Β-carotene Production Research Articles

Fermentative Production of β-Carotene from Sugarcane Bagasse Hydrolysate by Rhodotorula glutinis CCT-2186.

Β-Carotene is a red-orange pigment that serves as a precursor to important pharmaceutical molecules like vitamin A and retinol, making it highly significant in the industrial sector. Consequently, there is an ongoing quest for more sustainable production methods. In this study, glucose and xylose, two primary sugars derived from sugarcane bagasse (SCB), were utilized as substrates for β-carotene production by Rhodotorula glutinis CCT-2186. To achieve this, SCB underwent pretreatment using NaOH, involved different concentrations of total solids (TS) (10%, 15%, and 20%) to remove lignin. Each sample was enzymatically hydrolyzed using two substrate loadings (5% and 10%). The pretreated SCB with 10%, 15%, and 20% TS exhibited glucose hydrolysis yields (%wt) of 93.10%, 91.88%, and 90.77%, respectively. The resulting hydrolysate was employed for β-carotene production under batch fermentation. After 72h of fermentation, the SCB hydrolysate yielded a β-carotene concentration of 118.56 ± 3.01mg/L. These findings showcase the robustness of R. glutinis as a biocatalyst for converting SCB into β-carotene.

Rhodotorula paludigena CM33 cultivation process development for high β-carotene single cell protein production

Rhodotorula yeast has potential in terms of biotechnology for the production of various compounds for the environmental, industrial, and medical sectors. It is well known to generate carotenoid pigments that are beneficially distinguished. This investigation aimed to optimize the growth of Rhodotorula paludigena CM33 and its production of β-carotene. Response Surface Methodology was employed to optimize temperature, ammonium sulfate, and ferric chloride concentrations in a 5L-bioreactor batch culture. The optimal conditions of 30 °C, 6.2 g/L of ammonium sulfate, and 75.0 mg/L of ferric chloride provided biomass of 17.71 g/L, a specific growth rate of 0.22 h−1, and β-carotene concentration of 40.29 mg/L. Upon scaling up the cultivation process, the 22L-bioreactor displayed similar growth patterns to those of the 5L-bioreactor. However, the 22L-bioreactor exhibited higher product yields in a shorter time period. The maximum biomass production of 18.96 ± 1.31 g/L and the maximum β-carotene production of 42.81 ± 0.61 mg/L were achieved at 28 h. This scaling-up strategy resulted in improved production efficiency. Additionally, the nutritional value of the cells was evaluated, suggesting their potential as animal feed supplements. R. paludigena CM33 demonstrated a β-carotene single-cell protein content, making it a valuable source for aquaculture and livestock feed. These findings highlight the potential of this yeast strain for large-scale production of β-carotene and single-cell protein, offering nutritional benefits for animal feed.

Quantitative and modularized CRISPR/dCas9-dCpf1 dual function system in Saccharomyces cerevisiae.

Introduction: Both CRISPR/dCas9 and CRISPR/dCpf1 genome editing systems have shown exciting promises in modulating yeast cell metabolic pathways. However, each system has its deficiencies to overcome. In this study, to achieve a compensatory effect, we successfully constructed a dual functional CRISPR activation/inhibition (CRISPRa/i) system based on Sp-dCas9 and Fn-dCpf1 proteins, along with their corresponding complementary RNAs. Methods: We validated the high orthogonality and precise quantity targeting of selected yeast promoters. Various activating effector proteins (VP64, p65, Rta, and VP64-p65-Rta) and inhibiting effector proteins (KRAB, MeCP2, and KRAB-MeCP2), along with RNA scaffolds of MS2, PP7 and crRNA arrays were implemented in different combinations to investigate quantitative promoter strength. In the CRISPR/dCas9 system, the regulation rate ranged from 81.9% suppression to 627% activation in the mCherry gene reporter system. Studies on crRNA point mutations and crRNA arrays were conducted in the CRISPR/dCpf1 system, with the highest transcriptional inhibitory rate reaching up to 530% higher than the control. Furthermore, the orthogonal CRISPR/dCas9-dCpf1 inhibition system displayed distinct dual functions, simultaneously regulating the mCherry gene by dCas9/gRNA (54.6% efficiency) and eGFP gene by dCpf1/crRNA (62.4% efficiency) without signal crosstalk. Results and discussion: Finally, we established an engineered yeast cell factory for β-carotene production using the CRISPR/dCas9-dCpf1 bifunctional system to achieve targeted modulation of both heterologous and endogenous metabolic pathways in Saccharomyces cerevisiae. The system includes an activation module of CRISPRa/dCas9 corresponding to a gRNA-protein complex library of 136 plasmids, and an inhibition module of CRISPRi/dCpf1 corresponding to a small crRNA array library. Results show that this CRISPR/dCas9-dCpf1 bifunctional orthogonal system is more quantitatively effective and expandable for simultaneous CRISPRa/i network control compared to single-guide edition, demonstrating higher potential of future application in yeast biotechnology.

Improved production of β-carotene in light-powered Escherichia coli by co-expression of Gloeobacter rhodopsin expression

BackgroundProviding sufficient and usable energy for the cell factory has long been a heated issue in biosynthesis as solar energy has never been rooted out from the strategy for improvement, and turning Escherichia coli (E. coli) into a phototrophic host has multiple captivating qualities for biosynthesis. In this study, β-carotene was a stable compound for production in E. coli with the expression of four enzymes (CrtE, CrtB, CrtI, CrtY) for production due to its light-harvesting feature as an antenna pigment and as an antioxidant and important precursor for human health. The expression of Gloeobacter rhodopsin (GR) in microbial organisms was proved to have potential for application.ResultsThe expression of fusion protein, GR-GFP, in E. coli showed visible GFP signal under fluorescent microscopy, and its in vivo proton pumping activity signal can be detected in induced photocurrent by electrodes on the chip under intervals of illumination. To assess the phototrophic synthesis ability of the host strain compared to wild-type and vector control strain in chemostat batch with illumination, the expression of red fluorescent protein (RFP) as a target protein showed its yield improvement in protein assay and also reflected its early dominance in RFP fluorescence signal during the incubation, whereas the synthesis of β-carotene also showed yield increase by 1.36-fold and 2.32-fold compared with its wildtype and vector control strain. To investigate the effect of GR-GFP on E. coli, the growth of the host showed early rise into the exponential phase compared to the vector control strain and glucose turnover rate was elevated in increased glucose intake rate and upregulation of ATP-related genes in glycolysis (PtsG, Pgk, Pyk).ConclusionWe reported the first-time potential application of GR in the form of fusion protein GR-GFP. Expression of GR-GFP in E. coli improved the production of β-carotene and RFP. Our work provides a strain of E. coli harboring phototrophic metabolism, thus paving path to a more sustainable and scalable production of biosynthesis.

Optimized fed-batch cultivation of Rhodotorula toruloides in a bubble column bioreactor progressed the β-carotene production from corn steep liquor.

In this work, a new isolate yeast, namely Rhodotorula toruloides KP324973, was examined for β-carotene production from corn steep liquor (CSL) as a sole carbon source because CSL as the by-product of corn wet-milling process mainly enriched from the water-soluble carbohydrates. The studies were preliminary performed at the shaken flasks, and then developed at batch and fed-batch modes in a bubble column reactor (BCR). Application of the BCR improved the carotenogenesis of the cells in comparison with shaken flasks and the specific β-carotene production rate (Rp) and the yield of β-carotene production from the total reducing sugars (YP/TRS) reached 2.23 mg gcell-1 h-1 and 36.82 mg gTRS-1, respectively. Further studies were carried out to optimize the operational factors of the BCR for a fed-batch production by the response surface methodology. An optimal condition at a feed flow rate of 2.5 mL h-1, temperature 11.7°C, and initial pH of 6.1 obtained the highest Rp = 12.31 mg gcell-1 h-1 and YP/TRS = 97.18 mg gTRS-1.

A versatile regulatory toolkit of arabinose-inducible artificial transcription factors for Enterobacteriaceae

The Gram-negative bacteria Salmonella enterica and Escherichia coli are important model organisms, powerful prokaryotic expression platforms for biotechnological applications, and pathogenic strains constitute major public health threats. To facilitate new approaches for research and biotechnological applications, we here develop a set of arabinose-inducible artificial transcription factors (ATFs) using CRISPR/dCas9 and Arabidopsis-derived DNA-binding proteins to control gene expression in E. coli and Salmonella over a wide inducer concentration range. The transcriptional output of the different ATFs, in particular when expressed in Salmonella rewired for arabinose catabolism, varies over a wide spectrum (up to 35-fold gene activation). As a proof-of-concept, we use the developed ATFs to engineer a Salmonella two-input biosensor strain, SALSOR 0.2 (SALmonella biosenSOR 0.2), which detects and quantifies alkaloid drugs through a measurable fluorescent output. Moreover, we use plant-derived ATFs to regulate β-carotene biosynthesis in E. coli, resulting in ~2.1-fold higher β-carotene production compared to expression of the biosynthesis pathway using a strong constitutive promoter.

Integrated fermentative process for lipid and β-carotene production from acetogenic syngas fermentation using an engineered oleaginous Yarrowia lipolytica yeast

An engineered Yarrowia lipolytica strain was successfully employed to produce β-carotene and lipids from acetic acid, a product of syngas fermentation by Clostridium aceticum. The strain showed acetic acid tolerance up to concentrations of 20 g/L. Flask experiments yielded a peak lipid content of 33.7 % and β-carotene concentration of 13.6 mg/g under specific nutrient conditions. The study also investigated pH effects on production in bioreactors, revealing optimal lipid and β-carotene contents at pH 6.0, reaching 22.9 % and 44 mg/g, respectively. Lipid profiles were consistent across experiments, with C18:1 being the dominant compound at approximately 50 %. This research underscores a green revolution in bioprocessing, showing how biocatalysts can convert syngas, a potentially polluting byproduct, into valuable β-carotene and lipids with a Y. lipolytica strain.

Coproduction of Phase-Separated Carotenoids and β-Farnesene as a Yeast Biomass Valorization Strategy.

Valorization, the process whereby waste materials are converted into more valuable products, is rarely practiced in industrial fermentation. We developed a model valorization system whereby Saccharomyces cerevisiae that had previously been engineered to produce high concentrations (>100 g/L) of extracellular β-farnesene was further engineered to simultaneously produce intracellular carotenoids, both products being isoprenoids. Thus, a single fermentation generates two valuable products, namely, β-farnesene in the liquid phase and carotenoids in the solid biomass phase. Initial attempts to produce high levels of canthaxanthin (a ketocarotenoid used extensively in animal feed) in a β-farnesene production strain negatively impacted both biomass growth and β-farnesene production. A refined approach used a promoter titration strategy to reduce β-carotene production to a level that had minimal impact on growth and β-farnesene production in fed-batch fermentations and then engineered the resulting strain to produce canthaxanthin. Further optimization of canthaxanthin coproduction used a bioprospecting approach to identify ketolase enzymes that maximized conversion of β-carotene to canthaxanthin. Finally, we demonstrated that β-carotene is not present in the extracellular β-farnesene at a significant concentration and that which is present can be removed by a simple distillation, indicating that β-farnesene (the primary fermentation product) purity is unaffected by coproduction of carotenoids.

Comparative metabolomics analysis of the fermented plant-based meat analogue of soybean proteins

For the past few years, the research on plant-based meat analogue (PBMA) has gained increased attention, and the metabolites in the PBMA playing a crucial role in determining the quality of the products. In this study, PBMA was fermented using a complex strain starter comprising Rhodotorula mucilaginosa, Monascus purpureus and Lactiplantibacillus plantarum at an optimized ratio of 5%, 1.5% and 2%, respectively. By using metabolomics based on LC-MS/MS, the metabolites were investigated during their mixed fermentation, and significant metabolites related to the meaty flavor and color were detected in different strain-starter treated between a and d groups, such as the upregulation Glutathione and Methyl (indol-3-yl) acetate that produce meaty flavors in the fermentation process. Meat-like pigments, especially β-carotene accumulated significantly after fermentation with the addition of three strain starters at a ratio of R. mucilaginosa 5%, M. purpureus 1.5% and L. plantarum 2%, providing PBMA with a good meat-like color stimulation. This study suggests that soybean protein accumulates meaty flavor with acid and ester metabolites, and color stimulation through metabolic modulation by β-carotene production during the yeasts and Lactobacillus fermentation. In conclusion, this study provides a fermentation approach for producing PBMA with meat-like flavor and color.

Metabolic Engineering of Yarrowia lipolytica for Zeaxanthin Production.

Zeaxanthin is a carotenoid, a dihydroxy derivative of β-carotene. Zeaxanthin has antioxidant, anti-inflammatory, anticancer, and neuroprotective properties. In this study, Yarrowia lipolytica was used as a host for the efficient production of zeaxanthin. The strain Y. lipolytica PO1h was used to construct the following engineered strains for carotenoid production since it produced the highest β-carotene among the Y. lipolytica PO1h- and Y. lipolytica PEX17-HA-derived strains. By regulating the key nodes on the carotenoid pathway through wild and mutant enzyme comparison and successive modular assembly, the β-carotene concentration was improved from 19.9 to 422.0 mg/L. To provide more precursor mevalonate, heterologous genes mvaE and mvaSMT were introduced to increase the production of β-carotene by 27.2% to the yield of 536.8 mg/L. The β-carotene hydroxylase gene crtZ was then transferred, resulting in a yield of zeaxanthin of 326.5 mg/L. The oxidoreductase RFNR1 and CrtZ were then used to further enhance zeaxanthin production, and the yield of zeaxanthin was up to 775.3 mg/L in YPD shake flask.

Enhancement of β-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene β-cyclase

β-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of β-carotene and α-carotene. Aiming to develop the microalgae with enhanced β-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of β-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant β-carotene), confirming the superior activity of CrtY on β-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased β-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae.

Concomitant Production of Erythritol and β-Carotene by Engineered Yarrowia lipolytica.

While the expansion of the erythritol production industry has resulted in unprecedented production of yeast cells, it also suffers from a lack of effective utilization. β-Carotene is a value-added compound that can be synthesized by engineered Yarrowia lipolytica. Here, we first evaluated the production performance of erythritol-producing yeast strains under two different morphologies and then successfully constructed a chassis with yeast-like morphology by deleting Mhy1 and Cla4 genes. Subsequently, β-carotene synthesis pathway genes, CarRA and CarB from Blakeslea trispora, were introduced to construct the β-carotene and erythritol coproducing Y. lipolytica strain ylmcc. The rate-limiting genes GGS1 and tHMG1 were overexpressed to increase the β-carotene yield by 45.32-fold compared with the strain ylmcc. However, increased β-carotene accumulation led to prolonged fermentation time; therefore, transporter engineering through overexpression of YTH1 and YTH3 genes was used to alleviate fermentation delays. Using batch fermentation in a 3 L bioreactor, this engineered Y. lipolytica strain produced erythritol with production, yield, and productivity values of 171 g/L, 0.56 g/g glucose, and 2.38 g/(L·h), respectively, with a concomitant β-carotene yield of 47.36 ± 0.06 mg/g DCW. The approach presented here improves the value of erythritol-producing cells and offers a low-cost technique to obtain hydrophobic terpenoids.

Overexpression of S-R enhances the accumulation of biomass, fatty acids, and β-carotene in Schizochytrium

Strategies for enhancing biomass accumulation and increasing the production of fatty acids and β-carotene in Schizochytrium are hindered by the lack of suitable targets. In this study, S-R, a RING (really interesting new gene) finger domain-containing protein, was identified in Schizochytrium, with homologs found in the family Thraustochytriaceae. Transgenic strains overexpressing S-R showed a minor improvement in cell growth but a significant increase in total fatty acids content by 1.29- to 1.36-fold. Almost all individual saturated fatty acids exhibited significant increases, with the greatest increase observed in the C14:0 content, by 1.52- to 1.78-fold. Additionally, the β-carotene content of S-R strains was significantly upregulated. Overexpression of s-r conferred hypersaline tolerance in Schizochytrium, with a significant increase in dry cell weight, total fatty acids and β-carotene, likely due to the upregulation of glycerol and proline. This study provides a feasible strategy to engineer Thraustochytriaceae for efficient biomass and biochemical production.

Utilization of olive mill wastewater for selective production of lipids and carotenoids by Rhodotorula glutinis.

Olive mill wastewater (OMW) is a zero-cost substrate for numerous value-added compounds. Although several studies on the production of lipids and carotenoids by Rhodotorula glutinis in OMW exist, none of them has specifically focused on the conditions for a target lipid or carotenoid. This study presents cultivation conditions that selectively stimulate the cell biomass, individual carotenoids and lipids. It was found that supplemental carbon and nitrogen sources as well as illumination affected cell biomass the most. High temperature, low initial pH, illumination, lack of urea and presence of glycerol stimulated the lipid synthesis. The highest total lipid content obtained in undiluted OMW supplemented with urea was 11.08 ± 0.17% (w/w) whilst it was 41.40 ± 0.21% (w/w) when supplemented with glycerol. Moreover, the main fatty acid produced by R. glutinis in all media was oleic acid, whose fraction reached 63.94 ± 0.58%. Total carotenoid yield was significantly increased with low initial pH, high temperature, illumination, certain amounts of urea, glycerol and cultivation time. Up to 192.09 ± 0.16μg/g cell carotenoid yield was achieved. Torularhodin could be selectively produced at high pH, low temperature and with urea and glycerol supplementation. To selectively induce torulene synthesis, cultivation conditions should have low pH, high temperature and illumination. In addition, low pH, high temperature and urea supplementation served high production of β-carotene. Up to 85.40 ± 0.76, 80.67 ± 1.40 and 39.45 ± 0.69% of torulene, torularhodin and β-carotene, respectively, were obtained under selected conditions. KEY POINTS: • Cultivation conditions selectively induced target carotenoids and lipids • 41.40 ± 0.21% (w/w) lipid content and 192.09 ± 0.16μg/g cell carotenoid yield were achieved • Markedly high selectivity values for torularhodin and torulene were achieved.

Utilizing microalgal hydrolysate from dairy wastewater-grown Chlorella sorokiniana SU-1 as sustainable feedstock for polyhydroxybutyrate and β-carotene production by engineered Rhodotorula glutinis #100-29

The objective of this study was to explore the potential of utilizing Chlorella sorokiniana SU-1 biomass grown on dairy wastewater-amended medium as sustainable feedstock for the biosynthesis of β-carotene and polyhydroxybutyrate (PHB) by Rhodotorula glutinis #100-29. To break down the rigid cell wall, 100 g/L of microalgal biomass was treated with 3% sulfuric acid, followed by detoxification using 5% activated carbon to remove the hydroxymethylfurfural inhibitor. The detoxified microalgal hydrolysate (DMH) was used for flask-scale fermentation, which yielded a maximum biomass production of 9.22 g/L, with PHB and β-carotene concentration of 897 mg/L and 93.62 mg/L, respectively. Upon scaling up to a 5-L fermenter, the biomass concentration increased to 11.2 g/L, while the PHB and β-carotene concentrations rose to 1830 mg/L and 134.2 mg/L. These outcomes indicate that DMH holds promise as sustainable feedstock for the production of PHB and β-carotene by yeast.

Economic–Financial Assessment of Seawater Desalination Plants in Northern Chile to Reduce Hydric Scarcity and a Proposal for the Environmental and Sustainable Use of Brine Waste by Cultivating the Microalga Dunaliella salina to Produce β-Carotene

Climate change and global warming generate serious consequences and disturbances by drastically modifying historical temperature and precipitation patterns. Water scarcity is one of the most revealing phenomena of these instabilities. This transdisciplinary bibliometric and economic–financial research focuses on analyzing two aspects: first, the feasibility of implementing seawater desalination plants as a solution to water scarcity in northern Chile. Investment and amortization costs of the desalination plants were determined (NPV-IRR-IRP). NPV showed a positive value indicating a recovery of the initial investment and a surplus over profitability. The IRR was higher than the discount rate calculated for NPV, which showed that the investment project was accepted. The IRP indicated that the initial investment of the plant would be recovered in 3.7 years. Second, an innovative and environmentally sustainable solution to the brine (NaCl) waste generated by desalination plants is proposed through the cultivation of Dunaliella salina microalgae tolerant to high brine concentrations to produce β-carotene. The analyzed desalination plants and the sustainable use of brine residues offer interesting economic perspectives to a 10-year projection establishing a surplus over profitability. The SWOT analysis estimates an excellent production of β-carotene through the microalgae and alternatives to the problem of sea pollution by concentrated brine waste.

Engineering an Efficient Expression Using Heterologous GAL Promoters and Transcriptional Activators in Saccharomyces cerevisiae.

Galactose-inducible (GAL) promoters have been widely used in metabolic engineering in Saccharomyces cerevisiae for production of valuable products. Endogenous GAL promoters and GAL transcription factors have often been engineered to improve GAL promoter activities. Heterologous GAL promoters and GAL activator (Gal4p-like transcriptional activators), although existing in other yeasts or fungi, have not been well explored. In this study, we comprehensively characterized the activation effects of Gal4p activators from different yeasts or fungi on a variant of GAL promoters. Overexpressing endogenous Gal4p driven by PHHF1 increased the activities of native PGAL1 and heterologous PSkGAL2 by 131.20% and 72.45%, respectively. Furthermore, eight transcriptional activators from different organisms were characterized and most of them exhibited functions that were consistent with ScGal4p. Expression of KlLac9p from Kluyveromyces lactis further increased the activity of PScGAL1 and PSkGAL2 by 41.56% and 100.63%, respectively, compared to ScGal4p expression, and was able to evade Gal80p inhibition. This optimized GAL expression system can be used to increase the production of β-carotene by 9.02-fold in S. cerevisiae. Our study demonstrated that a combination of heterologous transcriptional activators and GAL promoters provided novel insights into the optimization of the GAL expression system.

Strategies for the Microbial Carotenoids Production Competitiveness Improvement

The research and development of carotenoids production have a long history, and interest in this group of pigments has not decreased to this day. Among all existing carotenoids, six are considered industrially important: astaxanthin, β-carotene, lutein, zeaxanthin, canthaxanthin, and lycopene. These carotenoids have a wide range of application and are used as additives in food and beverage, feed, nutraceuticals, pharmaceuticals, and cosmetics due to their bioactive and colour properties. An undisputed leader in the global pigment market is chemically synthesized carotenoids. To a lesser extent, carotenoids derived from natural sources as plants and microorganisms. Currently, the market of natural carotenoids is mainly represented by microalgae Haematococcus pluvialis, Dunaliella salina, Botryococcus braunii, fungus Blakeslea trispora, yeast Phaffia rhodozyma and bacteria Paracoccus carotinifaciens. These microorganisms afford the production of astaxanthin, βcarotene, canthaxanthin, and lycopene. In turn, lutein is obtained by extracting marigold flowers Tagetes eracta L. and there is no other competitive source yet. Therefore, the potential of microorganisms to synthesize and accumulate lutein and other equally important carotenoids in their cells has been actively studied. Several yeast and bacteria species from Rhodosporidium, Rhodotorula, Sporobolomyces, Sphingomonas, Gordonia, and Sphingobacterium genus have a potential to replenish the diversity of sources of industrially important natural pigments, but available technologies still need improving. This paper reviews strategies for increasing of competitiveness of fungal and bacterial carotenoids production. Strategies such as selection of carotenogenic strain, use of low-cost substrates, simultaneous production of carotenoids and other value-added compounds, and optimization of fermentation medium and conditions are considered.

Screening of Structurally Distinct Lycopene β-Cyclases for Production of the Cyclic C40 Carotenoids β-Carotene and Astaxanthin by Corynebacterium glutamicum

Lycopene β-cyclase (EC 5.5.1.19) is one of the key enzymes in the biosynthesis of β-carotene and derived carotenoids. It catalyzes isomerase reactions to form β-carotene from lycopene by β-cyclization of both of its ψ-ends. Lycopene β-cyclases are widespread in nature. We systematically analyzed the phylogeny of lycopene β-cyclases from all kingdoms of life and predicted their transmembrane structures. To this end, a collection of previously characterized lycopene β-cyclase polypeptide sequences served as bait sequences to identify their closest homologues in a range of bacteria, archaea, fungi, algae, and plant species. Furthermore, a DeepTMHMM scan was applied to search for the presence of transmembrane domains. A phylogenetic tree suggests at least five distinct clades, and the DeepTMHMM scan revealed that lycopene β-cyclases are a group of structurally different proteins: membrane-bound and cytosolic enzymes. Representative lycopene β-cyclases were screened in the lycopene-overproducing Corynebacterium glutamicum strain for β-carotene and astaxanthin production. This systematic screening facilitates the identification of new enzymes for carotenoid production. Higher astaxanthin production and less reduction of total carotenoids were achieved with the cytosolic lycopene β-cyclase CrtL from Synechococcus elongatus and the membrane-bound heterodimeric lycopene β-cyclase CrtYcd from Brevibacterium linens.

Production of bioactive β-carotene by the endophytic bacterium Citricoccus parietis AUCs with multiple in vitro biological potentials

BackgroundAlthough microalgae and plants are traditionally used for obtaining natural pigments, overexploitation and overharvesting threaten them. Bacteria represent a superior alternative for the production of pigments due to their ability to produce greater amounts in a short time without seasonal restrictions; furthermore, bacterial pigments have a wide range of uses and are safe and biodegradable. This study is the first on the production of ß-carotene as a promising bioactive agent from endophytic bacteria.ResultsThe yellow pigment produced by the endophytic bacterium Citricoccus parietis AUCs (NCBI accession number: OQ448507.1) was extracted by methanol and then purified and identified. One band was obtained by TLC analysis, which was identified as ß-carotene based on its spectroscopic and chromatographic characteristics. The pigment exhibited remarkable antibacterial, antioxidant and antidiabetic activities.ConclusionsThis research may serve as a valuable starting point for exploiting C. parietis AUCs as a potent source of ß-carotene for biomedical therapies. To validate the findings of this research, in vivo studies must be performed.