Identification and optimization of the key growth parameters involved in carotenoids production of the marine microalga Pavlova gyrans.

In this work, a multivariate analysis was carried out, using a Plackett-Burman (PB) design involving seventeen growth parameters, on carotenoids production of Pavlova gyrans (p < 0.10). Each assay was analysed regarding its content (mg g-1) of fucoxanthin (Fx), diatoxanthin, diadinoxanthin, ß-carotene (ßCar), α-carotene, and the sum of all carotenoids analysed individually (TCar). According to the statistical analysis, modified medium formulations were developed for the particular cases of Fx, ßCar, and TCar. The study showed that Fx content was positively affected by nitrogen supplementation and lower light intensities. Higher concentrations of nitrogen and iron increased the final content of ßCar as well. Similarly, salinity, light intensity, nitrogen, iron, and cobalt were identified as key factors in TCar production. The PB-based formulations showed significant improvements (p < 0.05) for TCar (11.794 mg g-1) and Fx (6.153 mg g-1) when compared to the control conditions (Walne's medium-2.010 mg g-1). Furthermore, effective control of key variables (e.g., light intensity) throughout P. gyrans growth proved successful (p < 0.05), increasing the productivity of Fx (0.759 mg L-1 d-1) and TCar (1.615 mg L-1 d-1).

Development of a metabolic engineering technology to simultaneously suppress the expression of multiple genes in yeast and application in carotenoid production.

In yeast metabolic engineering, there is a need for technologies that simultaneously suppress and regulate the expression of multiple genes and improve the production of target chemicals. In this study, we aimed to develop a novel technology that simultaneously suppresses the expression of multiple genes by combining RNA interference with global metabolic engineering strategy. Furthermore, using ß-carotene as the target chemical, we attempted to improve its production by using the technology. First, we developed a technology to suppress the expression of the target genes with various strengths using RNA interference. Using this technology, total carotenoid production was successfully improved by suppressing the expression of a single gene out of 10 candidate genes. Then, using this technology, RNA interference strain targeting 10 candidate genes for simultaneous suppression was constructed. The total carotenoid production of the constructed RNA interference strain was 1.7 times compared with the parental strain. In the constructed strain, the expression of eight out of the 10 candidate genes was suppressed. We developed a novel technology that can simultaneously suppress the expression of multiple genes at various intensities and succeeded in improving carotenoid production in yeast. Because this technology can suppress the expression of any gene, even essential genes, using only gene sequence information, it is considered a useful technology that can suppress the formation of by-products during the production of various target chemicals by yeast.

Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.

ß-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve ß-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for ß-carotene production. Point and structural mutations were introduced into ß-carotene-producing engineered yeast. The resulting mutants showed higher ß-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of ß-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and ß-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with ß-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.

ß-Carotene production from sugarcane molasses by a newly isolated Rhodotorula toruloides L/24-26-1.

Production of carotenoids by yeast fermentation is an advantaged technology due to its easy scaling and safety. Nevertheless, carotenoid production needs an economic culture medium and other efficient yeast stains. The study aims to isolate and identify a yeast strain capable of producing carotenoids using a cost-effective substrate. A new strain was identified as Rhodotorula toruloides L/24-26-1, which can produce carotenoids at different pretreated and unpretreated sugarcane molasses concentrations (40 and 80 g/L). The highest biomass concentration (18.6 ± 0.6 g/L) was reached in the culture using 80 g/L of hydrolyzed molasses. On the other hand, the carotenoid accumulation reached the maximum value using pretreated molasses at 40 g/L (715.4 ± 15.1 µg/g d.w). In this case, the ß-carotene was 1.5 times higher than that on the control medium. The yeast growth in molasses was not correlated with carotenoid production. The most outstanding production of The DPPH, ABTS, and FRAP tests demonstrated the antioxidant activity of the obtained carotenogenic extracts. This research demonstrated the R. toruloides L/24-26-1 strain biotechnological potential for carotenoid compounds. The yeast produces carotenoids with antioxidant activity in an inexpensive medium, such as sulfuric acid pretreated and unpretreated molasses.

Enhanced high ß-carotene yeast cell production by Rhodotorula paludigena CM33 and in vitro digestibility in aquatic animals.

This study assessed Rhodotorula paludigena CM33's growth and ß-carotene production in a 22-L bioreactor for potential use as an aquatic animal feed supplement. Optimizing the feed medium's micronutrient concentration for high-cell-density fed-batch cultivation using glucose as the carbon source yielded biomass of 89.84 g/L and ß-carotene concentration of 251.64 mg/L. Notably, using sucrose as the carbon source in feed medium outperforms glucose feeds, resulting in a ß-carotene concentration of 285.00 mg/L with a similar biomass of 87.78 g/L. In the fed-batch fermentation using Sucrose Feed Medium, R. paludigena CM33 exhibited high biomass production rates (Qx) of 0.91 g/L.h and remarkable ß-carotene production rates (Qp) of 2.97 mg/L.h. In vitro digestibility assays showed that R. paludigena CM33, especially when cultivated using sucrose, enhances protein digestibility affirming its suitability as an aquatic feed supplement. Furthermore, R. paludigena CM33's nutrient-rich profile and probiotic potential make it an attractive option for aquatic nutrition. This research highlights the importance of cost-effective carbon sources in large-scale ß-carotene production for aquatic animal nutrition.

Simultaneous Production of Cellulase and ß-Carotene in the Filamentous Fungus Trichoderma reesei.

ß-Carotene is an indispensable additive in beverage, cosmetic, feed, and pharmaceutical production. The fermentation industry annually generates abundant waste mycelia from Trichoderma reesei (T. reesei), a pivotal industrial strain for cellulase and heterologous protein production. In this study, we constructed a T. reesei cell factory for ß-carotene production for the first time. Four key enzymes, CarRP, CarB, GGS1/CrtE, and HMG1, were overexpressed in T. reesei. The concentrations of medium components, including tryptone and glucose, were optimized. The modified strain accumulated ß-carotene at a titer of 218.8 mg/L in flask culture. We achieved cellulase production (FPase, 22.33 IU/mL) with the concomitant production of ß-carotene (286.63 mg/L) from T. reesei in a jar. Overall, this study offers a novel and unique approach to address the costly waste mycelium management process using T. reesei industrial strains that simultaneously produce proteins and carotenoids.

Dual regulation of lipid droplet-triacylglycerol metabolism and ERG9 expression for improved ß-carotene production in Saccharomyces cerevisiae.

BACKGROUND: The limitation of storage space, product cytotoxicity and the competition for precursor are the major challenges for efficiently overproducing carotenoid in engineered non-carotenogenic microorganisms. In this work, to improve ß-carotene accumulation in Saccharomyces cerevisiae, a strategy that simultaneous increases cell storage capability and strengthens metabolic flux to carotenoid pathway was developed using exogenous oleic acid (OA) combined with metabolic engineering approaches. RESULTS: The direct separation of lipid droplets (LDs), quantitative analysis and genes disruption trial indicated that LDs are major storage locations of ß-carotene in S. cerevisiae. However, due to the competition for precursor between ß-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on ß-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in ß-carotene content. The transcriptome analysis was adopted to mine OA-repressible promoters and IZH1 promoter was used to replace native ERG9 promoter to dynamically down-regulate ERG9 expression, which diverted the metabolic flux to ß-carotene pathway and achieved additional 31.7% increase in ß-carotene content without adversely affecting cell growth. By inducing an extra constitutive ß-carotene synthesis pathway for further conversion precursor farnesol to ß-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of ß-carotene, which is 107.3% and 49.5% increase respectively over the parent strain. CONCLUSIONS: This strategy can be applied in the overproduction of other heterogeneous FPP-derived hydrophobic compounds with similar synthesis and storage mechanisms in S. cerevisiae.

AgZDS, a gene encoding ζ-carotene desaturase, increases lutein and ß-carotene contents in transgenic Arabidopsis and celery.

ζ-Carotene desaturase (ZDS) is one of the key enzymes regulating carotenoids biosynthesis and accumulation. Celery transgenic efficiency is low and it is difficult to obtain transgenic plants. The study on ZDS was limited in celery. Here, the AgZDS gene was cloned from celery and overexpressed in Arabidopsis thaliana and celery to verify its function. The AgZDS has typical characteristic of ZDS protein and is highly conserved in higher plants. Phylogenetic analysis showed that AgZDS has the closest evolutionary relationship with ZDSs from Solanum lycopersicum, Capsicum annuum and Tagetes erecta. Overexpression of AgZDS gene in A. thaliana and celery resulted in increased accumulations of lutein and ß-carotene and up-regulated the expression levels of the genes involved in carotenoids biosynthesis. The contents of lutein and ß-carotene in two lines, AtL1 and AgL5, were the highest in transgenic A. thaliana and celery, respectively. The relative expression levels of 5 genes (AtPDS, AtZISO, AtZEP, AtNCED3, and AtCCD4) were up-regulated compared to the wild type plants. The relative expression levels of most genes in carotenoids biosynthesis pathway, such as AgPDS, AgCRTISO1, and AgZISO, were up-regulated in transgenic celery plants. The antioxidant capacity of A. thaliana and photosynthetic capacity of celery were also enhanced. This research is the first report on the function of structure gene related to carotenoid biosynthesis in transgenic celery plants. The findings in this study demonstrated the roles of AgZDS in regulating carotenoids metabolism of celery, which laid a potential foundation for quality improvement of celery.

Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a.

CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous ß-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.

In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii.

Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (ß-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled ß-carotene synthesis (194 µg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional ß-carotene recovered in feces was 56-fold higher than the ß-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.

Pathway Engineering for Beta-Carotene and Carotenoid Biosynthesis in Y. lipolytica.

ß-carotene is an increasingly sought-after organic pigment with antioxidant properties and a vitamin precursor. Yarrowia lipolytica, though unable to naturally synthesize carotenoids, can produce high amounts of the precursor acetyl-CoA making it a promising host for metabolic engineering towards novel biotechnological production of carotenoids. Here, we describe a synthetic biology methodology for Y. Lipolytica metabolic engineering based on Golden Gate DNA assembly for the generation of a multigene cassette, subsequent transformation enabling ß-carotene biosynthesis, and quantification of the compound.

Robust and flexible platform for directed evolution of yeast genetic switches.

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.

Protoplast fusion between Blakeslea trispora 14,271 (+) and 14,272 (-) enhanced the yield of lycopene and ß-carotene.

Blakeslea trispora, a heterothallic Zygomycota with two mating types (termed "plus" and "minus"), is an ideal source of lycopene and ß-carotene. The lycopene and ß-carotene yields when the two type strains are used for fermentation separately are lower than those when they are joint together. To enhance the yield of lycopene and ß-carotene in B. trispora, protoplast fusion technology was carried out between ATCC 14,271 (+) and ATCC 14,272 (-). After protoplast preparation, protoplast fusion, fusion sorting, fusion regeneration, and high-throughput screening, two fusions (Fu-1and Fu-2) with high lycopene and ß-carotene yields were obtained. The lycopene yields of Fu-1 and Fu-2 were increased to 0.60 mg/gDW and 0.90 mg/gDW, which were respectively 3.62- and 5.44-fold those of 14,271 and 1.76- and 2.64-fold those of 14,272. The ß-carotene yields of Fu-1 and Fu-2 were increased to 22.07 mg/gDW and 36.93 mg/gDW, which were respectively 1.72- and 2.89-fold those of 14,271 and 1.23- and 2.06-fold those of 14,272. In this study, the protoplast fusion technique was successfully used in Blakeslea trispora, providing new ideas for improving lycopene and ß-carotene production.

Development and characterization of GR2E Golden rice introgression lines.

Golden Rice with ß-carotene in the grain helps to address the problem of vitamin A deficiency. Prior to commercialize Golden Rice, several performance and regulatory checkpoints must be achieved. We report results of marker assisted backcross breeding of the GR2E trait into three popular rice varieties followed by a series of confined field tests of event GR2E introgression lines to assess their agronomic performance and carotenoid expression. Results from confined tests in the Philippines and Bangladesh have shown that GR2E introgression lines matched the performance of the recurrent parents for agronomic and yield performance, and the key components of grain quality. Moreover, no differences were observed in terms of pest and disease reaction. The best performing lines identified in each genetic background had significant amounts of carotenoids in the milled grains. These lines can supply 30-50% of the estimated average requirements of vitamin A.

Analysis of carotenoid profile changes and carotenogenic genes transcript levels in Rhodosporidium toruloides mutants from an optimized Agrobacterium tumefaciens-mediated transformation method.

Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium-mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and ß-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and ß-carotene, and the proportion of γ-carotene and ß-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.

Metabolic engineering of Saccharomyces cerevisiae for production of ß-carotene from hydrophobic substrates.

ß-Carotene is a yellow-orange-red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based process for ß-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of ß-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding ß-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L ß-carotene in yeast peptone dextrose (YPD) medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest ß-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

Dissolved-oxygen feedback control fermentation for enhancing ß-carotene in engineered Yarrowia lipolytica.

The DO-stat fed-batch fermentation was carried out to explore the volumetric productivity of ß-carotene in engineered Yarrowia lipolytica C11 strain. Using DO-stat fed-batch fermentation, we achieved 94 g/L biomass and 2.01 g/L ß-carotene. Both biomass and ß-carotene were about 1.28-fold higher than that in fed-batch fermentation. The ATP, NADP+/NADPH, and gene expression levels of tHMG, GGS1, carRA, and carB were promoted as compared to that in fed-batch fermentation. As for as the kinetic parameters in DO-stat fed-batch fermentation, µm', Yx/s', and Yp/s' was 0.527, 0.353, and 0.158, respectively. The µm' was elevated 4.66-fold than that in fed-batch fermentation. These data illustrate that more dissolved oxygen increased the biomass. The Yx/s' and Yp/s' were increased 1.15 and 22.57-fold, which suggest that the DO-stat fed-batch fermentation reduced the Crabtree effect and improved the utilization rate of glucose. Therefore, DO-stat fed-batch fermentation is a promising strategy in the industrialized production of ß-carotene.

Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.

Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.

Light dependent accumulation of ß-carotene enhances photo-acclimation of Euglena gracilis.

Carotenoids are essential components of photosynthetic organisms including land plants, algae, cyanobacteria, and photosynthetic bacteria. Although the light-mediated regulation of carotenoid biosynthesis, including the light/dark cycle as well as the dependence of carotenoid biosynthesis-related gene translation on light wavelength, has been investigated in land plants, these aspects have not been studied in microalgae. Here, we investigated carotenoid biosynthesis in Euglena gracilis and found that zeaxanthin accumulates in the dark. The major carotenoid species in E. gracilis, namely ß-carotene, neoxanthin, diadinoxanthin and diatoxanthin, accumulated corresponding to the duration of light irradiation under the light/dark cycle, although the translation of carotenoid biosynthesis genes hardly changed. Irradiation with either blue or red-light (3 µmol photons m-2 s-1) caused a 1.3-fold increase in ß-carotene content compared with the dark control. Blue-light irradiation (300 µmol photons m-2 s-1) caused an increase in the cellular content of both zeaxanthin and all trans-diatoxanthin, and this increase was proportional to blue-light intensity. In addition, pre-irradiation with blue-light of 3 or 30 µmol photons m-2 s-1 enhanced the photosynthetic activity and tolerance to high-light stress. These findings suggest that the accumulation of ß-carotene is regulated by the intensity of light, which may contribute to the acclimation of E. gracilis to the light environment in day night conditions.

A multifocal approach towards understanding the complexities of carotenoid biosynthesis and accumulation in rice grains.

Carotenoids are mostly C40 terpenoids that participate in several important functions in plants including photosynthesis, responses to various forms of stress, signal transduction and photoprotection. While the antioxidant potential of carotenoids is of particular importance for human health, equally important is the role of ß-carotene as the precursor for vitamin A in the human diet. Rice, which contributes upto 40% of dietary energy for mankind, contains very low level of ß-carotene, thereby making it an important crop for enhancing ß-carotene accumulation in its grains and consequently targeting vitamin A deficiency. Biosynthesis of carotenoids in the endosperm of white rice is blocked at the first enzymatic step wherein geranylgeranyl diphosphate is converted to phytoene by the action of phytoene synthase (PSY). Strategies aimed at enhancing ß-carotene levels in the endosperm of white rice identified Narcissus pseudonarcissus (npPSY) and bacterial CRT1 as the regulators of the carotenoid biosynthetic pathway in rice. Besides transcriptional regulation of PSY, posttranscriptional regulation of PSY expression by OR gene, molecular synergism between ε-LCY and ß-LCY and epigenetic control of CRITSO through SET DOMAIN containing protein appear to be the other regulatory nodes which regulate carotenoid biosynthesis and accumulation in rice grains. In this review, we elucidate a comprehensive and deeper understanding of the regulatory mechanisms of carotenoid metabolism in crops that will enable us to identify an effective tool to alleviate carotenoid content in rice grains.