Portable, On-Demand Biomolecular Manufacturing.

Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world.

Architecting a transcriptional repressor-based genetic inverter for tryptophan derived pathway regulation in Escherichia coli.

Efficient microbial cell factories require intricate and precise metabolic regulations for optimized production, which can be significantly aided by implementing regulatory genetic circuits with versatile functions. However, constructing functionally diverse genetic circuits in host strains is challenging. Especially, functional diversification based on transcriptional repressors has been rarely explored due to the difficulty in inverting their repression properties. To address this, we proposed a design logic to create transcriptional repressor-based genetic inverters for functional enrichment. As proof of concept, a tryptophan-inducible genetic inverter was constructed by integrating two sets of transcriptional repressors, PtrpO1-TrpR1 and PtetO1-TetR. In this genetic inverter, the repression of TetR towards PtetO1 could be alleviated by the tryptophan-TrpR1 complex in the presence of tryptophan, leading to the activated output. Subsequently, we optimized the dynamic performance of the inverter and constructed tryptophan-triggered dynamic activation systems. Further coupling of the original repression function of PtrpO1-TrpR1 with inverter variants realized the tryptophan-triggered bifunctional regulation system. Finally, the dynamic regulation systems enabled tryptophan production monitoring. These systems also remarkably increased the titers of the tryptophan derivatives tryptamine and violacein by 2.0-fold and 7.4-fold, respectively. The successful design and application of the genetic inverter enhanced the applicability of transcriptional repressors.

Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

Screening and characterization of Chromobacterium piscinae AMA-5 for enhanced production of violacein and its ability of textile dyeing.

A violet pigment (violacein) bacterial isolate AMA-5 was isolated from soil samples collected from Achanakmar Biosphere Reserve, Mungeli district, Chhattisgarh, India. The yield of biocolor from this isolate was screened in minimal medium after 48 h of incubation at 37°C ± 2°C temperature. The violet pigment was extracted in ethanol. It was also observed that ammonium chloride (2.5 g/1000 mL) as a nitrogen source is the best to enhance AMA-5 pigment production among other nitrogen sources (ammonium sulfate, tryptophan, ammonium iron sulfate, and peptone). The Sanger sequencing of 16S rDNA of strain AMA-5 showed similarity with Chromobacterium piscinae. From the available literature and research articles, it was assumed that this violet color pigment is violacein. It was further verified by conducting high-performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance (1H-NMR) analysis. The violet biocolor that extracted was used in cotton and polyester fabric dyeing. After the fabrics treated with sodium chloride as a mordant were completely dried, it was identified that the color was solidifying. Overall study showed that C. piscinae AMA-5 has good potential for production of violacein, which is the most important industrial natural dye used to add color to textile products.

Improving the medium composition for the production of the natural blue-violet pigment violacein by a new Janthinobacterium sp. isolate.

The interest in natural compounds has increased primarily due to their beneficial health and environmental aspects. However, natural sources of some compounds, such as blueish pigments, are limited, requiring the development of efficient processes to meet commercial demands. This study isolated a blue-violet bacterium from spoiled cooked rice and identified it as a potential new species of Janthinobacterium through 16S rDNA analysis. UHPLC-MS/MS analyses confirmed that the blue-violet pigment violacein was responsible for the blueish color. In laboratory conditions, different carbon and nitrogen sources were evaluated in submerged culture media to enhance pigment production. Glycerol did not result in significant pigment production by this strain, as expected from previous reports. Instead, a culture medium composed of yeast extract and fructose yielded higher pigment production, reaching about 113.68 ± 16.68 mg L-1 after 120 hours. This result provides crucial insights for future studies aiming for sustainable and commercially viable violacein production. Based on a bioeconomy concept, this approach has the potential to supply natural and economic bluish pigments for various industrial sectors, including pharmaceutical, cosmetic, and food.

Synthetic co-culture in an interconnected two-compartment bioreactor system: violacein production with recombinant E. coli strains.

The concept of modular synthetic co-cultures holds considerable potential for biomanufacturing, primarily to reduce the metabolic burden of individual strains by sharing tasks among consortium members. However, current consortia often show unilateral relationships solely, without stabilizing feedback control mechanisms, and are grown in a shared cultivation setting. Such 'one pot' approaches hardly install optimum growth and production conditions for the individual partners. Hence, novel mutualistic, self-coordinating consortia are needed that are cultured under optimal growth and production conditions for each member. The heterologous production of the antibiotic violacein (VIO) in the mutually interacting E. coli-E. coli consortium serves as an example of this new principle. Interdependencies for growth control were implemented via auxotrophies for L-tryptophan and anthranilate (ANT) that were satisfied by the respective partner. Furthermore, VIO production was installed in the ANT auxotrophic strain. VIO production, however, requires low temperatures of 20-30 °C which conflicts with the optimum growth temperature of E. coli at 37 °C. Consequently, a two-compartment, two-temperature level setup was used, retaining the mutual interaction of the cells via the filter membrane-based exchange of medium. This configuration also provided the flexibility to perform individualized batch and fed-batch strategies for each co-culture member. We achieved maximum biomass-specific productivities of around 6 mg (g h)-1 at 25 °C which holds great promise for future applications.

Improvement of violacein production using abiotic stresses and microbial adaptation.

The aim of the current research was to improve violacein production with Janthinobacterium lividum using abiotic stresses and bacterial adaptation against stress. Initially, the effect of carbon sources and the medium volume: air ratio on violacein production was assessed. Then, the production of violacein under hydrogen peroxide (H2O2) and ampicillin (Amp) stresses and acyl homoserine lactone (AHL) was evaluated. In the next step, J. lividum was adapted against increased concentrations of Amp. Finally, the production of violacein was analyzed in adapted bacterium cultivated in the presence of optimal amounts of H2O2, Amp, and AHL. The alterations in the expression of some of genes involved in violacein production was evaluated using Real-time PCR (RT-PCR). The highest amount of violacein was achieved using medium volume: air ratio of 10% v/v (in 100 ml flasks) and glycerol as carbon source. Also, H2O2 (103 mg/l) and Amp (130 mg/l) stresses increased the production of violacein significantly compared to normal conditions (57 mg/l) and violacein production in the presence of crude AHL increased from 56 mg/l to 210 mg/l. The production of violacein with adapted bacterium under the above-mentioned stresses and AHL was about 1.3 g/l. RT-PCR results showed that the expression of the AHL encoding gene (luxI) was repressed in the presence of stresses and glycerol. Also, the expression of vioA increased in the presence of Amp but H2O2 had no significant effect on vioA expression. Totally, we showed that microbial adaptation and abiotic stresses are cost-effective methods to generate significant improvement in violacein production.

Structural basis for substrate binding and catalytic mechanism of the key enzyme VioD in the violacein synthesis pathway.

Violacein is a pigment synthesized by gram-negative bacteria with various biological activities such as antimicrobial, antiviral, and anticancer activities. VioD is a key oxygenase converting protodeoxyviolaceinic acid to protoviolaceinic acid in violacein biosynthesis. To elucidate the catalytic mechanism of VioD, here, we resolved two crystal structures of VioD, a binary complex structure containing VioD and a FAD and a ternary complex structure composed of VioD, a FAD and a 2-ethyl-1-hexanol (EHN). Structural analysis revealed a deep funnel like binding pocket with wide entrance, this pocket is positively charged. The EHN is located at the deep bottom of the binding pocket near isoalloxazine ring. Further docking simulation help us to propose the mechanism of the hydroxylation of the substrate catalyzed by VioD. Bioinformatic analysis suggested and emphasized the importance of the conserved residues involved in substrate binding. Our results provide a structural basis for the catalytic mechanism of VioD.

Study on extraction and characterization of new antibiotics violacein from engineered Escherichia coli VioABCDE-SD.

To better understand the characteristic properties of violacein biosynthesized by engineered Escherichia coli VioABCDE-SD, a convenient and simplified method was designed to extract violacein and its stability, antimicrobial activity, and antioxidant capacity were analyzed. Different from the traditional extraction methods, our new method is easier and less time consuming and can directly obtain violacein dry powder product with a higher extraction rate. Low temperature, dark condition, neutral pH, reducing agents, Ba2+ , Mn2+ , Ni2+ , Co2+ , and some food additives of sucrose, xylose, and glucose were conducive to maintaining its stability. The violacein also exhibited surprisingly high bacteriostatic action against Gram-positive Bacillus subtilis, Deinococcus radiodurans R1, and Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, but no effect on E. coli. The violacein of VioABCDE-SD exhibited strong antioxidant activity, with the scavenging rate of 1,1-diphenyl-2-picrylhydrazyl free radicals reaching 60.33%, the scavenging efficiency of hydroxyl radical scavenging reaching 56.34%, and the total antioxidant capacity reaching 0.63 U/mL. Violacein from VioABCDE-SD can be synthesized directionally with better stability, antibacterial, and antioxidant properties compared with that from the original strain Janthinobacterium sp. B9-8. Therefore, our study indicated that violacein from engineered E. coli VioABCDE-SD was a kind of new antibiotic with potential biological activities, which may have potential utility in multiple areas such as pharmacological, cosmetics, and healthy food industries.

Purification of Natural Pigments Violacein and Deoxyviolacein Produced by Fermentation Using Yarrowia lipolytica.

Violacein and deoxyviolacein are bis-indole pigments synthesized by a number of microorganisms. The present study describes the biosynthesis of a mixture of violacein and deoxyviolacein using a genetically modified Y. lipolytica strain as a production chassis, the subsequent extraction of the intracellular pigments, and ultimately their purification using column chromatography. The results show that the optimal separation between the pigments occurs using an ethyl acetate/cyclohexane mixture with different ratios, first 65:35 until both pigments were clearly visible and distinguishable, then 40:60 to create a noticeable separation between them and recover the deoxyviolacein, and finally 80:20, which allows the recovery of the violacein. The purified pigments were then analyzed by thin-layer chromatography and nuclear magnetic resonance.

Violacein negatively modulates the colorectal cancer survival and epithelial-mesenchymal transition.

Violacein is a secondary metabolite produced by several microorganisms including Chromobacterium violaceum, and it is already used in food and cosmetics. However, due to its potent anticancer and low side effects, its molecular action needs to be deeply scrutinized. Therefore, the main objective of this study was to evaluate the violacein's ability to interfere with three cancer hallmarks: growth factors receptor-dependent signaling, proliferation, and epithelial-mesenchymal transition (EMT). Violacein has been associated with the induction of apoptosis in colorectal cancer (CRC) cells. Here, we demonstrate that this molecule is also active in CRC spheroids and inhibits cell migration. Violacein treatment reduced the amount of EGFR and AXL receptors in the HT29 cell line. Accordingly, the inhibition of the AKT, ERK, and PKCδ kinases, which are downstream mediators of the signaling pathways triggered by EGFR and AXL, is detected. Another interesting finding was that even when the cells were stimulated with transforming growth factor-ß, the EMT marker (N-cadherin) decreased. Therefore, this study provides further evidence that reinforces the potential of violacein as an antitumor agent, once this biomolecule can "switch off" properties associated with cancer plasticity.

Development of Methylorubrum extorquens AM1 as a promising platform strain for enhanced violacein production from co-utilization of methanol and acetate.

Violacein, a blue-violet compound with a wide range of beneficial bioactivities, is an attractive product for microbial production. Currently, violacein production has been demonstrated in several sugar heterotrophs through metabolic engineering; however, the cost of production remains an obstacle for business ventures. To address this issue, the development of host strains that can utilize inexpensive alternative substrates to reduce production costs would enable the commercialization of violacein. In this study, we engineered a facultative methylotroph, Methylorubrum extorquens AM1, to develop a methanol-based platform for violacein production. By optimizing expression vectors as well as inducer concentrations, 11.7 mg/L violacein production was first demonstrated using methanol as the sole substrate. Considering that unidentified bottlenecks for violacein biosynthesis in the shikimate pathway of M. extorquens AM1 would be difficult to address using generic metabolic engineering approaches, random mutagenesis and site-directed mutagenesis were implemented, and a 2-fold improvement in violacein production was achieved. Finally, by co-utilization of methanol and acetate, a remarkable enhancement of violacein production to 118 mg/L was achieved. Our results establish a platform strain for violacein production from non-sugar feedstocks, which may contribute to the development of an economically efficient large-scale fermentation system for violacein production.

Enhanced production of violacein by Chromobacterium violaceum using agro-industrial waste soybean meal.

AIMS: The research is aimed at developing an economic and sustainable growth medium using abundantly available and highly nutritive agro-industrial waste soybean meal as the substrate for the production of violacein by Chromobacterium violaceum. METHODS AND RESULTS: Violacein produced using soybean meal medium was compared with the commercial complex growth media. Upon utilization of 2% w/v soybean meal (SM2 ) medium, 496 mg/L crude violacein was achieved after 48-hr incubation time, which was 1.62-fold higher than the crude violacein produced in Luria-Bertani (LB) broth. Additionally, supplementation of 100 mg/L L-tryptophan to 1% and 2% w/v soybean meal (SMT1 and SMT2 ) medium yielded 1217 mg/L (3.96-fold higher as compared to LB) and 1198 mg/L (3.90-fold higher as compared to LB) crude violacein respectively. Optimization of culture conditions and concentration of L-tryptophan using Box-Behnken design (BBD) model produced as high as 1504.5 mg/L crude violacein. To the best of our knowledge, this is the highest crude violacein produced to date using agro-industrial-based waste as a substrate with minimal supplementation in a shake flask. CONCLUSIONS: The study signifies the potentiality of soybean meal as a cost-effective growth medium for the production of violacein. Optimization of the fermentation parameters clearly demonstrated a surge in violacein production. SIGNIFICANCE AND IMPACT OF THE STUDY: Utilization of soybean meal as an alternative to the expensive commercial media would surely promote the large-scale synthesis of this multifaceted compound.

Violacein switches off low molecular weight tyrosine phosphatase and rewires mitochondria in colorectal cancer cells.

In the last decade, emerging evidence has shown that low molecular weight protein tyrosine phosphatase (LMWPTP) not only contributes to the progression of cancer but is associated with prostate low survival rate and colorectal cancer metastasis. We report that LMWPTP favors the glycolytic profile in some tumors. Therefore, the focus of the present study was to identify metabolic enzymes that correlate with LMWPTP expression in patient samples. Exploratory data analysis from RNA-seq, proteomics, and histology staining, confirmed the higher expression of LMWPTP in CRC. Our descriptive statistical analyses indicate a positive expression correlation between LMWPTP and energy metabolism enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In addition, we examine the potential of violacein to reprogram energetic metabolism and LMWPTP activity. Violacein treatment induced a shift of glycolytic to oxidative metabolism associated with alteration in mitochondrial efficiency, as indicated by higher oxygen consumption rate. Particularly, violacein treated cells displayed higher proton leak and ATP-linked oxygen consumption rate (OCR) as an indicator of the OXPHOS preference. Notably, violacein is able to bind and inhibit LMWPTP. Since the LMWPTP acts as a hub of signaling pathways that offer tumor cells invasive advantages, such as survival and the ability to migrate, our findings highlight an unexplored potential of violacein in circumventing the metabolic plasticity of tumor cells.

Functional characterization of a novel violacein biosynthesis operon from Janthinobacterium sp. B9-8.

Violacein is a secondary metabolite mainly produced by Gram-negative bacteria that is formed from tryptophan by five enzymes encoded by a single operon. It is a broad-spectrum antibacterial pigment with various important biological activities such as anti-tumor, antiviral, and antioxidative effects. The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8. The key enzyme-encoding genes were cloned to construct the multigene coexpression plasmids pET-vioAB and pRSF-vioCDE. The violacein biosynthesis pathway was heterologously introduced into engineered Escherichia coli VioABCDE and VioABCDE-SD. The factors affecting violacein production, including temperature, pH, inoculum size, carbon and nitrogen source, precursor, and inducers were investigated. The violacein titer of VioABCDE-SD reached 107 mg/L in a two-stage fermentation process, representing a 454.4% increase over the original strain. The violacein operon from B9-8 provides a new microbial gene source for the analysis of the violacein synthesis mechanism, and the constructed engineering E. coli strains lay a foundation for the efficient and rapid synthesis of other natural products.Key points• The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8.• The violacein synthesis pathway was reconstructed in E. coli using two compatible plasmids.• A two-stage fermentation process was optimized for improved violacein accumulation.

Violacein and its antifungal activity: comments and potentialities.

Violacein is an important natural antimicrobial pigment that is mainly produced by Chromobacterium violaceum and Janthinobacterium lividum. It presents a significant range of effects against phytopathogenic and human fungi, besides being featured as having low toxicity, and by its important ecological role in protecting amphibian species and applications in dyed medical fabric. The hypothesis about violacein's action mechanisms against mucormycosis (Rhizopus arrhizus) and candidiasis (Candida auris) is herein discussed based on data available in the scientific literature.

Violacein-embedded nanofiber filters with antiviral and antibacterial activities.

Most respiratory masks are made of fabrics, which only capture the infectious virus carriers into the matrix. However, these contagious viruses stay active for a long duration (∼7 days) within the fabric matrix possibly inducing post-contact transmissions. Moreover, conventional masks are vulnerable to bacterial growth with prolonged exposure to exhaled breaths. Herein, we combined violacein, a naturally-occurring antimicrobial agent, with porous nanofiber membranes to develop a series of functional filters that autonomously sterilizes viruses and bacteria. The violacein-embedded membrane inactivates viruses within 4 h (99.532 % reduction for influenza and 99.999 % for human coronavirus) and bacteria within 2 h (75.5 % reduction). Besides, its nanofiber structure physically filters out the nanoscale (<0.8 µm) and micron-scale (0.8 µm - 3 µm) particulates, providing high filtration efficiencies (99.7 % and 100 % for PM 1.0 and PM 10, respectively) with long-term stability (for 25 days). In addition, violacein provides additional UV-resistant property, which protects the skin from sunlight. The violacein-embedded membrane not only proved the sterile efficacy of microbe extracted pigments for biomedical products but also provided insights to advance the personal protective equipment (PPE) to fight against contagious pathogens.

Investigation of potential inhibitor properties of violacein against HIV-1 RT and CoV-2 Spike RBD:ACE-2.

A violacein-producing bacterium was isolated from a mud sample collected near a hot spring on Kümbet Plateau in Giresun Province and named the GK strain. According to the phylogenetic tree constructed using 16S rRNA gene sequence analysis, the GK strain was identified and named Janthinobacterium sp. GK. The crude violacein pigments were separated into three different bands on a TLC sheet. Then violacein and deoxyviolacein were purified by vacuum liquid column chromatography and identified by NMR spectroscopy. According to the inhibition studies, the HIV-1 RT inhibition rate of 1 mM violacein from the GK strain was 94.28% and the CoV-2 spike RBD:ACE2 inhibition rate of 2 mM violacein was 53%. In silico studies were conducted to investigate the possible interactions between violacein and deoxyviolacein and three reference molecules with the target proteins: angiotensin-converting enzyme 2 (ACE2), HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain. Ligand violacein binds strongly to the receptor ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of -9.94 kcal/mol, -9.32 kcal/mol, and -8.27 kcal/mol, respectively. Deoxyviolacein strongly binds to the ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of -10.38 kcal/mol, -9.50 kcal/mol, and -8.06 kcal/mol, respectively. According to these data, violacein and deoxyviolacein bind to all the receptors quite effectively. SARS-CoV-2 spike protein and HIV-1-RT inhibition studies with violacein and deoxyviolacein were performed for the first time in the literature.

Molecular signatures of Janthinobacterium lividum from Trinidad support high potential for crude oil metabolism.

BACKGROUND: Janthinobacterium lividum is considered to be a psychrotrophic bacterial species. For the first time in the literature, J. lividum strains were isolated from Trinidad presenting with atypical features - hydrocarbonoclastic and able to survive in a tropical environment. METHODS: Identification of the Trinidad strains was carried out through 16S rRNA phylogenetic analysis. Gene-specific primers were designed to target the VioA which encodes violacein pigment and the EstA/B gene which encodes secreted extracellular lipase. Bioinformatics analyses were carried out on the nucleotide and amino acid sequences of VioA and EstA/B genes of the Trinidad Janthinobacterium strains to assess functionality and phylogenetic relatedness to other Janthinobacterium sequences specifically and more broadly, to other members of the Oxalobacteraceae family of betaproteobacteria. RESULTS: 16S rRNA confirmed the identity of the Trinidad strains as J. lividum and resolved three of the Trinidad strains at the intra-specific level. Typical motility patterns of this species were recorded. VioAp sequences were highly conserved, however, synonymous substitutions located outside of the critical sites for enzyme function were detected for the Trinidad strains. Comparisons with PDB 6g2p model from aa231 to aa406 further indicated no functional disruption of the VioA gene of the Trinidad strains. Phylogeny of the VioA protein sequences inferred placement of all J. lividum taxa into a highly supported species-specific clade (bs = 98%). EstA/Bp sequences were highly conserved, however, synonymous substitutions were detected that were unique to the Trinidad strains. Phylogenetic inference positioned the Trinidad consensus VioA and EstA protein sequences in a clearly distinct branch. CONCLUSIONS: The findings showed that the primary sequence of VioAp and EstA/Bp were unique to the Trinidad strains and these molecular signatures were reflected in phylogenetic inference. Our results supported chemotaxis, possible elective inactivation of VioA gene expression and secreted lipase activity as survival mechanisms of the Trinidad strains in petrogenic conditions.

Microbial synthesis of violacein pigment and its potential applications.

Violacein is a pigment synthesized by Gram-negative bacteria such as Chromobacterium violaceum. It has garnered significant interest owing to its unique physiological and biological activities along with its synergistic effects with various antibiotics. In addition to C. violaceum, several microorganisms, including: Duganella sp., Pseudoalteromonas sp., Iodobacter sp., and Massilia sp., are known to produce violacein. Along with the identification of violacein-producing strains, the genetic regulation, quorum sensing mechanism, and sequence of the vio-operon involved in the biosynthesis of violacein have been elucidated. From an engineering perspective, the heterologous production of violacein using the genetically engineered Escherichia coli or Citrobacter freundii host has also been attempted. Genetic engineering of host cells involves the heterologous expression of genes involved in the vio operon and the optimization of metabolic pathways and gene regulation. Further, the crystallography of VioD and VioE was revealed, and mass production by enzyme engineering has been accelerated. In this review, we highlight the biologically assisted end-use applications of violacein (such as functional fabric development, nanoparticles, functional polymer composites, and sunscreen ingredients) and violacein activation mechanisms, production strains, and the results of mass production with engineered methods. The prospects for violacein research and engineering applications have also been discussed.