Sphingobacterium tenebrionis sp. nov., isolated from intestine of mealworm.

A bacterial strain designated PU5-4T was isolated from the mealworm (the larvae of Tenebrio molitor) intestines. It was identified to be Gram-stain-negative, strictly aerobic, rod-shaped, non-motile, and non-spore-forming. Strain PU5-4T was observed to grow at 10-40 °C, at pH 7.0-10.0, and in the presence of 0-3.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain PU5-4T should be assigned to the genus Sphingobacterium. The 16S rRNA gene sequence similarity analysis showed that strain PU5-4T was closely related to the type strains of Sphingobacterium lactis DSM 22361T (98.49 %), Sphingobacterium endophyticum NYYP31T (98.11 %), Sphingobacterium soli NCCP 698T (97.69 %) and Sphingobacterium olei HAL-9T (95.73 %). The predominant isoprenoid quinone is MK-7. The major fatty acids were identified as iso-C15 : 0, iso-C17 : 03-OH and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 9 (iso-C17 : 0 ω9c). The polar lipids are phosphatidylethanolamine, one unidentified phospholipid, and six unidentified lipids. The genomic DNA G+C content of strain PU5-4T is 40.24 mol%. The average nucleotide identity of strain PU5-4T exhibited respective values of 73.88, 73.37, 73.36 and 70.84 % comparing to the type strains of S. lactis DSM 22361T, S. soli NCCP 698T, S. endophyticum NYYP31T and S. olei HAL-9T, which are below the cut-off level (95-96 %) for species delineation. Based on the above results, strain PU5-4T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium temoinsis sp. nov. is proposed. The type strain is PU5-4T (=CGMCC 1.61908T=JCM 36663T).

Comprehensive transcriptomic and metabolomic insights into simultaneous CO2 sequestration and nitrate removal by the Chlorella vulgaris and Pseudomonas sp. consortium.

Simultaneous CO2 sequestration and nitrate removal can be achieved by co-cultivation of Chlorella vulgaris with Pseudomonas sp. However, a comprehensive understanding of the synergistic mechanism between C. vulgaris and Pseudomonas sp. remains unknown. In this study, transcriptomics and metabolomics analysis were employed to elucidate the synergistic mechanism of C. vulgaris and Pseudomonas sp. Transcriptomic and metabolomic analyses identified 3664 differentially expressed genes and 314 metabolites. Transcriptome analysis revealed that co-culture with Pseudomonas sp. promoted the photosynthesis of C. vulgaris by promoting the synthesis of photosynthetic pigments and photosynthesis-antenna proteins. Furthermore, it stimulated pathways associated with energy metabolism from carbon sources, such as the Calvin cycle, glycolytic pathway, and TCA cycle. Additionally, Pseudomonas sp. reduced nitrate levels in the co-culture system by denitrification, and microalgae regulated nitrate uptake by down-regulating the transcript levels of nitrate transporter genes. Metabolomic analysis indicated that nutrient exchange was conducted between algae and bacteria, and amino acids, phytohormones, and organic heterocyclic compounds secreted by the bacteria promoted the growth metabolism of microalgae. After supplementation with differential metabolites, the carbon fixation rate and nitrate removal rate of the co-culture system reached 0.549 g L-1 d-1 and 135.4 mg L-1 d-1, which were increased by 20% and 8%, respectively. This study provides a theoretical insight into microalgae-bacteria interaction and its practical application, as well as a novel perspective on flue gas treatment management.

Screening and identification of genes involved in ß-alanine biosynthesis in Bacillus subtilis.

ß-alanine is the only naturally occurring ß-amino acid, which is widely used in medicine, food, and feed fields, and generally produced through synthetic biological methods based on engineered strains of Escherichia coli or Corynebacterium glutamicum. However, the ß-alanine biosynthesis in Bacillus subtilis, a traditional industrial model microorganism of food safety grade, has not been thoroughly explored. In this study, the native l-aspartate-α-decarboxylase was overexpressed in B. subtilis 168 to obtain an increase of 842% in ß-alanine production. A total of 16 single-gene knockout strains were constructed to block the competitive consumption pathways to identify a total of 6 genes (i.e., ptsG, fbp, ydaP, yhfS, mmgA, and pckA) involved in ß-alanine synthesis, while the multigene knockout of these 6 genes obtained an increased ß-alanine production by 40.1%. Ten single-gene suppression strains with the competitive metabolic pathways inhibited revealed that the inhibited expressions of genes glmS, accB, and accA enhanced the ß-alanine production. The introduction of heterologous phosphoenolpyruvate carboxylase increased the ß-alanine production by 81.7%, which was 17-fold higher than that of the original strain. This was the first study using multiple molecular strategies to investigate the biosynthetic pathway of ß-alanine in B. subtilis and to identify the genetic factors limiting the excessive synthesis of ß-alanine by microorganisms.

Sustainable production of astaxanthin in microorganisms: the past, present, and future.

Astaxanthin (3,3'-dihydroxy-4,4'-diketo-ß-carotene) is a type of C40 carotenoid with remarkable antioxidant characteristics, showing significant application prospects in many fields. Traditionally, the astaxanthin is mainly obtained from chemical synthesis and natural acquisition, with both approaches having many limitations and not capable of meeting the growing market demand. In order to cope with these challenges, novel techniques, e.g., the innovative cell engineering strategies, have been developed to increase the astaxanthin production. In this review, we first elaborated the biosynthetic pathway of astaxanthin, with the key enzymes and their functions discussed in the metabolic process. Then, we summarized the conventional, non-genetic strategies to promote the production of astaxanthin, including the methods of exogenous additives, mutagenesis, and adaptive evolution. Lastly, we reviewed comprehensively the latest studies on the synthesis of astaxanthin in various recombinant microorganisms based on the concept of microbial cell factory. Furthermore, we have proposed several novel technologies for improving the astaxanthin accumulation in several model species of microorganisms.

Improved biosynthesis of heme in Bacillus subtilis through metabolic engineering assisted fed-batch fermentation.

BACKGROUND: Heme is an iron/porphyrin complex compound, widely used in the health care, food, and pharmaceutical industries. It is more advantageous and attractive to develop microbial cell factories to produce heme by fermentation, with lower production costs and environmentally more friendly procedures than those of the traditional extraction based on animal blood. In this study, Bacillus subtilis, a typical industrial model microorganism of food safety grade, was used for the first time as the host to synthesize heme. RESULTS: The heme biosynthetic pathway was engineered as four modules, the endogenous C5 pathway, the heterologous C4 pathway, the uroporphyrinogen (urogen) III synthesis pathway, and the downstream synthesis pathway. Knockout of hemX encoding the negative effector of the concentration of HemA, overexpression of hemA encoding glutamyl-tRNA reductase, and knockout of rocG encoding the major glutamate dehydrogenase in the C5 pathway, resulted in an increase of 427% in heme production. Introduction of the heterologous C4 pathway showed a negligible effect on heme biosynthesis. Overexpression of hemCDB, which encoded hydroxymethylbilane synthase, urogen III synthase, and porphobilinogen synthase participating in the urogen III synthesis pathway, increased heme production by 39%. Knockouts of uroporphyrinogen methyltransferase gene nasF and both heme monooxygenase genes hmoA and hmoB in the downstream synthesis pathway increased heme production by 52%. The engineered B. subtilis produced 248.26 ± 6.97 mg/L of total heme with 221.83 ± 4.71 mg/L of extracellular heme during the fed-batch fermentation in 10 L fermenter. CONCLUSIONS: Strengthening endogenous C5 pathway, urogen III synthesis pathway and downstream synthesis pathway promoted the biosynthesis of heme in B. subtilis. The engineered B. subtilis strain has great potential as a microbial cell factory for efficient industrial heme production.

Promoting Heme and Phycocyanin Biosynthesis in Synechocystis sp. PCC 6803 by Overexpression of Porphyrin Pathway Genes with Genetic Engineering.

Due to their unique biochemical and spectroscopic properties, both heme and phycocyanobilin are widely applied in the medical and food industries. Synechocystis sp. PCC 6803 contains both heme and phycocyanin, and is capable of synthesizing phycocyanin using heme as a precursor. The aim of this study was to uncover viable metabolic targets in the porphyrin pathway from Synechocystis sp. PCC 6803 to promote the accumulation of heme and phycocyanin in the recombinant strains of microalgae. A total of 10 genes related to heme synthesis pathway derived from Synechococcus elongatus PCC 7942 and 12 genes related to endogenous heme synthesis were individually overexpressed in strain PCC 6803. The growth rate and pigment content (heme, phycocyanin, chlorophyll a and carotenoids) of 22 recombinant algal strains were characterized. Quantitative real-time PCR technology was used to investigate the molecular mechanisms underlying the changes in physiological indicators in the recombinant algal strains. Among the 22 mutant strains, the mutant overexpressing the haemoglobin gene (glbN) of strain PCC 6803 had the highest heme content, which was 2.5 times higher than the wild type; the mutant overexpressing the gene of strain PCC 7942 (hemF) had the highest phycocyanin content, which was 4.57 times higher than the wild type. Overall, the results suggest that genes in the porphyrin pathway could significantly affect the heme and phycocyanin content in strain PCC 6803. Our study provides novel crucial targets for promoting the accumulation of heme and phycocyanin in cyanobacteria.

Plastid Engineering of a Marine Alga, Nannochloropsis gaditana, for Co-Expression of Two Recombinant Peptides.

The purpose of this study was to establish a plastid transformation system for expressing recombinant proteins in Nannochloropsis gaditana. On the basis of the sequenced plastid genome, the homologous flanking region, 16S-trnI/trnA-23S, and the endogenous regulatory fragments containing the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator were amplified from N. gaditana as elements of a plastid transformation vector. Then, the herbicide-resistant gene (bar) was used as a selectable marker, regulated by the psbA promoter and rbcL terminator. Finally, two codon-optimized antimicrobial peptide-coding genes linked by endogenous ribosome binding site (RBS) in a polycistron were inserted into the constructed vector under the regulation of the rbcL promoter and psbA terminator. After microparticle bombardment, the positive clones were detected using polymerase chain reaction (PCR), and Southern and Western blotting were used to assess the co-expression of the two antimicrobial peptides from the plastid. Nannochloropsis gaditana showed the potential to express recombinant proteins for biotechnological applications, for example, for the development of oral vaccines in aquaculture.

Transcriptomic and Proteomic Characterizations of the Molecular Response to Blue Light and Salicylic Acid in Haematococcus pluvialis.

Haematococcus pluvialis accumulates a large amount of astaxanthin under various stresses, e.g., blue light and salicylic acid (SA). However, the metabolic response of H. pluvialis to blue light and SA is still unclear. We investigate the effects of blue light and SA on the metabolic response in H. pluvialis using both transcriptomic and proteomic sequencing analyses. The largest numbers of differentially expressed proteins (DEPs; 324) and differentially expressed genes (DEGs; 13,555) were identified on day 2 and day 7 of the treatment with blue light irradiation (150 µmol photons m-2s-1), respectively. With the addition of SA (2.5 mg/L), a total of 63 DEPs and 11,638 DEGs were revealed on day 2 and day 7, respectively. We further analyzed the molecular response in five metabolic pathways related to astaxanthin synthesis, including the astaxanthin synthesis pathway, the fatty acid synthesis pathway, the heme synthesis pathway, the reactive oxygen species (ROS) clearance pathway, and the cell wall biosynthesis pathway. Results show that blue light causes a significant down-regulation of the expression of key genes involved in astaxanthin synthesis and significantly increases the expression of heme oxygenase, which shows decreased expression by the treatment with SA. Our study provides novel insights into the production of astaxanthin by H. pluvialis treated with blue light and SA.

Genome-wide identification and expression profiling of HD-ZIP gene family in Medicago truncatula.

The homeodomain-leucine zipper (HD-ZIP) transcription factors are important regulators in various developmental processes and responses to environmental stimuli. Currently, little information is available for HD-ZIP gene family in Medicago truncatula. Here we perform a genome-wide analysis of HD-ZIP gene family in M. truncatula. Totally 52 M. truncatula HD-ZIPs (MtHDZs) were identified and classified into four distinctive subfamilies (I to IV). Members clustered in the same subfamily shared similar gene structure and protein motifs. Fifty-one MtHDZs were non-evenly distributed on eight chromosomes. Segmental duplication and purifying selection mainly contributed to the expansion and retention of M. truncatula HD-ZIP gene family. Expression profiling using the publicly available microarray data revealed that MtHDZ genes exhibited distinctive tissue-specific patterns and divergent responses to drought and salt stresses. In addition, the expression profile between each paralogous pair diverged differentially. Our results identified potential targets for the genetic improvement of abiotic stress tolerance in Medicago.

Effects of Methanol on Carotenoids as Well as Biomass and Fatty Acid Biosynthesis in Schizochytrium limacinum B4D1.

Schizochytrium is a promising source for the production of docosahexaenoic acid and astaxanthin. The effects of different methanol concentrations on astaxanthin, biomass, and production of the lipids, squalene, and total sterol in Schizochytrium limacinum B4D1 were investigated. Astaxanthin began to accumulate when the methanol concentration reached 3.2% and peaked at 5.6% methanol, with a 2,000-fold increase over that in the control. However, under cultivation with 5.6% methanol, the biomass, lipids, squalene, and total sterol decreased to various degrees. Transcriptomic analysis was performed to explore the effects of different methanol concentrations (0%, 3.2%, and 5.6%) on the expression profile of B4D1. Three key signaling pathways were found to play important roles in regulating cell growth and metabolism under cultivation with methanol. Five central carbon metabolism-associated genes were significantly downregulated in response to 5.6% methanol and thus were expected to result in less ATP and NADPH being available for cell growth and synthesis. High methanol conditions significantly downregulated three genes involved in fatty acid and squalene/sterol precursor biosynthesis but significantly upregulated geranylgeranyl diphosphate synthase, lycopene ß-cyclase, and ß-carotene 3-hydroxylase, which are involved in astaxanthin synthesis, thus resulting in an increase in the levels of precursors and the final production of astaxanthin. Additionally, the transcriptional levels of three stress response genes were upregulated. This study investigates gene expression profiles in the astaxanthin producer Schizochytrium when grown under various methanol concentrations. These results broaden current knowledge regarding genetic expression and provide important information for promoting astaxanthin biosynthesis in SchizochytriumIMPORTANCESchizochytrium strains are usually studied as oil-producing strains, but they can also synthesize other secondary metabolites, such as astaxanthin. In this study, methanol was used as an inducer, and we explored its effects on the production of astaxanthin, a highly valuable substance in Schizochytrium Methanol induced Schizochytrium to synthesize large amounts of astaxanthin. Transcriptomic analysis was used to investigate the regulation of signaling and metabolic pathways (mainly relative gene expression) in Schizochytrium grown in the presence of various concentrations of methanol. These results contribute to the understanding of the underlying molecular mechanisms and may aid in the future optimization of Schizochytrium for astaxanthin biosynthesis.

Effects of zinc on the production of alcohol by Clostridium carboxidivorans P7 using model syngas.

Renewable energy, including biofuels such as ethanol and butanol from syngas bioconversed by Clostridium carboxidivorans P7, has been drawing extensive attention due to the fossil energy depletion and global eco-environmental issues. Effects of zinc on the growth and metabolites of C. carboxidivorans P7 were investigated with model syngas as the carbon source. The cell concentration was doubled, the ethanol content increased 3.02-fold and the butanol content increased 7.60-fold, the hexanol content increased 44.00-fold in the medium with 280 µM Zn2+, when comparing with those in the control medium [Zn2+, (7 µM)]. Studies of the genes expression involved in the carbon fixation as well as acid and alcohol production in the medium with 280 µM Zn2+ indicated that fdhII was up-regulated on the second day, acs A, fdhII, bdh35 and bdh50 were up-regulated on the third day and bdh35, acsB, fdhI, fdhIII, fdhIV, buk, bdh10, bdh35, bdh40 and bdh50 were up-regulated on the fourth day. The results indicated that the increased Zn2+ content increased the alcohol production through increase in the gene expression of the carbon fixation and alcohol dehydrogenase.

Oral Administration of Antimicrobial Peptide NZ2114 Through the Microalgal Bait Tetraselmis subcordiformis (Wille) Butcher for Improving the Immunity and Gut Health in Turbot (Scophthalmus maximus L.).

Antibiotics are widely used in aquaculture to treat the bacterial diseases. However, the improper use of antibiotics could lead to environmental pollution and development of resistance. As a safe and eco-friendly alternative, antimicrobial peptides (AMPs) are commonly explored as therapeutic agents. In this study, a mutant strain of Tetraselmis subcordiformis containing AMP NZ2114 was developed and used as an oral drug delivery system to reduce the use of antibiotics in turbot (Scophthalmus maximus) aquaculture. The gut, kidney, and liver immune-related genes and their effects on gut digestion and bacterial communities in turbot fed with NZ2114 were evaluated in an 11-day feeding experiment. The results showed that compared with the group fed with wild-type T. subcordiformis, the group fed with T. subcordiformis transformants containing NZ2114 was revealed with decreased levels of both pro-inflammatory factors (TNF-α and IL-1ß), inhibitory effect on Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio splendidus demonstrated by the in vitro simulation experiments, and increased richness and diversity of the gut microbiota of turbot. In conclusion, our study provided a novel, beneficial, and low-cost method for controlling bacteria in turbot culture through the oral drug delivery systems.

Analysis of mRNA expression profiles of carotenogenesis and astaxanthin production of Haematococcus pluvialis under exogenous 2, 4-epibrassinolide (EBR).

The fresh-water green unicellular alga Haematococcus pluvialis is known to accumulate astaxanthin under stress conditions. In the present study, transcriptional expression of eight genes involved in astaxanthin biosynthesis exposed to EBR (25 and 50 mg/L) was analyzed using qRT-PCR. The results demonstrated that both 25 and 50 mg/L EBR could increase astaxanthin productivity and the eight carotenogenic genes were up-regulated by EBR with different expression profiles. Moreover, EBR25 induction had a greater influence on the transcriptional expression of ipi-1, ipi-2, crtR-B, lyc and crtO (> 5- fold up-regulation) than on psy, pds, bkt; EBR50 treatment had a greater effect on the transcriptional expression of ipi-2, pds, lyc, crtR-B, bkt and crtO than on ipi-1 and psy. Furthermore, astaxanthin biosynthesis under EBR was up-regulated mainly by ipi1Ö¾ and psy at the post-transcriptional level, pds, lyc, crtR-B, bkt and crtO at the transcriptional level and ipi-2 at both levels.

Carotenoid genes transcriptional regulation for astaxanthin accumulation in fresh water unicellular alga Haematococcus pluvialis by gibberellin A3 (GA3).

The fresh water unicellular alga Haematococcus pluvialis is a promising natural source of astaxanthin. The present study investigated the transcriptional expression of carotenoid genes for astaxanthin accumulation in H. pluvialis using real-time fluorescence quantitative PCR (qRT-PCR). With treatments of 20 and 40 mg/L of gibberllin A3 (GA3), five genes ipi-1, ipi-2, psy, pds and bkt2 were up-regulated with different expression profiles. GA20 (20 mg/L of GA3) treatment had a greater effect on transcriptional expression of bkt2 than on ipi-1 ipi-2, psy and pds (> 4-fold up-regulation). However, GA40 (40 mg/L of GA3) induced more transcriptional expression of ipi-2, psy and bkt2 than both ipi-1 and pds. The expression of lyc, crtR-B and crtO for astaxanthin biosynthesis was not affected by GA3 in H. piuvialis. In the presence of GA3, astaxanthin biosynthesis genes of ipi-1, pds and bkt2 were up-regulated at transcriptional level, psy at post-transcriptional level, whereas ipi-2 was up-regulated at both levels. The study could potentially lead to a scale application of exogenous GA3 in astaxanthin production with H. pluvialis just like GAs perform in increasing crops production and it would provide new insight about the multifunctional roles of carotenogenesis in response to GA3.

Lipid accumulation mechanism of Amphora coffeaeformis under nitrogen deprivation and its application as a feed additive in Carassius auratus aquaculture.

BACKGROUND: Amphora coffeaeformis, a unicellular diatom, can significantly accumulate lipids under nitrogen (N) limitation. However, the molecular mechanism underlying lipid accumulation in A. coffeaeformis remains unknown and its application development is lagging. RESULTS: This work analyzed the lipid composition of A. coffeaeformis under N deprivation and investigated its mechanism underlying lipid accumulation using RNA-seq. The results showed that the total lipid content of A. coffeaeformis increased from 28.22 to 44.05% after 5 days of N deprivation, while the neutral lipid triacylglycerol (TAG) content increased from 10.41 to 25.21%. The transcriptional profile showed that N deprivation induced wide-ranging reprogramming of regulation and that most physiological activities were repressed, while the upregulation of glycerol-3-phosphate acyltransferase directly determined TAG accumulation. Moreover, we explored the effect of A. coffeaeformis as a food additive on the lipid composition of crucian carp. The results showed that the contents of unsaturated fatty acids in the meat of fish supplemented with A. coffeaeformis were significantly increased, indicating its potential application in animal nutrition for improving meat quality indicators. CONCLUSION: The findings shed light on the molecular mechanisms of neutral lipid accumulation and revealed the key genes involved in lipid metabolism in A. coffeaeformis. Moreover, we also confirmed that A. coffeaeformis can be used as feed additive for improving the lipid composition of crucian carp meat, which provided evidence for the biotechnology application of this high-oil microalgae.

Phycobiliproteins from algae: Current updates in sustainable production and applications in food and health.

Phycobiliproteins are light-harvesting complexes found mainly in cyanobacteria and red algae, playing a key role in photosynthesis. They are extensively applied in food, cosmetics, and biomedical industry due to bright color, unique fluorescence characteristics and diverse physiological activities. They have received much attention in the past few decades because of their green and sustainable production, safe application, and functional diversity. This work aimed to provide a comprehensive summary of parameters affecting the whole bioprocess with a special focus on the extraction and purification, which directly determines the application of phycobiliproteins. Food grade phycobiliproteins are easy to prepare, whereas analytical grade phycobiliproteins are extremely complex and costly to produce. Most phycobiliproteins are denatured and inactivated at high temperatures, severely limiting their application. Inspired by recent advances, future perspectives are put forward, including (1) the mutagenesis and screening of algal strains for higher phycobiliprotein productivity, (2) the application of omics and genetic engineering for stronger phycobiliprotein stability, and (3) the utilization of synthetic biology and heterologous expression systems for easier phycobiliprotein isolation. This review will give a reference for exploring more phycobiliproteins for food and health application development.

Synergistic degradation of Azure B and sulfanilamide antibiotics by the white-rot fungus Trametes versicolor with an activated ligninolytic enzyme system.

The treatment of complex polluted wastewater has become an increasingly critical concern for the various types of hazardous organic compounds, including synthetic dyes and pharmaceuticals. Due to their efficient and eco-friendly advantages, the white-rot fungi (WRF) have been applied to degrade environmental pollutants. This study aimed to investigate the removal ability of WRF (i.e., Trametes versicolor WH21) in the co-contamination system composed of Azure B dye and sulfacetamide (SCT). Our study discovered that the decolorization of Azure B (300 mg/L) by strain WH21 was significantly improved (from 30.5% to 86.5%) by the addition of SCT (30 mg/L), while the degradation of SCT was also increased from 76.4% to 96.2% in the co-contamination system. Transcriptomic and biochemical analyses indicated that the ligninolytic enzyme system was activated by the enhanced enzymatic activities of MnPs and laccases, generating higher concentration of extracellular H2O2 and organic acids in strain WH21 in response to SCT stress. Purified MnP and laccase of strain WH21 were revealed with remarkable degradation effect on both Azure B and SCT. These findings significantly expanded the existing knowledge on the biological treatment of organic pollutants, indicating the strong promise of WRF in the treatment of complex polluted wastewater.

Metabolic engineering and synthetic biology strategies for producing high-value natural pigments in Microalgae.

Microalgae are microorganisms capable of producing bioactive compounds using photosynthesis. Microalgae contain a variety of high value-added natural pigments such as carotenoids, phycobilins, and chlorophylls. These pigments play an important role in many areas such as food, pharmaceuticals, and cosmetics. Natural pigments have a health value that is unmatched by synthetic pigments. However, the current commercial production of natural pigments from microalgae is not able to meet the growing market demand. The use of metabolic engineering and synthetic biological strategies to improve the production performance of microalgal cell factories is essential to promote the large-scale production of high-value pigments from microalgae. This paper reviews the health and economic values, the applications, and the synthesis pathways of microalgal pigments. Overall, this review aims to highlight the latest research progress in metabolic engineering and synthetic biology in constructing engineered strains of microalgae with high-value pigments and the application of CRISPR technology and multi-omics in this context. Finally, we conclude with a discussion on the bottlenecks and challenges of microalgal pigment production and their future development prospects.

Cytochrome P450s in algae: Bioactive natural product biosynthesis and light-driven bioproduction.

Algae are a large group of photosynthetic organisms responsible for approximately half of the earth's total photosynthesis. In addition to their fundamental ecological roles as oxygen producers and as the food base for almost all aquatic life, algae are also a rich source of bioactive natural products, including several clinical drugs. Cytochrome P450 enzymes (P450s) are a superfamily of biocatalysts that are extensively involved in natural product biosynthesis by mediating various types of reactions. In the post-genome era, a growing number of P450 genes have been discovered from algae, indicating their important roles in algal life-cycle. However, the functional studies of algal P450s remain limited. Benefitting from the recent technical advances in algae cultivation and genetic manipulation, the researches on P450s in algal natural product biosynthesis have been approaching to a new stage. Moreover, some photoautotrophic algae have been developed into "photo-bioreactors" for heterologous P450s to produce high-value added pharmaceuticals and chemicals in a carbon-neutral or carbon-negative manner. Here, we comprehensively review these advances of P450 studies in algae from 2000 to 2021.

Transcriptome Analysis of the Accumulation of Astaxanthin in Haematococcus pluvialis Treated with White and Blue Lights as well as Salicylic Acid.

Haematococcus pluvialis is the most commercially valuable microalga for the production of natural astaxanthin, showing enhanced production of astaxanthin with the treatments of high-intensity light and hormones. The molecular mechanisms regulating the biosynthesis of astaxanthin in H. pluvialis treated with white light, blue light, and blue light with salicylic acid (SA) were investigated based on the transcriptome analysis. Results showed that the combined treatment with both blue light and SA generated the highest production of astaxanthin. A total of 109,443 unigenes were identified to show that the genes involved in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), and the astaxanthin biosynthesis were significantly upregulated to increase the production of the substrates for the synthesis of astaxanthin, i.e., pyruvate and glyceraldehyde-3-phosphate generated in the TCA cycle and PPP, respectively. Results of transcriptome analysis were further verified by the quantitative real-time PCR (qRT-PCR) analysis, showing that the highest content of astaxanthin was obtained with the expression of the bkt gene significantly increased. Our study provided the novel insights into the molecular mechanisms regulating the synthesis of astaxanthin and an innovative strategy combining the exogenous hormone and physical stress to increase the commercial production of astaxanthin by H. pluvialis.