crtI Genes Research Articles

Enhanced torularhodin production in Rhodosporidium toruloides A1-15 under salt stress: Insights from multi-omics analysis

The carotenoid-producing strain Rhodosporidium toruloides NP11 and its mutant strain A1-15 were cultivated under salt stress using chemostat cultivation. A multi-omics approach revealed distinct mechanisms between NP11 and A1-15 under stress conditions. Salt stress significantly reduced carotenoid production in NP11 but increased it in A1-15, with torularhodin being the predominant carotenoid in A1-15. NP11 exhibited enhanced lipid synthesis under salt stress, suggesting this as its primary adaptive response. In A1-15, reactive oxygen species (ROS) pressure upregulated the CRTI and CRTY genes, with a more pronounced upregulation of CRTI, likely contributing to increased torularhodin production. Additionally, upregulation of the COX7A gene in both strains may support energy demands and mitigate oxidative stress. Notably, this study is the first to mention and explore COX7A in research related to R. toruloides.

Gene editing technology combined with response surface optimization to improve the synthesis ability of lycopene in Pantoea dispersa MSC14.

The aim of this study is to engineer Pantoea dispersa MSC14 into a strain capable of producing lycopene and to enhance its lycopene content. Our laboratory isolated the strain P. dispersa MSC14 from petroleum-contaminated soil in a mining area. Whole-genome sequencing confirmed the existence of a carotenoid synthesis pathway in this strain. This study employed an optimized CRISPR/Cas9 system to perform a traceless gene knockout of the lycopene cyclase gene crtY and to overexpress the octahydrolycopene dehydrogenase gene crtI in the P. dispersa MSC14. This strategic genetic modification successfully constructed the lycopene-producing strain MSC14-LY, which exhibited a notable lycopene content with a biomass productivity of 553μg of lycopene per gram dry cell weight (DCW). Additionally, the components of the lycopene fermentation medium were optimized using Plackett-Burman design and response surface methodology. The average lycopene content was increased to 5.13mg g -1 DCW in the optimized LY fermentation medium. Through genetic engineering, P. dispersa MSC14 was transformed into a strain capable of producing lycopene, achieving a yield of 5.13mg g-1 DCW after medium optimization. Genetic engineering successfully transformed P. dispersa MSC14 into a strain capable of producing lycopene, achieving a yield of 5.13mg g-1 DCW after medium optimization.

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.

Extended one generation reproductive toxicity study and effect on gut flora of genetically modified rice rich in β-carotene in wistar rats

To evaluate the reproductive toxicity of gene modified rice generated by introducing phytoene synthase (Psy) and bacterial phytoene desaturase (CrtI) from maize and Erwinia uredovora, Wistar rats were allocated into 3 groups and fed with Psy and CrtI gene modified rice mixture diet (GM group), non-gene modified rice mixture diet (non-GM group), and AIN-93 diet (Blank control group) from parental generation (F0) to the offsprings (F1). GM rice, Heijinmi (HJM) and Non-GM rice, Heishuai (HS), were both formulated into diets at ratios of 73.5% and 75.5% according to the AIN93 diet for rodent animals, respectively. Relative to the non-GM group, no biologically relevant differences were observed in GM group rats concerning reproductive performance such as fertility rate, gestation rate, mean duration, hormone level, and reproductive organ pathology. The developmental parameters results were not significantly different from the non-GM group such as body weight, food consumption, developmental neurotoxicity, behavior, hematology, and serum chemistry. In terms of immunotoxicity, the IgG indicators of offspring from the GM group improved in contrast with the non-GM group. Additional gut flora analysis of F0 generation rats resulted as that the treatment elicited an increased gut microflora diversity of F0 rats. And no horizontal gene transfer of Psy and CrtI genes in rats fed a GM rice HJM diet. In conclusion, we found no adverse effects related to GM rice in the extended one-generation reproductive toxicity study, indicating that GM rice is a safe alternative for its counterpart rice regarding reproductive toxicity.

Evaluation of Various Escherichia coli Strains for Enhanced Lycopene Production.

Lycopene is a carotenoid widely used as a food and feed supplement due to its antioxidant, anti-inflammatory, and anti-cancer functions. Various metabolic engineering strategies have been implemented for high lycopene production in Escherichia coli, and for this purpose it was essential to select and develop an E. coli strain with the highest potency. In this study, we evaluated 16 E. coli strains to determine the best lycopene production host by introducing a lycopene biosynthetic pathway (crtE, crtB, and crtI genes cloned from Deinococcus wulumuqiensis R12 and dxs, dxr, ispA, and idi genes cloned from E. coli). The 16 lycopene strain titers diverged from 0 to 0.141 g/l, with MG1655 demonstrating the highest titer (0.141 g/l), while the SURE and W strains expressed the lowest (0 g/l) in an LB medium. When a 2 × YTg medium replaced the MG1655 culture medium, the titer further escalated to 1.595 g/l. These results substantiate that strain selection is vital in metabolic engineering, and further, that MG1655 is a potent host for producing lycopene and other carotenoids with the same lycopene biosynthetic pathway.

Multi-omics analysis reveals the mechanism of torularhodin accumulation in the mutant Rhodosporidium toruloides A1-15 under nitrogen-limited conditions.

A carotenoid production strain Rhodosporidium toruloides NP11 and its mutant strain R. toruloides A1-15 were studied under chemostat nitrogen-limited cultivation. Multi-omics analysis (metabolomics, lipidomics and transcriptomics) was used to investigate the different mechanisms of torularhodin accumulation between NP11 and A1-15. The results showed that the carotenoid synthesis pathway was significantly enhanced in A1-15 compared to NP11 under nitrogen limitation, due to the significant increase of torularhodin. Under nitrogen-limited conditions, higher levels of β-oxidation were present in A1-15 compared to those in NP11, which provided sufficient precursors for carotenoid synthesis. In addition, ROS stress accelerated the intracellular transport of iron ions, promoted the expression of CRTI and CRTY genes, and reduced the transcript levels of FNTB1 and FNTB2 in the bypass pathway, and these factors may be responsible for the regulation of high torularhodin production in A1-15. This study provided insights into the selective production of torularhodin.

GATA transcription factor WC2 regulates the biosynthesis of astaxanthin in yeast Xanthophyllomyces dendrorhous.

Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one‐hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two‐hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild‐type strain did not largely modify the carotenoid profile, indicating translational and/or post‐translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild‐type strain and the XdWC2‐disrupted strain apparently improved the production of monocyclic carotenoid 3‐hydroxy‐3′, 4′‐didehydro‐β, ψ‐carotene‐4‐one (HDCO) rather than β‐carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over‐producing strains.

Gene repression using synthetic small regulatory RNA in Methylorubrum extorquens.

Genetic tools are a prerequisite for engineering cell factories for synthetic biology and biotechnology. Methylorubrum extorquens is an important platform for a future one-carbon (C1) bioeconomy, but its application is currently limited by the availability of genetic tools. Small regulatory RNA (sRNA) is an important regulatory factor in bacteria and has been applied for gene repression in several strains. This study aimed to construct a synthetic sRNA system based on the MicC scaffold and the chaperone Hfq to control gene expression in M. extorquens. Initially, the exogenous lacZ gene was transposed into the M. extorquens chromosome as a reporter, and corresponding β-galactosidase was measured to assess the knockdown efficiency of lacZ. A synthetic sRNA containing a 24-nt antisense RNA targeting lacZ and an Escherichia coli MicC scaffold were constructed, and different Hfqs from E. coli, M. extorquens AM1 and PA1 were further identified. The results showed that the expression of endogenous hfqs from the chromosome in M. extorquens strains was inadequate, and only when it was overexpressed via the plasmid did the colonies show a colour change and a corresponding decrease in β-galactosidase expression. More specifically, M. extorquens strains with overexpressing their own Hfq showed the best gene repression efficiency. Furthermore, this E. coli MicC scaffold and AM1 Hfq system were combined to knock down crtI gene expression in AM1, leading to an 86% decrease in carotenoid production (0·09 mg g-1 ) compared to that (0·65 mg g-1 ) in the wild-type strain. A functional synthetic sRNA system combined with E. coli MicC and endogenous Hfq was constructed in M. extorquens strains, which was able to interfere with the target crtI gene and reduce carotenoid production. The synthetic sRNA system reported in this study provides a genetic tool for the manipulation of M. extorquens. The present findings might be helpful for achieving high-throughput gene knockdown expression.

Subchronic Oral Toxicity Study of Genetically Modified Rice Rich in β-Carotene in Wistar Rats.

(1) Background: a hybrid black rice rich in β-carotene carrying the psy and crtI genes (HJM) was evaluated in Wistar rats by a 90-day feeding study, aiming to assess its dietary safety. (2) Methods: the HJM rice and its parental line HS were included in rats’ diets at levels of 73.5% and 75.5%, respectively. The AIN-93 diet was administered as a nutritional control. No adverse effects on animal behavior or weight gain were observed during the study. Blood samples were collected and analyzed, and standard hematological and biochemical parameters were compared. (3) Results: Some parameters were found to be significantly different, though they remained within the normal range for rats of this breed and age. In addition, upon sacrifice, various organs were weighed, and macroscopic and histopathological examinations were performed, with only minor changes to report. (4) Conclusions: HJM rice exhibited no adverse or toxic effects in Wistar rats in this 90-day study.

Carotenoid biosynthesis and the evolution of carotenogenesis genes in rust fungi

Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl–geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the “scabrum” rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi.

Identification and Characterization of Carotenoids, Vitamin E and Minerals of Biofortified Sorghum

Abstract Objectives Sorghum is a critical staple crop in Sub-Saharan Africa and has been included in biofortification efforts to enhance shortfall micronutrient content including provitamin A carotenoids (pVA), zinc and iron. The purpose of this study was to evaluate progress in enhancement of pVA, tocochromanol (vitamin E), iron and zinc content as well as improvements in stability from a new generation of transgenic biofortified sorghum events in order to prioritize selection for translation to food systems. Methods Transgenic sorghum events (n = 16) from three different genotypic backgrounds developed to increase provitamin A biosynthesis (PSY1 + CRTI genes, +/− CRTB gene) and increase tocochromanol accumulation (HGGT gene) for vitamin A stability were characterized for carotenoid and tocochromanol profile using liquid chromatography coupled with diode array detection. Events were compared against their respective null segregants and wild type (WT) sorghum. Mineral content of sorghum lines and those developed to increase iron and zinc accumulation (YSL2 + NAS2 genes) was assessed by inductively coupled plasma - optical emission spectrometry. Storage stability of carotenoids and tocochromanols was assessed under controlled conditions (25°C; 57%RH) over 3 months. Results Total pVA content from transgenic sorghum (5.9 ± 0.1 – 28.6 ± 0.6 mg/gFW) was significantly different (P < 0.05) from null (0.4 ± 0 – 1.2 ± 0 mg/gFW) or WT (0.73 ± 0.2 mg/gFW). A general increase in total tocochromanol accumulation was observed in transgenic sorghum events (14.9 ± 0.8 – 36.2 ± 1.3 mg/gFW) compared to null/WT (14.2 ± 0.5 – 32.4 ± 4.4 mg/gFW) with significant differences observed among transgenic events across different sorghum genotypes (P < 0.05). Mineral accumulation varied among sorghum events with levels ranging from 28.44 ± 1.9 – 48.85 ± 4.9 mg/g and 27.65 ± 4.5 – 63.59 ± 13.4 mg/g FW for zinc and iron respectively. Highest level of zinc and iron was observed in Tx430 sorghum event. Conclusions Findings from this study highlight progress in pVA levels of transgenic biofortified sorghum and the potential for increased vitamin E levels to improve pVA stability over storage. Studies are underway to characterize the bioaccessibility of pVA carotenoids, Fe and Zn from these events. Funding Sources Pioneer Foundation.

Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.

The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.

Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides.

The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) has been explored as a promising host for the production of lipids and carotenoids. However, the rational manipulation of this yeast remains difficult due to lack of efficient genetic tools. Here, the development of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas9) system for genome editing in R. toruloides is described. First, R. toruloides strains are generated with sufficient production of the Cas9 protein of Staphylococcus aureus origin by integrating a cassette containing a codon-optimized Cas9 gene into the genome. In parallel, two U6 genes are identified, predicting two U6 promoters and confirming better transcription of single-guide RNA (sgRNA) with the U6b promoter. Next, sgRNA cassettes are designed targeting CRTI, CAR2, and CLYBL gene, respectively, transforming into those Cas9-expressed strains, and finding over 60% transformants with successful insertion and deletion (indel) mutations. Furthermore, when the sgRNA cassette includes donor DNA flanked by two homologous arms of the gene CRTI, gene knockout occurs via homologous recombination. Thus, the CRISPR/Cas9 system is now established as a powerful genome-editing tool in R. toruloides, which should facilitate functional genomic study and advanced cell factory development.

The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae.

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

Metabolic Engineering of Deinococcus radiodurans for the Production of Phytoene.

A metabolically-engineered Deinococcus radiodurans R1 strain capable of producing phytoene, a colorless C40 carotenoid and a promising antioxidant, has been developed. To make this base strain, first, the crtI gene encoding phytoene desaturase was deleted to block the conversion of phytoene to other carotenoids such as lycopene and γ-carotene. This engineered strain produced 0.413 ± 0.023 mg/l of phytoene from 10 g/l of fructose. Further enhanced production of phytoene up to 4.46 ± 0.19 mg/l was achieved by overexpressing the crtB gene encoding phytoene synthase and the dxs genes encoding 1-deoxy-D-xylulose-5-phosphate synthase gene, and by deleting the crtD gene. High cell-density culture of our final engineered strain allowed production of 10.3 ± 0.85 mg/l of phytoene with the yield and productivity of 1.04 ± 0.05 mg/g and 0.143 ± 0.012 mg/l/h, respectively, from 10 g/l of fructose. Furthermore, the antioxidant potential of phytoene produced by the final engineered strain was confirmed by in vitro DPPH radical-scavenging assay.

Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production.

Lycopene is a red carotenoid pigment with strong antioxidant activity. Saccharomyces cerevisiae is considered a promising host to produce lycopene, but lycopene toxicity is one of the limiting factors for high-level production. In this study, we used heterologous lycopene biosynthesis genes crtE and crtI from Xanthophyllomyces dendrorhous and crtB from Pantoea agglomerans for lycopene production in S. cerevisiae. The crtE, crtB, and crtI genes were integrated into the genome of S. cerevisiae CEN.PK2-1C strain, while deleting DPP1 and LPP1 genes to inhibit a competing pathway producing farnesol. Lycopene production was further improved by inhibiting ergosterol production via downregulation of ERG9 expression and by deleting ROX1 or MOT3 genes encoding transcriptional repressors for mevalonate and sterol biosynthetic pathways. To further increase lycopene production, CrtE and CrtB mutants with improved activities were isolated by directed evolution, and subsequently, the mutated genes were randomly integrated into the engineered lycopene-producing strains via delta-integration. To relieve lycopene toxicity by increasing unsaturated fatty acid content in cell membranes, the OLE1 gene encoding stearoyl-CoA 9-desaturase was overexpressed. In combination with the overexpression of STB5 gene encoding a transcription factor involved in NADPH production, the final strain produced up to 41.8mg/gDCW of lycopene, which is approximately 74.6-fold higher than that produced in the initial strain.

Agroinfiltration of Phytoene Desaturase and Lycopene Β-Cyclase Genes from Bacterial Source in Tomato (Solanum Lycopersicum L.) Enhances Nutritional and Processing Quality of its Juice

ABSTRACTThere has been considerable interest in the value of carotenoids due to their high nutritional and nutraceutical properties. We have earlier shown that agroinfiltration with Agrobacterium tumifaciens GV 3101 containing binary vector pRI 101(I) harboring CrtI gene (Phytoene desaturase) and binary vector pRI 101(Y) harboring CrtY gene (Lycopene β-cyclase) from bacterial sources increases carotenoids in tomatoes. In the present study, the juice prepared from fruits having CrtY gene had higher content of β-carotene (0.60 mg 100 mL−1), lycopene (0.40 mg 100 mL−1) and total phenolics (11.41 μg GAE mL−1) compared to juice prepared from control fruits (0.47 mg 100 mL−1, 0.32 mg 100 mL−1 and 11.22 μg GAE mL−1, respectively), and it showed better antioxidant potential (DPPH IC50 value of 27.6) than that of non-transgenic fruit juice (56.4 μL mL−1). The juice from fruits having CrtY gene also had lower Bostwick value (7.5 cm min−1) than control fruits (9.8 cm min−1), indicating improvement in processing quality because of gene integration. Juice prepared from agroinfiltrated tomatoes may benefit both consumers as well as juice processing industry due to their high nutritional value and better processing quality.

Molecular Cloning and Overexpression of Phytoene Desaturase (CrtI) from Paracoccus haeundaensis

Among the carotenoid biosynthesis genes, crtI gene encodes the phytoene desaturase (CrtI) enzyme, and phytoene desaturase convert phytoene to lycopene. Phytoene desaturase is involved in the dehydrogenation reaction, in which four single bonds in the phytoene are introduced into a double bond, eliminating eight hydrogen atoms in the process. Phytoene desaturase is one of the key regulating enzyme in carotenoid biosynthetic pathway of various carotenoid biosynthetic organisms. The crtI gene in genomic DNA of Paracoccus haeundaensis was amplified and cloned into a T-vector to analyze the nucleotide sequence. As a result, the crtI gene coding for phytoene desaturase from P. haeundaensis consists of 1,503 base pairs encoding 501 amino acids residues. An expression plasmid containing the crtI gene was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 55 kDa, equivalent to the molecular weight of phytoene desaturase. The expressed protein in cell lysate showed enzymatic activity similar to phytoene desaturase. Phytoene and lycopene were analyzed by HPLC and measured at wavelength of 280 nm and 470 nm, respectively. The Km values for phytoene and NADPH were 11.1 μM and 129.3 μM, respectively.

Structural and functional analysis of the carotenoid biosynthesis genes of a Pseudomonas strain isolated from the excrement of Autumn Darter.

There are many reports about carotenoid-producing bacteria and carotenoid biosynthesis genes. In databases for Pseudomonas genome sequences, there are genes homologous to carotenoid biosynthesis genes, but the function of these genes in Pseudomonas has not been elucidated. In this study, we cloned the carotenoid biosynthesis genes from a Pseudomonas sp. strain, named Akiakane, which was isolated from the excrement of the Autumn Darter dragonfly. Using an Escherichia coli functional expression system, we confirmed that the idi, crtE, crtB, crtI, and crtY gene products of the Akiakane strain show predictable catalytic activities. A cluster of six genes was also found, which was comparable to other carotenoid-producing bacteria that belong to the α-Proteobacteria or γ-Proteobacteria class.

Effect of codon optimization on the enhancement of the β-carotene contents in rice endosperm

A β-carotene is the most well-known dietary source as provitamin A carotenoids. Among β-carotene-producing Golden Rice varieties, PAC (Psy:2A:CrtI) rice has been previously developed using a bicistronic recombinant gene that linked the Capsicum Psy and Pantoea CrtI genes by a viral 2A sequence. To enhance β-carotene content by improving this PAC gene, its codon was optimized for rice plants (Oryza sativa L.) by minimizing the codon bias between the transgene donor and the host rice and was then artificially synthesized as stPAC (stPsy:2A:stCrtI) gene. The GC content (58.7 from 50.9%) and codon adaptation index (0.85 from 0.77) of the stPAC gene were increased relative to the original PAC gene with 76% DNA identity. Among 67 T1 seeds of stPAC transformants showing positive correlations between transgene copy numbers (up to three) and carotenoid contents, three stPAC lines with a single intact copy were chosen to minimize unintended insertional effects and compared to the representative line of the PAC transgene with respect to their codon optimization effects. Translation levels were stably increased in all three stPAC lines (3.0-, 2.5-, 2.9-fold). Moreover, a greater intensity of the yellow color of stPAC seeds was correlated with enhanced levels of β-carotene (4-fold, 2.37 μg/g) as well as total carotenoid (2.9-fold, 3.50 μg/g) relative to PAC seeds, suggesting a β-branch preference for the stPAC gene. As a result, the codon optimization of the transgene might be an effective tool in genetic engineering for crop improvement as proven at the enhanced levels of translation and carotenoid production.