Spectral characteristics and fluorescence of lycopene from a purple-colored Microbacterium albipurpureum ET2.

Cas13-based RNA effectors may enable dynamic, multiplexed and reversible gene regulation in bacteria. Yet, their widespread adoption is hindered by inherent cytotoxicity and collateral cleavage. Here we present a rational protein engineering strategy to generate attenuated Cas13d variants with tunable RNase activity through targeted truncation of flexible regions. This permits effective transcript knockdown while greatly reducing toxicity as reflected by a 2.2-fold higher growth optical density. By introducing proximal mismatches at the 5' end of CRISPR RNA spacers, our system allows functional switching between translation inhibition, polycistronic mRNA degradation and IF3-fusion-based translation-level CRISPR activation. We demonstrate programmable, orthogonal and multiplexed regulation of individual genes within polycistronic mRNAs and synthetic circuits. Application to lycopene biosynthesis optimization shows robust pathway rewiring and improved yields alongside fine-tuned modulation of essential and competing pathways in Escherichia coli. Our work provides a versatile RNA-regulatory toolkit for next-generation microbial synthetic biology and RNA-based biotechnology.

Fruit coloration is a key determinant of tomato quality, yet how light and temperature interact to regulate pigmentation during ripening remains unclear. Using a semi-in-fruit experimental system, we demonstrate that while high light accelerates chlorophyll degradation and lycopene accumulation at 25 °C, supra-optimal temperature (40 °C) completely abolishes lycopene biosynthesis irrespective of light conditions, primarily through transcriptional suppression of SlPSY1 and SlGGPS2. Elevated postharvest temperatures (≥30 °C) not only change the carotenoid composition but also reduce the antioxidant capacity and vitamin C content in fruit. Temperature-switch experiments revealed a critical developmental window, days 2–4 after ethylene treatment, during which temperature exerts dominant control over carotenoid metabolism. Exposure to high temperature within this window irreversibly shifts pigment accumulation from lycopene to yellow/orange carotenoids. These findings identify a temporally precise regulatory nexus integrating environmental signals with the ripening program, offering a framework for targeted temperature management to optimize tomato color and nutritional quality.

In synthetic metabolic pathways, the intracellular level of enzymes is a critical determinant of pathway efficiency and, thus, short-lived enzymes create bottlenecks and limit overall metabolic productivity due to their low abundance. However, since studies on protein half-life remain limited in bacteria, its accurate prediction is a significant challenge. To address this, we developed a machine learning model, ProHL, for the classification of short-lived and long-lived proteins. ProHL employs a multimodal strategy, integrating ProteinBERT encodings (at both residue and sequence levels) with physicochemical encodings of the protein sequences. This integration enables the effective capture of both local and global sequence features, thereby ensuring accurate half-life classification. When evaluated on an independent test dataset of E. coli proteins, ProHL achieved an accuracy of 0.818 and a Matthew's correlation coefficient of 0.624. To demonstrate its practical utility in metabolic engineering, we classified CrtE, CrtB, and CrtI enzymes involved in lycopene biosynthesis and identified that only CrtB as short-lived. Consistent with this prediction, when CrtB was additionally expressed in a lycopene-producing base strain, lycopene production in E. coli increased up to 25%. Our computational framework, ProHL, identifies short-lived, rate-limiting enzymes by employing in silico prediction of enzyme half-life. This approach provides a viable strategy for alleviating metabolic bottlenecks, ultimately enhancing metabolic productivity.

Microbial electrosynthesis (MES) systems aim to use electroactive microorganisms (EAMs) to achieve electricity-driven CO2 fixation for biosynthesis of multicarbon chemicals. However, the low efficiencies of extracellular electron transfer (EET) and CO2 assimilation of EAMs remain the essential limiting factors that restrict performance of MES systems. Herein, we developed an electrosynthetic biohybrid system to synergistically supply electrons and CO2 to Rhodopseudomonas palustris (an EAM) for lycopene biosynthesis. Intracellular carbon and energy fluxes were redirected by strengthening the lycopene biosynthesis pathway and blocking the nitrogen-fixation pathway, resulting in 23-fold increase in lycopene yield than that of the wild-type R. palustris. To enhance extracellular transfer of CO2 and electrons to R. palustris, metal-organic frameworks (MOFs) with high CO2 adsorption capacity were assembled with polydopamine on cell membrane to construct a biohybrid MES system, which produced 3.55mg/L lycopene in two consecutive MES cycles, the highest lycopene production from CO2. Electrochemical and transcriptomic analyses revealed that the biohybrid MES system stimulated microbial metabolism including EET, the Calvin-Benson-Bassham cycle and lycopene biosynthesis, thereby improving CO2-to-chemical conversion. This study demonstrated directional supply of electrons and CO2 to EAMs enabled high-performance MES systems, which also offered insights into the mechanisms underlying efficient CO2 fixation and carbon-negative biomanufacturing.

ABSTRACT Microbial electrosynthesis (MES) systems aim to use electroactive microorganisms (EAMs) to achieve electricity‐driven CO 2 fixation for biosynthesis of multicarbon chemicals. However, the low efficiencies of extracellular electron transfer (EET) and CO 2 assimilation of EAMs remain the essential limiting factors that restrict performance of MES systems. Herein, we developed an electrosynthetic biohybrid system to synergistically supply electrons and CO 2 to Rhodopseudomonas palustris (an EAM) for lycopene biosynthesis. Intracellular carbon and energy fluxes were redirected by strengthening the lycopene biosynthesis pathway and blocking the nitrogen‐fixation pathway, resulting in 23‐fold increase in lycopene yield than that of the wild‐type R. palustris . To enhance extracellular transfer of CO 2 and electrons to R. palustris , metal‐organic frameworks (MOFs) with high CO 2 adsorption capacity were assembled with polydopamine on cell membrane to construct a biohybrid MES system, which produced 3.55 mg/L lycopene in two consecutive MES cycles, the highest lycopene production from CO 2 . Electrochemical and transcriptomic analyses revealed that the biohybrid MES system stimulated microbial metabolism including EET, the Calvin‐Benson‐Bassham cycle and lycopene biosynthesis, thereby improving CO 2 ‐to‐chemical conversion. This study demonstrated directional supply of electrons and CO 2 to EAMs enabled high‐performance MES systems, which also offered insights into the mechanisms underlying efficient CO 2 fixation and carbon‐negative biomanufacturing.

Jasmonates influence carotenoids biosynthesis, the pigments responsible for tomato fruit coloration, but their effect on carotenoids synthesis remains controversial. Lycopene is the predominant carotenoid in ripe tomato fruits, accounting for more than 90% of the total carotenoid content in the fruit. Our study clarified this paradox by demonstrating that methyl jasmonate (MeJA) affects post-harvest tomato lycopene accumulation differently depending on light conditions and we identified SlPIF1a as the central regulatory factor mediating this light-JA crosstalk. In light, MeJA enhances lycopene synthesis by directly activating the expression of the SlPSY1 gene through the SlMYC2 transcription factor. SlMYC2 also inhibits the expression of SlPIF1a, encoding a negative regulator of light signal that degrades in light and accumulates in darkness. In dark conditions, the accumulated SlPIF1a interacts with SlMYC2 inhibiting its activation on SlPSY1 expression. Additionally, the MeJA-induced acetyltransferase SlNATA1 interacts with and acetylates SlPIF1a, enhancing its repression on SlPSY1. Our research uncovers a new mechanism for the dual regulation of lycopene synthesis by jasmonic acid under different light conditions.

Engineering microbial cell factories for the production of lycopene: Advances and perspectives.

Microbial cell factories represent the primary approach for heterologous lycopene synthesis, where gene source selection and pathway regulation have been demonstrated to have a significant impact on lycopene titer. In this study, key lycopene biosynthesis genes (crtE, crtB and crtI) derived from the extremophile Deinococcus wulumuqiensis R12 were introduced into Escherichia coli, generating the chassis strain H0. Fermentation optimization revealed sodium pyruvate significantly enhanced lycopene production and cell growth. Quantitative polymerase chain reaction (qPCR) analysis revealed that sodium pyruvate upregulated the expression of dxr, ispA, crtE, crtB and crtI genes, while downregulating the expression of dxs and idi genes. Consequently, different sources of dxs, dxr, idi and ispA genes were screened and co-expressed to reinforce the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in E. coli. The optimized combination of dxs from E. coli MG1655 with idi from D. wulumuqiensis R12 achieved maximal lycopene titer of 293.70 mg/L (112.49 mg/g DCW), which was 33.88-fold higher than that of the initial strain H0. This study offers genetic resources for heterologous carotenoid synthesis and establishes a reference framework for the synthesis of analogous complex isoprenoid metabolites.

Construction of a CRISPR-Cas6-mediated lycopene synthase assembly regulation method

Tomato (Solanum lycopersicum L.) is rich in the antioxidant lycopene, which often degrades postharvest. Pulsed light shows promise in preserving lycopene, yet its molecular mechanisms remain unclear. This study integrates transcriptomics, proteomics, and metabolomics to elucidate how pulsed light affects lycopene synthesis in tomatoes. The results showed that lycopene content increased significantly in pulsed light-treated tomatoes. Transcriptomic analysis identified 1092 significantly differentially expressed genes (DEGs), proteomic analysis identified 1046 significantly differentially accumulated proteins (DAPs), and metabolomic analysis identified 272 significantly differentially accumulated metabolites (DEMs). These were significantly enriched in pathways such as terpenoid backbone biosynthesis, carotenoid biosynthesis, the tricarboxylic acid cycle (TCA), and photosynthesis. The upregulation of eight key genes central to lycopene biosynthesis was validated by qRT-PCR, confirming their involvement in the observed accumulation. Integrated multi-omics analysis revealed coordinated regulation of photosynthesis, carbohydrate metabolism, and terpenoid synthesis, highlighting the reprogramming of energy metabolism and secondary metabolite synthesis in lycopene accumulation. This study provides a comprehensive understanding of the molecular mechanisms by which pulsed light enhances lycopene content in tomatoes. The findings suggest that pulsed light treatment activates key metabolic pathways, leading to increased lycopene synthesis. This research offers a theoretical basis for optimizing pulsed light technology and developing new preservation strategies to maintain and enhance the nutritional quality of tomatoes during postharvest storage.

Development of a highly efficient p-coumaric acid-responsive biosensor in Saccharomyces cerevisiae.

Lycopene, a natural carotenoid with antioxidant and health-promoting properties, has attracted attention as a valuable compound for the food, pharmaceutical, and cosmetic industries. Conventional production methods based on plant extraction or chemical synthesis are limited by low yields, high costs, and environmental concerns. In this study, the oleaginous yeast Yarrowia lipolytica was engineered as an alternative microbial cell factory for sustainable lycopene biosynthesis using short-chain fatty acids (SCFAs)—such as acetate, butyrate, and propionate—as inexpensive, renewable carbon sources. Four heterologous genes from Pantoea agglomerans (crtI, crtB, crtE, and idi) were codon-optimized and integrated into the Y. lipolytica genome using different expression systems, including the Golden Gate Assembly strategy. Among the tested strains, PS05/4lyc/GGA, characterized by enhanced phospholipid biosynthesis, demonstrated the highest lycopene yield of 462.9 mg/g dry cell weight and a titer of 3.41 g/L on butyrate medium—values comparable to or exceeding those reported for bioreactor-scale fermentations. The results indicate that co-activation of phospholipid and carotenoid biosynthesis pathways creates favorable intracellular conditions for hydrophobic pigment accumulation. Moreover, the use of SCFAs improved acetyl-CoA availability and redirected carbon flux through the mevalonate pathway, enhancing productivity. Strains with elevated membrane lipid biosynthesis also exhibited higher metabolic stability and stress tolerance.

Escherichia coli Nissle 1917(EcN), a non-pathogenic probiotic strain isolated from the human gut, exhibits intrinsic intestinal colonization, tumor-targeting capability, and proven biosafety, which make it an ideal live delivery system. This study engineered EcN to achieve dual functionality in intestinal colonization and controlled release of active compounds. Starting with wild-type EcN, this study used lycopene biosynthesis as a proof-of-concept. First, lycopene biosynthetic genes under arabinose-inducible promoters were integrated into the EcN genome via CRISPR/Cas9, which generated the base strain EcN-C1. Subsequent overexpression of rate-limiting genes in the endogenous methylerythritol phosphate(MEP) pathway: including isopentenyl diphosphate isomerase(idi), 1-deoxy-D-xylulose-5-phosphate synthase(dxs), and farnesyl diphosphate synthase(ispA): enhanced lycopene production by 4.28-fold. Further integration of a heterologous mevalonate(MVA) pathway augmented precursor supply, resulting in a further 2.58-fold increase in yield, elevating titer to(15.24±0.87)mg·L~(-1)(11.04-fold over initial strain). Dose-and time-dependent arabinose induction enabled precise control of lycopene release across engineered strains: EcN-C1 to EcN-C4 exhibited titers spanning 0.02-1.38, 0.22-2.81, 0.67-5.90, and 1.24-15.24 mg·L~(-1), respectively. This system achieved an 800-fold dynamic range(0.02-15.24 mg·L~(-1)), demonstrating fine-tuned control over compound delivery. This work lays an important foundation for the development of novel delivery systems based on probiotics for active compound release.

Bacillus subtilis a Generally Recognized As Safe (GRAS) microorganism, is an attractive chassis for producing high-value compounds in a safe and sustainable way. However, its potential for producing the C40 carotenoid lycopene has been limited by inefficient precursor supply and enzyme incompatibility. This study demonstrates that lycopene production in B. subtilis can be significantly enhanced through systematic metabolic engineering by rewiring the lycopene and methylerythritol phosphate (MEP) pathways. A synthetic lycopene biosynthesis pathway expressing the crtE gene from Pantoea agglomerans, which is commonly used for microbial lycopene production, failed to yield lycopene production in B. subtilis. However, replacing crtE with a multifunctional geranylgeranyl diphosphate synthase (GGPPS) from Archaeoglobus fulgidus successfully enabled lycopene synthesis. The optimization of the fermentation medium demonstrated that a combined carbon supply of glucose and glycerol markedly enhanced both cell growth and lycopene production in comparison with separate carbon sources. To further boost production, the methylerythritol phosphate (MEP) pathway was engineered by overexpressing the rate-limiting enzyme, 1-deoxy-D-xylulose-5-phosphate synthase (dxs), which resulted in a five-fold increase in lycopene titer after 72 h. Screening of various GGPPS enzymes revealed that idsA from Corynebacterium glutamicum was the most efficient, further increasing the yield. The final engineered strain achieved a lycopene titer of 55 mg/L in shake-flask cultivation, a significant improvement over the previously reported level in B. subtilis. These results demonstrate that targeted GGPPS selection and precursor pathway engineering are critical strategies for developing B. subtilis into a robust and sustainable platform for carotenoid production.

Efficient multi-gene expression in Escherichia coli is critical for advancing metabolic engineering and synthetic biology. However, existing strategies for combinatorial optimization remain labor-intensive and low-throughput. In addressing this challenge, a high-throughput platform was developed, encompassing the engineering of standardized genetic elements (promoters and 5' UTRs) with fluorescent reporters (e.g. eGFP, mCherry, TagBFP) to quantify expression variability. Libraries of single-, dual-, and tri-gene (dual-plasmid) constructs were assembled via Golden Gate, validated by IPTG induction, and applied to lycopene biosynthesis by replacing fluorescent genes with crtE, crtI, and crtB using Gibson assembly. The optimized tri-gene library was used to generate E. coli BL21(DE3) strains exhibiting variable levels of lycopene production, thereby demonstrating the platform's capacity to balance multi-gene pathways. Subsequent quantitative analysis by qPCR confirmed the uniformity of promoter-UTR combinations across the plasmid library. This modular platform, featuring reusable libraries and a dual-plasmid system, enables rapid exploration of multi-gene expression landscapes, offering a scalable tool for metabolic engineering and multi-enzyme co-expression.

The production of β-alanine from fatty acid feedstocks presents a promising synthetic strategy due to its high carbon yield. However, the excessive reducing power generated during fatty acid utilization disrupts cellular redox balance, adversely affecting metabolism and limiting the efficiency and final yield of β-alanine production. To address this challenge, we engineered a co-production system in which excess reducing equivalents generated during fatty acid β-oxidation and β-alanine biosynthesis were consumed by growth-coupled lycopene biosynthesis. The resulting dual-pathway strain, SA01, achieved 44.78 g/L β-alanine and 3.07 g/L lycopene in bioreactor fermentation, representing a 21.45% increase in β-alanine production compared to the β-alanine-producing strain WA01, and a 74.43% increase in lycopene production compared to the lycopene-producing strain LA01. Further optimization in strain SA06, involving cofactor engineering to shift redox flow from NADH to NADPH, enhanced the titers to 52.78 g/L β-alanine and 3.61 g/L lycopene. Metabolite analysis confirmed a decrease in intracellular NADH and FADH2 levels in SA06, indicating restoration of redox balance during the late fermentation phase. Additional improvements in the fermentation process, including gradual carbon source switching, optimization of the induction strategy, and fine-tuning of conditions during both growth and bioconversion phases, resulted in further increases in product titers, reaching 72 g/L β-alanine and 6.15 g/L lycopene. This study offers valuable insights into the development of microbial co-production systems, highlighting the critical role of dynamic cofactor and redox balance management, as well as process optimization, in improving production efficiency.

Lycopene is one of the key carotenoids in tomatoes (Lycopersicon esculentum Mill.). It is known for its strong antioxidant activity and its role in preventing cardiovascular diseases, cancer, and other chronic conditions. A complex interplay of genetic factors, agronomic practices, and environmental conditions determines its accumulation in tomato fruits. Breeding for high-lycopene tomato varieties is actively advancing through traditional breeding methods and modern molecular markers, which facilitate the identification of promising genotypes and accelerate the breeding process. Beyond genetic traits, external factors significantly impact lycopene synthesis. Temperature regimes, solar radiation levels, photoperiod, and temperature fluctuations on the fruit surface can all influence its accumulation. Agronomic practices, such as growth regulators, organic amendments, and potassium fertilizers, also contribute to increased lycopene content. Potassium enhances carbon transport into the fruits, while organic fertilizers stimulate the enzymatic activity of the carotenoid biosynthesis pathway. Growth regulators can activate gene expression related to lycopene accumulation, offering opportunities for targeted control of its levels. This review uniquely integrates insights from molecular genetics, environmental factors, and agronomic strategies to comprehensively understand lycopene biosynthesis in tomatoes. Systematically connecting molecular mechanisms with practical cultivation approaches addresses a significant gap in the existing literature. Additionally, the ripening stage and storage conditions further affect lycopene content. Thus, an integrated approach- combining molecular marker-assisted breeding, optimized agronomic techniques, and environmental factor management- can significantly enhance lycopene concentration in tomatoes, improving their nutritional value and functional properties. The findings presented offer actionable guidance for future breeding programs and cultivation practices to produce functionally enriched tomato varieties for the health-oriented food market. Keywords: lycopene, tomato, genetic factors, environmental factors, potassium fertilizers, organic fertilizers

Constructions of self-assembled protein nanocages for enzyme immobilization and cargo transport are very promising in biotechnology fields such as natural product biosynthesis. Here, we present an engineered isopentenyl pyrophosphate (IPP) synthetic nanocage with multiple enzymes for lycopene production in bacteria. The enzymes involved in IPP biosynthesis (ScCK, AtIPK, and MxanIDI) were assembled onto the exterior of an engineered protein cage based on α-carboxysome. The IPP synthetic nanocage was co-expressed with CrtE/CrtB/CrtI in Escherichia coli. This approach increased the metabolic flux and resulted in a 1.7-fold increase in lycopene production in the engineered E. coli compared with the control strain. The results provide insights into the immobilization and assembling of IPP biosynthetic enzymes in protein nanocages, which serve as a powerful tool for achieving efficient synthesis of lycopene.

Lycopene is a natural carotenoid with antioxidation properties. The objective of the present study was to investigate the roles of glutamate and proline in lycopene biosynthesis in a newly isolated purple non-sulfur bacterium Cereibacter sphaeroides DT.1, under aerobic conditions. This strain contained a distinct CrtI4 capable of converting phytoene to lycopene via four-step desaturation. In order to enhance lycopene production, a crtC knockout mutant was constructed via homologous recombination. Supplementation with glutamate or proline to fermentative medium significantly enhanced intracellular lycopene accumulation in wildtype strain by a respective 99.40 ± 0.54% and 101.70 ± 0.49% and in a ΔcrtC mutant strain by 38.13 ± 0.15% and 39.83 ± 0.27%, respectively. Differential transcriptomic and metabolomic analyses showed that these promoting effects were associated with downregulation of the expression of the acyclic carotenoid 1,2-hydratase gene, and increased accumulation of lycopene precursors such as pyruvate and acetyl-CoA. The fermentation conditions for lycopene production were optimized through shake flask experiments. Feasibility for lycopene production was confirmed in a fed-batch cultivation process and a high yield of 151.10 ± 0.13 mg/L was achieved. This ΔcrtC mutant strain exhibited advantages, such as relatively lower oxygen demand and no need for illumination, making it a potentially useful strain for lycopene production under aerobic conditions.