well-defined structure. For decades, carotenoid research and industrial development have been dominated by C40 carotenoids, leaving longer-chain carotenoids largely overlooked. Recent discoveries, particularly from microorganisms inhabiting Antarctic and other extreme environments, have repositioned DPXT as an adaptive pigment shaped by intense environmental pressures. Its extended polyene chain and membrane-associated behavior suggest roles in membrane stabilization and protection against ultraviolet radiation and oxidative stress, features that may hold relevance for food and biotechnological applications. This review integrates historical and recent knowledge on DPXT, covering its structural characteristics, biosynthetic pathways, ecological function, and emerging technological relevance. Special attention is given to microbial sources, particularly Actinomycetota from extreme environments, and to recent advances in microbial genomics, metabolic engineering, and sustainable bioprocess development that enable the production and exploration of C50 carotenoids beyond their native extremophilic context. The analysis highlights DPXT as a representative example of stress-resilient carotenoids, with physicochemical and membrane-interacting properties that may offer advantages for future food and biotechnological systems. Although significant challenges remain in cultivation strategies, yield optimization, and downstream recovery, advances in microbial cell factories and green extraction technologies open new opportunities for valorizing C50 carotenoids. This review bridges extremophile microbiology, carotenoid biochemistry, and sustainable food innovation, positioning DPXT as an emerging molecule that may expand the functional and structural landscape of carotenoids relevant to food science.
In leguminous plants such as the forage legume alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms (flavonoid inducers of the Rhizobium symbiosis), and in defense against pathogens (isoflavonoid phytoalexins). In addition, the phenylpropane polymer lignin is a major structural component of secondary vascular tissue and fibers in higher plants. the recent isolation of genes encoding key enzymes of the various phenylpropanoid branch pathways opens up the possibility of engineering important crop plants such as alfalfa for: (a) improved forage digestibility, by modification of lignin composition and/or content; (b) increased or broader-spectrum disease resistance, by introducing novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways; and (c) enhanced nodulation efficiency, by engineering over-production of flavonoid nod gene inducers. The basic biochemistry and molecular biology underlying these strategies is briefly reviewed, and recent progress with transgenic plants summarized. The potential importance of metabolic compartmentation for attempts to engineer phenylpropanoid biosynthetic pathways is also discussed. Over-expression of an alfalfa glucanase-encoding gene confers significant protection against Phytophthora in alfalfa, possibly via indirect effects on phenylpropanoid metabolism.
Isoflavonoids are key secondary metabolites in leguminous plants that play essential roles in plant physiology and provide significant health benefits to humans. In the isoflavone biosynthetic pathway, isoflavone synthase (IFS) catalyzes the conversion of naringenin and liquiritigenin into the bioactive isoflavones genistein and daidzein. This study aimed to enhance genistein and daidzein accumulation in soybean seeds through genetic engineering. Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301 containing GmIFS under the control of a seed-specific promoter (Gmβ-conglycinin) was used to transform modified half-seed explants of soybean cv. JS335. Hygromycin-B-resistant plants were regenerated, hardened, and confirmed by histochemical GUS assay. Molecular analysis by PCR validated the presence of the GmIFS transgene, yielding a 700 bp amplicon. Biochemical analysis revealed that seeds of T₀ transgenic plants showed a 1.53-fold increase in total phenolic content and a 3.67-fold increase in flavonoid content compared to non-transformed controls. Antioxidant assays demonstrated significantly higher DPPH radical-scavenging activity and ferric-reducing antioxidant power (FRAP) in GmIFS-overexpressing plants. HPLC analysis further indicated that transgenic seeds accumulated, on average, 4.07-fold higher daidzein and 1.75-fold higher genistein levels relative to control plants. qRT-PCR analysis showed significantly elevated GmIFS expression in immature cotyledons, mature cotyledons, and seeds of transgenic plants. Overall, these results demonstrate that GmIFS overexpression effectively enhances isoflavone production in soybean seeds, highlighting the potential of metabolic engineering of biosynthetic pathway genes to improve nutritional quality.
The biosynthesis of beta-lactams is one of the most thoroughly studied antibiotic pathways. The availability of the characteristics and the time profiles of activities of enzymes involved in the biosynthesis allows one to critically evaluate the potential rate-limiting steps in its production. Our approach to understanding the control of beta-lactam biosynthesis has been pursued using a two-stage strategy: (1) to predict the rate-limiting steps using a kinetic model and (2) to relax the rate-limiting steps by engineering the biosynthetic pathway or by altering the kinetic parameters of the predicted key rate-limiting enzyme. Kinetic analysis of the pathway dynamics of cephamycin C production in Streptomyces clavuligerus was performed using data obtained from wild type. Sensitivity analysis revealed that the availability of precursor alpha-aminoadipic acid and activity of ACV synthetase were the potential rate-limiting steps. Relaxation of the precursor limitation was accomplished by integration of an additional copy of the gene encoding lysine-epsilon-aminotransferase (lat) into the chromosome. The recombinant strain showed an increased level of cephamycin C production as expected. The intracellular levels of different intermediates in the pathway in batch cultures were analyzed.
In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0-9.5-fold increase for DHPS. T1 seeds of DHPS transformants showed the same changes in free amino acids as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.
Recombinant DNA technology has facilitated a rapid increase in our knowledge of beta-lactam antibiotic biosynthesis. Using the tools of this technology, beta-lactam biosynthetic genes and proteins have been characterized at the molecular level, cephalosporin-C production has been improved, new beta-lactams produced, and novel beta-lactam biosynthetic pathways have been constructed.
Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.
Paeoniflorin is a pharmacologically important cage-like monoterpene glycoside characteristic of Paeonia plants, yet its biosynthetic pathway has remained largely unresolved, hindering sustainable production. Here, we confirmed that paeoniflorin biosynthesis originates from α-pinene and identified three novel cytochrome P450 enzymes that catalyze pinene oxidation. CYP71AN126 catalyzes the hydroxylation of α-pinene at positions C4 and C10, followed by further oxidation of the alcohol to a ketone at C4, whereas CYP76A225/226 exclusively catalyze C10 hydroxylation. Virus-induced gene silencing (VIGS) assays demonstrated that silencing CYP71AN126, but not CYP76A225 and CYP76A226, significantly reduced the paeoniflorin content, indicating that C4 hydroxylation plays an important role in paeoniflorin biosynthesis, whereas C10 hydroxylation is not. Through the analysis of natural sequence and activity divergence among CYP71AN126 and CYP76A225/226, combined with protein structure prediction and site-directed mutagenesis, we identified L493 as a critical residue involved in regulating catalytic site specificity and substrate specificity of CYP71AN126. Mutation of L493 reduced or eliminated the formation of undesired C10 hydroxylation side-product and enhanced substrate specificity. These findings establish C4 oxidation of α-pinene as the critical committed step in paeoniflorin biosynthesis. Our study lays a foundation for elucidating the complete biosynthetic pathway of paeoniflorin in Paeonia and provides a target for enzyme engineering of CYP71AN126 aimed at the efficient production of paeoniflorin via synthetic biology approaches.
The past year has seen the fruits of biotechnological manipulation of forest trees approach commercial application. Advances in somatic embryogenesis have brought mass clonal propagation of the top commercial trees closer to reality, and efficient gene transfer systems have been developed for a number of conifers and hardwoods. Radical alterations in the quantity and quality of lignin in wood have been shown to be possible in softwoods and hardwoods through identification of naturally occurring mutants, as well as by engineering the lignin biosynthetic pathway with transgenes. The potential environmental and social impacts of the release of transgenic trees have become an increasingly contentious issue that will require more attention if we are to use these technologies to their full advantage.