Optimized wideband and compact multifunctional photonic device based on Sb2S3 phase change material.

In this paper, a 1 × 2 photonic switch is designed based on a silicon-on-insulator (SOI) platform combined with the phase change material (PCM), Sb2S3, assisted by the direct binary search (DBS) algorithm. The designed photonic switch exhibits an impressive operating bandwidth ranging from 1450 to 1650 nm. The device has an insertion loss (IL) from 0.44 dB to 0.70 dB (of less than 0.7 dB) and cross talk (CT) from -26 dB to -20 dB (of less than -20 dB) over an operating bandwidth of 200 nm, especially an IL of 0.52 dB and CT of -24 dB at 1550 nm. Notably, the device is highly compact, with footprints of merely 3 × 4 µm2. Furthermore, we have extended the device's functionality for multifunctional operation in the C-band that can serve as both a 1 × 2 photonic switch and a 3 dB photonic power splitter. In the photonic switch mode, the device demonstrates an IL of 0.7 dB and a CT of -13.5 dB. In addition, when operating as a 3 dB photonic power splitter, the IL is less than 0.5 dB.

Multifunctional metalens optical tweezers for optical information recognition.

Traditional optical information recognition (OIR), particle capture and manipulation require many optical devices or mechanical moving system components to achieve a specific function, which is difficult to achieve integration. This paper proposes a new method to realize these functions by using multi-focus metalens combining spectrum and polarization selection. The design incorporates three spectral bands, namely 500 nm, 580 nm, and 660 nm, within the visible light range. Additionally, it utilizes either left-handed or right-handed circularly polarized (LCP/RCP) light, resulting in six distinct focus focusing effects on a single focal plane. By analyzing the normalized light intensity (NLI) at the corresponding focus position, the OIR of any wavelength and polarization detection in the design can be realized, and the particle capture at different focusing positions can be realized. Our work can provide a new idea for the high integration of on-chip light recognition and operation and inspire the design of a highly integrated optical system with a smaller size and more substantial function.

Generation of human induced pluripotent stem cell line (XWHNi004-A) from a male with APOE gene mutation.

Genetic polymorphism of apolipoprotein E (APOE) confers differential susceptibility to Alzheimer's disease (AD), and APOE ɛ4 variants is the most powerful risk factor for this disease. Here, we report the generation of a human induced pluripotent stem cell (iPSC) line carrying the APOE ɛ4/ɛ4 genotype from peripheral blood mononuclear cells (PBMCs) isolated from a male with a family history of AD utilizing non-integrative Sendai virus vector. The iPSC maintains their original genotype, highly express endogenous pluripotency markers, displays a normal karyotype, and retains the ability to differentiate into cells representative of the three germ layers.

Fed-batch fermentation of Mucor circinelloides reveals significant improvement in biomass and lipid accumulation through performance evaluation, chemical analysis, and expression profiling.

This study aimed to improve the lipid and biomass yields of Mucor circinelloides WJ11 by implementing four different fed-batch fermentation strategies, varied in time and glucose concentration (S1-S4). The S1 fermentation strategy yielded the highest biomass, lipid, and fatty acid content (22 ± 0.7 g/L, 53 ± 1.2 %, and 28 ± 1.6 %) after 120 and 144 h, respectively. The γ-linolenic acid titer of 0.75 ± 0.0 g/L was greatest in S3 after 48 h. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the transcription of key genes involved in lipid accumulation. The glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase genes showed increased expression levels. Fourier-transform infrared (FTIR) spectroscopy was used to analyze the biochemical profile during fermentation strategies. Optimal abiotic factors for production efficiency included pH 6.5, 25-26 °C, 15 % (v/v) inoculum, 500 rpm, 20 %-30 % dissolved oxygen, and 120 h fermentation. Glucose co-feeding offers valuable insights to develop effective fermentation strategies for lipid production.

The role of areA in lipid accumulation in high lipid-producing fungus Mucor circinelloides WJ11.

In the oleaginous fungus Mucor circinelloides, lipid accumulation is regulated by nitrogen metabolism, which is regulated by the areA gene, a member of the GATA zinc finger transporter family and a major regulator for nitrogen metabolism. However, the role of areA in lipid accumulation in this fungus has not been reported. In order to explore the regulatory effect of areA gene on nitrogen metabolism and lipid accumulation in M. circinelloides, we constructed areA gene knockout and overexpression strains. Then, the recombinant strains were cultured and their biochemical indexes were measured. Simultaneously, transcriptomic studies on the recombinant strains were conducted to infer the regulatory mechanism of areA. The results showed that the areA knockout strain accumulated more lipid, which is 42 % higher than the control. While the areA overexpressing strain obtained the higher biomass accumulation (23 g/L) and used up the nitrogen source in the medium earlier than the control strain and knockout strain. Transcriptome data analysis showed that nr and nit-6 genes related to nitrogen metabolism were up-regulated. And the expression levels of key genes acc and aclY were higher in the areA knockout strain than others, which was positively correlated with the increased lipid accumulation. In addition, in knockout strains, protein catabolism tended to provide substrates for the lipid production, and the expression levels of the related genes were also higher than others. These results indicated that the areA gene not only controls the transcription level of genes related to nitrogen metabolism but also affects lipid accumulation.

AMP deaminase: A crucial regulator in nitrogen stress and lipid metabolism in Mucor circinelloides.

Lipid biosynthesis is a significant metabolic response to nitrogen starvation in oleaginous fungi. The oleaginous fungus Mucor circinelloides copes with nitrogen stress by degrading AMP through AMP deaminase (AMPD). However, the mechanism of AMPD in regulating lipogenesis remains largely unclear. To elucidate the mechanism of AMPD in lipid synthesis in this M. circinelloides, we identified two genes (ampd1 and ampd2) encoding AMPD and constructed an ampd double knockout mutant. The engineered M. circinelloides strain elevated cell growth and lipid accumulation, as well as the content of oleic acid (OA) and gamma-linolenic acid (GLA). In addition to the expected increase in transcription levels of genes associated with lipid and TAG synthesis, we observed suppression of lipid degradation and reduced amino acid biosynthesis. This suggested that the deletion of AMPD genes induces the redirection of carbon towards lipid synthesis pathways. Moreover, the pathways related to nitrogen metabolism, including nitrogen assimilation and purine metabolism (especially energy level), were also affected in order to maintain homeostasis. Further analysis discovered that the transcription factors (TFs) related to lipid accumulation were also regulated. This study provides new insights into lipid biosynthesis in M. circinelloides, indicating that the trigger for lipid accumulation is not entirely AMPD-dependent and suggest that there may be additional mechanisms involved in the initiation of lipogenesis.

Structure-activity strategies for mechanically responsive fluorescent materials: a molecular perspective.

Mechanical response luminescence (MRL) describes the photophysical properties triggered by mechanical stimulation. Usually, MRL can be regulated by intermolecular interactions, molecular conformation or molecular packing, to achieve the desirable optical properties. Herein, at the molecular level, this review covers the factors that influence mechanically responsive fluorescent materials, involving the single- or multifactorial modulation of aliphatic chains, donor-receptor switch, substituent adjustment, and position isomerism. According to these factors, the structure-activity strategies can be summarized as: (i) the self-recovery of optical properties, from the final to initial state, can be regulated by introducing long alkyl chains to a fluorophore. (ii) The sensitivity of MRL materials can be controlled by modifying the donor-acceptor structure via the changed ICT (intramolecular charge transfer) and intramolecular interaction. (iii) The electronic and steric effects of substituents can affect ICT and intermolecular interactions, thereby resulting in high quantum yield and high-contrast MRL materials via changing the molecular stacking of crystalline states. (iv) Intermolecular interaction is modulated by the position isomerism of the substituents, which results in switched molecular packing for the extended response toward a wide range of stimuli. It is anticipated that the molecular mechanisms of these structure-activity relationships will serve as a significant reference for developing novel, high contrast, recyclable mechanical response luminous materials.

Role of AMP Deaminase in Mucor circinelloides: Implications for Nitrogen Utilization and Lipid Biosynthesis.

Lipid accumulation in oleaginous organisms is initiated by AMP deaminase (AMPD) after nitrogen depletion because it mediates the concentration of intracellular adenosine monophosphate (AMP). However, the role of AMPD in lipogenesis in the oleaginous fungus Mucor circinelloides is largely unknown. Therefore, we identified the genes (ampd1 and ampd2) encoding AMPD and investigated the role of AMPD in lipid synthesis in this fungus by overexpressing and deleting ampd genes. Deletion of ampd1 and ampd2 caused 21 and 28% increments in lipid contents under N-limited conditions, respectively. These increases were correlated with the activation of enzymes involved in lipogenesis and the alteration of energy balance. Unexpectedly, overexpression of ampd genes affected nitrogen consumption in both N-limited and N-excess media, which resulted in an increase in cell growth and lipid accumulation compared with the control strain when nitrogen was available. Furthermore, the increased lipid accumulation in the ampd-overexpressing mutants in N-excess media was accompanied by enhanced activities of lipid biosynthetic enzymes. These data suggested that nitrogen metabolism and energy metabolism are affected by AMPD, and overexpression of ampd genes induced lipid accumulation under nitrogen-rich conditions by mimicking the nitrogen limitation response. This highlights an intriguing function of AMPD in M. circinelloides.

Comparative Transcriptome Analysis Revealed Key Genes Regulating Gossypol Synthesis in Tetraploid Cultivated Cotton.

Tetraploid cultivated cotton (Gossypium spp.) produces cottonseeds rich in protein and oil. Gossypol and related terpenoids, stored in the pigment glands of cottonseeds, are toxic to human beings and monogastric animals. However, a comprehensive understanding of the genetic basis of gossypol and gland formation is still lacking. We performed a comprehensive transcriptome analysis of four glanded versus two glandless tetraploid cultivars distributed in Gossypium hirsutum and Gossypium barbadense. A weighted gene co-expression network analysis (WGCNA) based on 431 common differentially expressed genes (DEGs) uncovered a candidate module that was strongly associated with the reduction in or disappearance of gossypol and pigment glands. Further, the co-expression network helped us to focus on 29 hub genes, which played key roles in the regulation of related genes in the candidate module. The present study contributes to our understanding of the genetic basis of gossypol and gland formation and serves as a rich potential source for breeding cotton cultivars with gossypol-rich plants and gossypol-free cottonseed, which is beneficial for improving food safety, environmental protection, and economic gains of tetraploid cultivated cotton.

Generation of induced pluripotent stem cell line (XWHNi002-A) from a female with APP gene mutation.

The ß-amyloid precursor protein (APP) is a crucial pathogenic gene linked to Alzheimer's disease (AD). A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) isolated from a female with APP gene mutation utilizing non-integrative Sendai virus. The iPSC line exhibits high expression of pluripotency markers, retains the APP mutation, displays a normal karyotype, and has the ability to differentiate into normal teratoma tissue. This iPSC line represents a valuable cell model for investigating the pathological mechanisms and therapeutic strategies of AD.

Industrially Important Fungal Carotenoids: Advancements in Biotechnological Production and Extraction.

Carotenoids are lipid-soluble compounds that are present in nature, including plants and microorganisms such as fungi, certain bacteria, and algae. In fungi, they are widely present in almost all taxonomic classifications. Fungal carotenoids have gained special attention due to their biochemistry and the genetics of their synthetic pathway. The antioxidant potential of carotenoids may help fungi survive longer in their natural environment. Carotenoids may be produced in greater quantities using biotechnological methods than by chemical synthesis or plant extraction. The initial focus of this review is on industrially important carotenoids in the most advanced fungal and yeast strains, with a brief description of their taxonomic classification. Biotechnology has long been regarded as the most suitable alternative way of producing natural pigment from microbes due to their immense capacity to accumulate these pigments. So, this review mainly presents the recent progress in the genetic modification of native and non-native producers to modify the carotenoid biosynthetic pathway for enhanced carotenoid production, as well as factors affecting carotenoid biosynthesis in fungal strains and yeast, and proposes various extraction methods to obtain high yields of carotenoids in an attempt to find suitable greener extraction methods. Finally, a brief description of the challenges regarding the commercialization of these fungal carotenoids and the solution is also given.

Lipid Accumulation by Snf-ß Engineered Mucor circinelloides Strains on Glucose and Xylose.

Sucrose non-fermenting 1 (SNF1) protein kinase plays the regulatory roles in the utilization of selective carbon sources and lipid metabolism. Previously, the role of ß subunit of SNF1 in lipid accumulation was evaluated by overexpression and knockout of Snf-ß in oleaginous fungus M. circinelloides. In the present study, the growth and lipid accumulation of Snf-ß overexpression and knockout strains were further analyzed and compared with glucose or xylose as a single or mixed carbon sources. The results showed that the lipid contents in Snf-ß knockout strain improved by 23.2% (for glucose), 28.4% (for xylose), and 30.5% (for mixed glucose and xylose) compared with that of the control strain, respectively. The deletion of Snf-ß subunit also altered the transcriptional level of acetyl-CoA carboxylase (ACC). The highest transcriptional levels of ACC1 in Snf-ß knockout strain at 24 h were increased by 2.4-fold (for glucose), 2.8-fold (for xylose), and 3.1-fold (for mixed glucose and xylose) compared with that of the control strain, respectively. Our results indicated that Snf-ß subunit enhanced lipid accumulation through the regulation of ACC1 in response to xylose or mixed sugars of glucose and xylose more significantly than that of response to glucose. This is the first study to explore the effect of Snf-ß subunit of M. circinelloides in regulating lipid accumulation responding to different carbon nutrient signals of glucose and xylose. This study provides a foundation for the future application of the Snf-ß engineered strains in lipid production from lignocellulose.

Fine mapping and candidate gene analysis of qFL-A12-5: a fiber length-related QTL introgressed from Gossypium barbadense into Gossypium hirsutum.

KEY MESSAGE: The fiber length-related qFL-A12-5 identified in CSSLs introgressed from Gossypium barbadense into Gossypium hirsutum was fine-mapped to an 18.8 kb region on chromosome A12, leading to the identification of the GhTPR gene as a potential regulator of cotton fiber length. Fiber length is a key determinant of fiber quality in cotton, and it is a key target of artificial selection for breeding and domestication. Although many fiber length-related quantitative trait loci have been identified, there are few reports on their fine mapping or candidate gene validation, thus hampering efforts to understand the mechanistic basis of cotton fiber development. Our previous study identified the qFL-A12-5 associated with superior fiber quality on chromosome A12 in the chromosome segment substitution line (CSSL) MBI7747 (BC4F3:5). A single segment substitution line (CSSL-106) screened from BC6F2 was backcrossed to construct a larger segregation population with its recurrent parent CCRI45, thus enabling the fine mapping of 2852 BC7F2 individuals using denser simple sequence repeat markers to narrow the qFL-A12-5 to an 18.8 kb region of the genome, in which six annotated genes were identified in Gossypium hirsutum. Quantitative real-time PCR and comparative analyses led to the identification of GH_A12G2192 (GhTPR) encoding a tetratricopeptide repeat-like superfamily protein as a promising candidate gene for qFL-A12-5. A comparative analysis of the protein-coding regions of GhTPR among Hai1, MBI7747, and CCRI45 revealed two non-synonymous mutations. The overexpression of GhTPR resulted in longer roots in Arabidopsis, suggesting that GhTPR may regulate cotton fiber development. These results provide a foundation for future efforts to improve cotton fiber length.

Genome-wide artificial introgressions of Gossypium barbadense into G. hirsutum reveal superior loci for simultaneous improvement of cotton fiber quality and yield traits.

INTRODUCTION: The simultaneous improvement of fiber quality and yield for cotton is strongly limited by the narrow genetic backgrounds of Gossypium hirsutum (Gh) and the negative genetic correlations among traits. An effective way to overcome the bottlenecks is to introgress the favorable alleles of Gossypium barbadense (Gb) for fiber quality into Gh with high yield. OBJECTIVES: This study was to identify superior loci for the improvement of fiber quality and yield. METHODS: Two sets of chromosome segment substitution lines (CSSLs) were generated by crossing Hai1 (Gb, donor-parent) with cultivar CCRI36 (Gh) and CCRI45 (Gh) as genetic backgrounds, and cultivated in 6 and 8 environments, respectively. The kmer genotyping strategy was improved and applied to the population genetic analysis of 743 genomic sequencing data. A progeny segregating population was constructed to validate genetic effects of the candidate loci. RESULTS: A total of 68,912 and 83,352 genome-wide introgressed kmers were identified in the CCRI36 and CCRI45 populations, respectively. Over 90 % introgressions were homologous exchanges and about 21 % were reverse insertions. In total, 291 major introgressed segments were identified with stable genetic effects, of which 66(22.98 %), 64(21.99 %), 35(12.03 %), 31(10.65 %) and 18(6.19 %) were beneficial for the improvement of fiber length (FL), strength (FS), micronaire, lint-percentage (LP) and boll-weight, respectively. Thirty-nine introgression segments were detected with stable favorable additive effects for simultaneous improvement of 2 or more traits in Gh genetic background, including 6 could increase FL/FS and LP. The pyramiding effects of 3 pleiotropic segments (A07:C45Clu-081, D06:C45Clu-218, D02:C45Clu-193) were further validated in the segregating population. CONCLUSION: The combining of genome-wide introgressions and kmer genotyping strategy showed significant advantages in exploring genetic resources. Through the genome-wide comprehensive mining, a total of 11 clusters (segments) were discovered for the stable simultaneous improvement of FL/FS and LP, which should be paid more attention in the future.

Homologous and Heterologous Expression of Delta(12)-Desaturase in Mucor circinelloides Enhanced the Production of Linolenic Acid.

Linolenic acid (LA) is gaining more interest within the scientific community. This is because it has a potential medical role in reducing the risk of inflammation, carcinogenesis, atherosclerosis and diabetes and is a valuable nutraceutical for human health. The oleaginous fungus Mucor circinelloides produces a high lipid content (36%), including valuable polyunsaturated fatty acids (PUFAs). However, the critical step in which oleic acid (OA) is converted into LA is not efficient at supplying enough substrates for PUFA synthesis. Hence, we propose a method to increase LA production based on genetic engineering. The overexpression of the Δ12-desaturase gene from M. circinelloides and Mortierella alpina increased the LA content and improved the lipid accumulation (from 14.9% to 21.6% in the Δ12-desaturase gene of the M. circinelloides overexpressing strain (Mc-D12MC) and from 14.9% to 18.7% in the Δ12-desaturase gene of M. alpina overexpressing strain (Mc-D12MA)). Additionally, the up-regulated expression levels of these genes targeted the genes involved in NADPH production, implying that the elevated Δ12-desaturase gene may function as a critical regulator of NADPH and lipid synthesis in M. circinelloides. This study provides the first evidence to support the design of metabolic engineering related to LA and PUFA production in M. circinelloides for potential industrial applications.

Identification and Functional Characterization of Adenosine Deaminase in Mucor circinelloides: A Novel Potential Regulator of Nitrogen Utilization and Lipid Biosynthesis.

Adenosine deaminase (ADA) is an enzyme distributed in a wide variety of organisms that cleaves adenosine into inosine. Since inosine plays an important role in nitrogen metabolism, ADA may have a critical function in the regulation of fatty acid synthesis. However, the role of ADA in oleaginous fungi has not been reported so far. Therefore, in this study, we identified one ada gene encoding ADA (with ID scaffold0027.9) in the high lipid-producing fungus, Mucor circinelloides WJ11, and investigated its role in cell growth, lipid production, and nitrogen metabolism by overexpressing and knockout of this gene. The results showed that knockout of the ada altered the efficiency of nitrogen consumption, which led to a 20% increment in the lipid content (25% of cell dry weight) of the engineered strain, while overexpression of the ada showed no significant differences compared with the control strain at the final growth stage; however, interestingly, it increased lipid accumulation at the early growth stage. Additionally, transcriptional analysis was conducted by RT-qPCR and our findings indicated that the deletion of ada activated the committed steps of lipid biosynthesis involved in acetyl-CoA carboxylase (acc1 gene), cytosolic malic acid enzyme (cme1 gene), and fatty acid synthases (fas1 gene), while it suppressed the expression of AMP-activated protein kinase (ampk α1 and ampk ß genes), which plays a role in lipolysis, whereas the ada-overexpressed strain displayed reverse trends. Conclusively, this work unraveled a novel role of ADA in governing lipid biosynthesis and nitrogen metabolism in the oleaginous fungus, M. circinelloides.

AAQSP increases mapping resolution of stable QTLs through applying NGS-BSA in multiple genetic backgrounds.

KEY MESSAGE: In this study, we present AAQSP as an extension of existing NGS-BSA applications for identifying stable QTLs at high resolution. GhPAP16 and GhIQD14 fine mapped on chromosome D09 of upland cotton are identified as important candidate genes for lint percentage (LP). Bulked segregant analysis combined with next generation sequencing (NGS-BSA) allows rapid identification of genome sequence differences responsible for phenotypic variation. The NGS-BSA approach applied to crops mainly depends on comparing two bulked DNA samples of individuals from an F2 population. Since some F2 individuals still maintain high heterozygosity, heterosis will exert complications in pursuing NGS-BSA in such populations. In addition, the genetic background influences the stability of gene expression in crops, so some QTLs mapped in one segregating population may not be widely applied in crop improvement. The AAQSP (Association Analysis of QTL-seq on Semi-homologous Populations) reported in our study combines the optimized scheme of constructing BSA bulks with NGS-BSA analysis in two (or more) different parental genetic backgrounds for isolating the stable QTLs. With application of AAQSP strategy and construction of a high-density linkage map, we have successfully identified a QTL significantly related to lint percentage (LP) in cultivated upland cotton, followed by map-based cloning to dissect two candidate genes, GhPAP16 and GhIQD14. This study demonstrated that AAQSP can efficiently identify stable QTLs for complex traits of interest, and thus accelerate the genetic improvement of upland cotton and other crop plants.

Identification and analysis of oil candidate genes reveals the molecular basis of cottonseed oil accumulation in Gossypium hirsutum L.

KEY MESSAGE: Based on the integration of QTL-mapping and regulatory network analyses, five high-confidence stable QTL regions, six candidate genes and two microRNAs that potentially affect the cottonseed oil content were discovered. Cottonseed oil is increasingly becoming a promising target for edible oil with its high content of unsaturated fatty acids. In this study, a recombinant inbred line (RIL) cotton population was constructed to detect quantitative trait loci (QTLs) for the cottonseed oil content. A total of 39 QTLs were detected across eight different environments, of which five QTLs were stable. Forty-three candidate genes potentially involved in carbon metabolism, fatty acid synthesis and triacylglycerol biosynthesis processes were further obtained in the stable QTL regions. Transcriptome analysis showed that nineteen of these candidate genes expressed during  the developing cottonseed ovules and may affect the cottonseed oil content. Besides, transcription factor (TF) and microRNA (miRNA) co-regulatory network analyses based on the nineteen candidate genes suggested that six genes, two core miRNAs (ghr-miR2949b and ghr-miR2949c), and one TF GhHSL1 were considered to be closely associated with the cottonseed oil content. Moreover, four vital genes were validated by quantitative real-time PCR (qRT-PCR). These results provide insights into the oil accumulation mechanism in developing cottonseed ovules through the construction of a detailed oil accumulation model.

Simultaneous overexpression of ∆6-, ∆12- and ∆9-desaturases enhanced the production of γ-linolenic acid in Mucor circinelloides WJ11.

Mucor circinelloides WJ11, an oleaginous filamentous fungus, produces 36% lipid of its cell dry weight when cultured in a high C/N ratio medium, however, the yield of γ-linolenic acid (GLA) is insufficient to make it competitive with other plant sources. To increase the GLA content in M. circinelloides WJ11, this fungus was engineered by overexpression of its key genes such as Δ6-, Δ12-, and Δ9-desaturases involved in GLA production. Firstly, we tried to overexpress two Δ6-desaturase isozymes to determine which one played important role in GLA synthesis. Secondly, Δ6-and Δ12-desaturase were co-overexpressed to check whether linoleic acid (LA), the precursor for GLA synthesis, is a limiting factor or not. Moreover, we tried to explore the effects of simultaneous overexpression of Δ6-, Δ12-, and Δ9-desaturases on GLA production. Our results showed that overexpression (1 gene) of DES61 promoted higher GLA content (21% of total fatty acids) while co-overexpressing (2 genes) DES61 and DES12 and simultaneous overexpressing (3 genes) DES61, DES12, and DES91 increased the GLA production of engineered strains by 1.5 folds and 1.9 folds compared to the control strain, respectively. This study provided more insights into GLA biosynthesis in oleaginous fungi and laid a foundation for further increase in GLA production into fungus such as M. circinelloides.

Genetic Modification of Mucor circinelloides for Canthaxanthin Production by Heterologous Expression of ß-carotene Ketolase Gene.

Canthaxanthin is a reddish-orange xanthophyll with strong antioxidant activity and higher bioavailability than carotenes, primarily used in food, cosmetics, aquaculture, and pharmaceutical industries. The spiking market for natural canthaxanthin promoted researchers toward genetic engineering of heterologous hosts for canthaxanthin production. Mucor circinelloides is a dimorphic fungus that produces ß-carotene as the major carotenoid and is considered as a model organism for carotenogenic studies. In this study, canthaxanthin-producing M. circinelloides strain was developed by integrating the codon-optimized ß-carotene ketolase gene (bkt) of the Haematococcus pluvialis into the genome of the fungus under the control of strong promoter zrt1. First, a basic plasmid was constructed to disrupt crgA gene, a negative regulator of carotene biosynthesis resulted in substantial ß-carotene production, which served as the building block for canthaxanthin by further enzymatic reaction of the ketolase enzyme. The genetically engineered strain produced a significant amount (576 ± 28 µg/g) of canthaxanthin, which is the highest amount reported in Mucor to date. Moreover, the cell dry weight of the recombinant strain was also determined, producing up to more than 9.0 g/L, after 96 h. The mRNA expression level of bkt in the overexpressing strain was analyzed by RT-qPCR, which increased by 5.3-, 4.1-, and 3-folds at 24, 48, and 72 h, respectively, compared with the control strain. The canthaxanthin-producing M. circinelloides strain obtained in this study provided a basis for further improving the biotechnological production of canthaxanthin and suggested a useful approach for the construction of more valuable carotenoids, such as astaxanthin.