Metabolic Engineering of Microorganisms to Increase Production of Violacein

— Violacein, an indole derivative, is a violent pigment which is extracted from the bacteria. It is considered to be an important aromatic compound as it exhibits essential antiparasitic, antimicrobial and antitumoral characteristics. One of the most eminent derivatives that is being induced by the biosynthetic pathway of violacein is deoxyviolacein. However, it is produced in an insignificant amount. By expunging the VioD protein from the violacein pathway, deoxyviolacein can be generated which is devoid of a hydroxyl group. Another derivative that was produced in addition to deoxyviolacein is oxyviolacein, generated by the derivative of tryptophan i.e. 5-hydroxytryptophan. In this review, our main focus is on different engineered microorganisms in increasing the production of the violacein. On undergoing genetic analysis and determining the basic mechanism of violacein production showed that, violacein is formed by the condensation of 2 tryptophan molecules in presence of vioABCD gene cluster. However, later on, the presence of another gene vioE was revealed to be involved in violacein biosynthesis and a new pathway was suggested. McClean reported the involvement of quorum sensing mechanism via AHL’s in violacein biosynthesis. Then using the above information and using violacein gene cluster vioABCDE, the violacein was produced in C. violaceum, Pseudoalteromonas sp. 520P1, V. natriegens, C. glutamicum, E. coli, Y. lipolytica and D. violaceinigra. Then the amount of violacein was increased by subjecting it to either batch or fed-batch fermentation. Then after its production, its anti-microbial activity was determined against Staphylococcus species. Also, its anti-cancerous activity was also determined on resistant leukemia cells.


I. INTRODUCTION
Metabolic engineering refers to the optimization of genetic and regulatory processes that occurs within the cell in order to increase the production of a specific substance in the cells. It includes series of biochemical reactions together with the enzymes to convert the raw materials into that specific substance and increase its amount. Metabolic engineering specifically deals with creating a mathematical model of these pathways, calculating its yield and blocking the path that will constrain the production of our desired substance (Yang, Bennett, & San, 1998). In this review, our main focus is on different engineered microorganisms in increasing the production of the violacein.

OXYVIOLACEIN AND DEOXYVIOLACEIN
Despite grabbing most of the attention, violacein is not only the substance produced within the bacterial hosts by proteins i.e. VioA, VioB, VioC, VioD and VioE. One of the most eminent derivatives that is being induced by the biosynthetic pathway of violacein is deoxyviolacein. However, deoxyviolacein, in comparison to violacein, is produced in an insignificant amount in Janthinobacterium lividum (Rodrigues et al., 2012).

Fig.1: Chemical structures of a) deoxyviolacein and b) violacein
Compatible results were also depicted in research with Duganella sp. B2 where also deoxyviolacein was produced in low concentration as compared to violacein which was evaluated by HPLC . Crude violacein extracts that are procured from the natural bacterial strains contain around ~ 10-20% of deoxyviolacein with 85% mostly violacein on the basis of HPLC evaluation.
By expunging the VioD protein from the violacein pathway, deoxyviolacein can be generated which is devoid of a hydroxyl group. A recent study by Xing group divulged the elevated production and characterization of deoxyviolacein using the chimeric Citrobacter freundii having a plasmid with VioD gene knocked out of it. Their research disclosed that deoxyviolacein showed comparatively better photostability as compared to the violacein under tests with either UV or natural light. However, both were proved to be toxic when are exposed to the 24h toxicity tests with HepG2 cell lines. However, deoxyviolacein impact was dose-independent as compared to violacein i.e. dose-dependent. The variance between violacein and deoxyviolacein was more conspicuous when the viability of HepG2 cell lines was determined after 48h . Another derivative that was produced in addition to deoxyviolacein is oxyviolacein, generated by the derivative of tryptophan i.e. 5-hydroxytryptophan (Sánchez, Braña, Méndez, & Salas, 2006

GENETIC ANALYSIS AND THE BASIC MECHANISM OF VIOLACEIN BIOSYNTHESIS
An intermediate of glycolysis i.e. phosphoenolpyruvate (PEP) and an intermediate of Hexose monophosphate pathway (HMP) i.e. erythrose-4-phosphate (E4P) together initiates the biosynthesis of the aromatic amino acids that leads through various steps to produce chorismate that lead to a branch that commences with anthranilate that finally leads to tryptophan pathway. The defective mutants in the HMP pathway can produce an enhanced mount of E4P that act as a limiting substance in the biosynthesis of violacein (Ikeda & Katsumata, 1999 Previous studies on the violacein biosynthetic pathway claimed 5-hydroxy-L-tryptophan to be the precursor in violacein synthesis (Hoshino & Ogasawara, 1990

QUORUM SENSING MODULATION OF SECONDARY METABOLITES
Quorum sensing processes via autoinducer molecules that the bacteria secrete modulates the production of violacein in C. violaceum. Quorum sensing is a mechanism that bacteria used in order to communicate with each other by releasing signaling substances (Waters & Bassler, 2005) (Williams, Winzer, Chan, & Camara, 2007). This quorum sensing mechanism regulates the production of many secondary metabolites like pigments, toxins, antibiotics, bioluminescence and biofilm formation. It was suggested that the cumulated AHL forms a complex by binding with receptor proteins which then activate the expression of gene cluster by interacting with transcriptional regulator site of violacein operon, which eventually leads to the synthesis of violacein. This alleged transcriptional promoter site is present in the intervening area between gene vioA and its upstream adjoining protein gene.

VIOLACEIN PRODUCTION AND REGULATION IN PSEUDOALTEROMONAS SP. 520P1
Zhang conducted an experiment to demonstrate the production and regulation of violacein by Pseudoalteromonas sp. 520P1 under quorum sensing systems via AHLs. This 520P1 strain is a gram-negative bacteria which was sequestered from the seawater of Cap Muroto in Japan (Yada et al., 2008). However, this strain showed violacein production under static culture circumstances only. However, previous studies showed that the 520P1 strain can produce violacein under the regulation of the quorum sensing process through AHLs (Y. Wang et al., 2008). In this research, they characterized and cloned the gene cluster of violacein and its upstream region using a fosmid library. It was used to create stable libraries using complex genomes (Huang et al., 2009) and sequestering genes (Schloss et al., 2010) from the genomic DNA. It contained ~ 13,000 clones developed from the 520P1 strain's genomic DNA. 5 clones were isolated containing violacein gene clusters. Thus, 5 ORF's cluster i.e. vioABCDE of 7383 total lengths for the biosynthesis of violacein was obtained. A highly conserved sequence was determined in 520P1 strain at ~ 200bp upstream of gene cluster having promoter sequences i.e. -10 and -35 box. ~700bp downstream and ~1500bp upstream are sequences that encodes for 2 putative proteins (figure: 8).

FEEDSTOCK FLEXIBILITY AND HETEROLOGOUS PRODUCTION OF VIOLACEIN BIOSYNTHESIS
V. natriegens having plasmid (pVio) was then tested in both minimal media and LBv2 rich media consisting of various carbon sources required for the biosynthesis of violacein and deoxyviolacein (by-product). UV-HPLC analysis showed that in rich media, V. natriegens produced 13.1 ± 0.9 mg/l and 24.9 ± 3.1 mg/l of violacein and deoxyviolacein respectively. It was observed that a higher amount of violacein is produced in mannitol followed by glucose, fructose and N-acetyl-glucosamine when absorbance is determined at OD600. A similar amount of violacein is produced in both LBv2 rich media and minimal media with 15.5 mg/l mannitol. However, the deoxyviolacein to violacein ratio was very interesting. Rich media produced 1.9X more deoxyviolacein than violacein but, more violacein is produced as compared to deoxyviolacein i.e. by 4.2X in minimal media with different carbon sources. Another significant finding was the exclusion of minimal media + arabinose for violacein biosynthesis because the findings will most likely to be confuted due to induced arabinose synthesis by phagemid-elements present in pVio plasmid. Violacein production by transformant containing pVio plasmid is comparatively less as compared to other carbon sources due to the synthesis of these proteins or lysis of V. natriengens due to activation of prophages (Ellis et al., 2019).

VIOLACEIN HYPER-PRODUCTION FROM ENGINEERED CORYNEBACTERIUM GLUTAMICUM
Because of the numerous advantages of Corynebacterium glutamicum as a microbial cell factory, it is basically identified as safes as compared to E. coli. C. glutamicum has dominated the fermentation processes of industrialscale to synthesize various amino acids and other products for food, animal feed, cosmetics and health (Pühler, Kalinowski, & Tauch, 2008

VIA GLUCOSE ENGINEERED WITH INTERACTIVE CONTROL TRYPTOPHAN AND VIOLACEIN SYNTHETIC PATHWAY
Fang and his co-workers conducted an experiment to engineer E. coli to increase the production of crude violacein by controlling trp and violacein synthetic pathways via glucose. Firstly, strains were generated that have a multivariate module for differed throughputs of trp. This was achieved by overexpression of 2 vital genes from the upstream trp metabolic pathway i.e. trpE fbr /trpD genes along with conjugational knockout of 3 genes i.e. pheA/tnaA/trpR genes ( fig. 12).

Fig.12: Plasmid construction and trp accumulation gene knockdown
To achieve this, trp metabolic pathway was thoroughly studied in C. glutamicum  pathway. There is also the presence of trp branches and phenylalanine which force chorismate to produce tyrosine and phenylalanine. Trp repression, attenuation and feedback inhibition controlled the flow of trp metabolic pathway (Ikeda, 2006  After the generation of trp biosynthetic pathway, the violacein synthetic gene cluster was instigated downstream. Maximum production of crude violacein directly via glucose was achieved in a cultural flask with a titer of 0.6 ± 0.01 g/l in E. coli B2/pED + pVio that was 3.98X more than control B1/pVio devoid of trp pathway upregulation i.e. 0.153 ± 0.005 g/l after collaborating these 2 pathways. The highest crude violacein productivity i.e. 36 mg/h/l and titer 1.75 g/l was evinced by recombinant E. coli B2/pED + pVio, which was 4.48X more than C. freundii (pCom10vio) (Xiao et al., 2011) when subjected to the same cultural conditions devoid of the addition of trp, using C-source glucose (glucose yield = 0.116 g-violacein/g-glucose and glucose consumption= 15.1 g/l) in a 5L bioreactor batch fermentation (Fang et al., 2015).

VIA GLUCOSE BY OVEREXPRESSING RATE-LIMITING VITAL ENZYMES
For the production of crude violacein using the economic industrial source of carbon, Zhou with his co-workers initiated the biosynthetic pathway of violacein in E. coli strain B8/pTRPH1, on which work had done in the previous study as described above to engineer this strain metabolically for trp accumulation via glucose. By using glucose as a carbon source in a medium, they were able to produce a higher amount of crude violacein of capacity 0.25 g/l/OD600. They disclosed VioE enzyme to be the rate-limiting step biosynthesis of violacein by further over-expression of all of the 5 genes i.e. vioABCDE that were associated with the biosynthetic pathways of violacein. In a 5L bioreactor undergoing fed-batch fermentation, the crude violacein productivity 98.7 mg/l/h along with the titer of 4.45 g/l was produced using the optimal E. coli strain i.e. B8/pTRPH1-pVio-VioE. It was revealed that this strain had produced the highest amount of crude violacein productivity and titer so far (Zhou, Fang, Li, Zhang, & Xing, 2018).

VIOLACEIN BIOSYNTHESIS BY ENGINEERING OLEAGINOUS YEAST YARROWIA LIPOLYTICA
In the food industry, Y. lipolytica, as a host, is extensively espoused for the production of β-ionone (Czajka et al., 2018), β-carotenoids (Larroude et al., 2018) (Gao et al., 2017) and citric acid (Fickers et al., 2005). It was found that both C. violacein and Y. lipolytica were collected from the marine surroundings that had high GC contents i.e. about 65%. Scientists argue that due to the GRAS status of the Y. lipolytica, it can provide a novel platform for the biosynthesis of violacein. In this study, the extraction of the violacein from yeast culture was optimized in order to improve the purity and recovery ratio of violacein from the culture by keeping in consideration incubation time, using cell wall degrading enzymes as the cell wall of Y. lipolytica is composed of thick polymer i.e. chitin having galactose and mannose (Liu, Ding, Sun, Boussetta, & Vorobiev, 2016), mechanical shear stress choice i.e. using glass beads and vortex (Jones et al., 2015) and organic solvent's variations i.e. methanol or ethyl acetate (Xu, Rizzoni, Sul, & Stephanopoulos, 2017). The quantitative relation between the microplate reader method and HPLC proved to be equivalent to measuring the production of violacein from the yeast culture. By using the extraction protocol, maximum production of violacein and deoxyviolacein was obtained in shake flasks i.e. 70.04 mg/l and 5.28 mg/l respectively. At 60 C/N ration with the incorporation of 10g/l of CaCO3 in order to optimize the pH of the media, the purity of violacein reached 86.92% (Tong, Zhou, Zhang, & Xu, 2019).

VIOLACEIN BIOSYNTHESIS BY DUGANELLA VIOLACEINIGRA
In this study, Choi and his collaborators isolated a violacein biosynthetic new strain that was identified to be the relative of D. violaceinigra YIM 31327 on the basis of a phylogenetic analysis by using FAME (fatty acid methyl ester) analysis, vioA and gyrB gene sequences and 16S rRNA sequencing. Along with its isolation and identification, within the last few years, cloning of vioABCDE genes and its heterogeneous expression and fermentation for the biosynthesis of violacein had been reported (Rodrigues et al., 2012) (Rodrigues et al., 2013). This newly isolated strain had been identified as D. violaceinigra NI28 strain. Though the phylogenetic analysis showed similarity between these 2 strains, N128 strain showed a different phenotype than YIM 31327 strain, as it was able to grow 25% faster than YIM 31327 on nutrient media and was able to produce 45X more violacein at a higher rate (Li et al., 2004) ( Similarly, in another study conducted by Dodou et al, the anti-microbial activity of violacein was determined on S. epidermidis and its symbiotic impact on the antibiotics. Excellent anti-microbial effect of violacein was revealed on both non-biofilm and biofilm-forming strains of S. epidermis i.e. ATCC 12228 and ATCC 35984 respectively. In short time period of exposure, both the bactericidal (for both strains, MBC = 20 μg/ml) and bacteriostatic effects (MIC = 10 μg/ml for ATCC 12228 and MIC = 20 μg/ml for ATCC 35984) were observed. After exposure to 2-3h, the bactericidal concentration of violacein led to the death of S. epidermidis. In addition, the violacein symbiotically optimized the action of various antimicrobial types on S. epidermidis strain ATCC 35984 (545%; n = 6) and strain ATCC 12228 (818%; n = 9), thus decreases the MIC up to 16X of these respective antibiotics (Dodou et al., 2017).

ANTI-CANCEROUS ACTIVITY INDUCED BY VIOLACEIN IN THE RESISTANT LEUKEMIA CELLS
Generally, it is recognized that the cancerous processes are intimately linked with the various modes of PCD (programmed cell death). But the problem is that there is not well-known PCD mechanism that is involved in the chemoprevention of cancer and it can differ between types of tumor cells involved and kinds of chemopreventive agents. Thus, according to pharmacologists, it is quite vital to describe the candidate's cellular specificity along with its bypass dysfunctional tumoral signaling pathway's capability to provide insensitivity to the death stimulus during the initial steps of drug development. While studying the violacein's cytotoxic effect, it was disclosed that the death that was induced in the leukemia progenitor cells i.e. CD34 + /c-Kit + /P-glycoprotein + /MRP1 + TF1 was not mediated by autophagy or apoptosis, as this compound did not significantly affect the biomarkers of both kinds of cell death. Working mechanisms of violacein were clarified by performing kinome profiling that used peptide arrays that determined the elaborated descriptions of activities of the cellular kinase. Activation of PDK, PKA and AKT along with the inhibition of DAPK1 and calpain carried out the pro-death activity of violacein that is accompanied the structural changes that are caused by Golgi apparatus collapse and ER stress, that lead to cellular quietus. The results declared that kinome reprogramming was induced by violacein that overcame death signaling affliction of tolerant leukemia cells (Queiroz et al., 2012).

II. CONCLUSION
This review is basically based on the study of the production of violacein and its derivatives from metabolically engineered microorganisms. Violacein being a secondary metabolite is found to have a high range of biological activities like anti-microbial activities and anti-cancerous activities. Due to these biological activities, scientists have developed an increased emphasis to study this compound and increasing the production via both wild-type strains and recombinant microbial strains. As discussed in this review, the production of violacein and its characterization can't be achieved without its struggles and obstacles and there is still more work that can be done. This, in particular, is based on the mode of action of violacein that needs to be studied in more in detail. The current tendency in the molecular genetic field has basically helped the researchers to genetically engineered the bacterial host that can undergo the overproduction of the violacein within the fermentation. A supplementary scheme in order to enhance the production abilities of genetically engineered strain should be kept in consideration including all the aspects of gene expression, optimization of bioprocessing and downstream processing of violacein and its derivative. Generally, bioprocess optimization for heterologous product formation entails the incrementation of metabolic abilities of engineered hot for the desired compound or product. The engineered hot of violacein and deoxyviolacein possess a high flux trp pathway enciphered in their genome that acts as a strong initiating point to obtain various high-value trp-bed therapeutic. In the future, violacein and its derivative will become readily accessible for clinical studies and the scientific community.