Complete microbial synthesis of crocetin and crocins from glycerol in Escherichia coli.

BACKGROUND: Crocin, a glycosylated apocarotenoid pigment predominantly found in saffron, has garnered significant interest in the field of biotechnology for its bioactive properties. Traditional production of crocins and their aglycone, crocetin, typically involves extraction from crocin-producing plants. This study aimed to develop an alternative biosynthetic method for these compounds by engineering the metabolic pathways of zeaxanthin, crocetin, and crocin in Escherichia coli strains. RESULTS: Employing a series of genetic modifications and the strategic overexpression of key enzymes, we successfully established a complete microbial pathway for synthesizing crocetin and four glycosylated derivatives of crocetin, utilizing glycerol as the primary carbon source. The overexpression of zeaxanthin cleavage dioxygenase and a novel variant of crocetin dialdehyde dehydrogenase resulted in a notable yield of crocetin (34.77 ± 1.03 mg/L). Further optimization involved the overexpression of new types of crocetin and crocin-2 glycosyltransferases, facilitating the production of crocin-1 (6.29 ± 0.19 mg/L), crocin-2 (5.29 ± 0.24 mg/L), crocin-3 (1.48 ± 0.10 mg/L), and crocin-4 (2.72 ± 0.13 mg/L). CONCLUSIONS: This investigation introduces a pioneering and integrated microbial synthesis method for generating crocin and its derivatives, employing glycerol as a sustainable carbon feedstock. The substantial yields achieved highlight the commercial potential of microbial-derived crocins as an eco-friendly alternative to plant extraction methods. The development of these microbial processes not only broadens the scope for crocin production but also suggests significant implications for the exploitation of bioengineered compounds in pharmaceutical and food industries.

Rapid Biodegradable Ionic Aggregates of Polyesters Constructed with Fertilizer Ingredients.

Synthetic biodegradable polyesters tend to undergo slow biodegradation under ambient natural conditions and, hence, have been rejected or even banned recently in ecofriendly applications. Here, we demonstrate the preparation of polyesters exhibiting enhanced biodegradability, which were generated through a combination of old controversial macromolecules and aggregate theories. H3PO4-catalyzed diacid/diol polycondensation afforded polyester chains bearing chain-end -CH2OP(O)(OH)2 and inner-chain (-CH2O)2P(O)(OH) groups, which were subsequently treated with M(2-ethylhexanoate)2 (M = Zn, Mg, Mn, and Ca) to form ionic aggregates of polyesters. The prepared ionic aggregates of polyesters, which were constructed with fertilizer ingredients (such as M2+ and phosphate), exhibit much faster biodegradability than that of the conventional polyesters under controlled soil conditions at 25 °C, while displaying comparable or superior rheological and mechanical properties.

Melanin biopolymer synthesis using a new melanogenic strain of Flavobacterium kingsejongi and a recombinant strain of Escherichia coli expressing 4-hydroxyphenylpyruvate dioxygenase from F. kingsejongi.

BACKGROUND: Melanins are a heterologous group of biopolymeric pigments synthesized by diverse prokaryotes and eukaryotes and are widely utilized as bioactive materials and functional polymers in the biotechnology industry. Here, we report the high-level melanin production using a new melanogenic Flavobacterium kingsejongi strain and a recombinant Escherichia coli overexpressing F. kingsejongi 4-hydroxyphenylpyruvate dioxygenase (HPPD). RESULTS: Melanin synthesis of F. kingsejongi strain was confirmed via melanin synthesis inhibition test, melanin solubility test, genome analysis, and structural analysis of purified melanin from both wild-type F. kingsejongi and recombinant E. coli expressing F. kingsejongi HPPD. The activity of F. kingsejongi HPPD was demonstrated via in vitro assays with 6 × His-tagged and native forms of HPPD. The specific activity of F. kingsejongi HPPD was 1.2 ± 0.03 µmol homogentisate/min/mg-protein. Bioreactor fermentation of F. kingsejongi produced a large amount of melanin with a titer of 6.07 ± 0.32 g/L, a conversion yield of 60% (0.6 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.03 g/L·h, indicating its potential for industrial melanin production. Additionally, bioreactor fermentation of recombinant E. coli expressing F. kingsejongi HPPD produced melanin at a titer of 3.76 ± 0.30 g/L, a conversion yield of 38% (0.38 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.04 g/L·h. CONCLUSIONS: Both strains showed sufficiently high fermentation capability to indicate their potential as platform strains for large-scale bacterial melanin production. Furthermore, F. kingsejongi strain could serve as a model to elucidate the regulation of melanin biosynthesis pathway and its networks with other cellular pathways, and to understand the cellular responses of melanin-producing bacteria to environmental changes, including nutrient starvation and other stresses.

Comparative Genomic Analysis of Agarolytic Flavobacterium faecale WV33T.

Flavobacteria are widely dispersed in a variety of environments and produce various polysaccharide-degrading enzymes. Here, we report the complete genome of Flavobacterium faecale WV33T, an agar-degrading bacterium isolated from the stools of Antarctic penguins. The sequenced genome of F. faecale WV33T represents a single circular chromosome (4,621,116 bp, 35.2% G + C content), containing 3984 coding DNA sequences and 85 RNA-coding genes. The genome of F. faecale WV33T contains 154 genes that encode carbohydrate-active enzymes (CAZymes). Among the CAZymes, seven putative genes encoding agarases have been identified in the genome. Transcriptional analysis revealed that the expression of these putative agarases was significantly enhanced by the presence of agar in the culture medium, suggesting that these proteins are involved in agar hydrolysis. Pangenome analysis revealed that the genomes of the 27 Flavobacterium type strains, including F. faecale WV33T, tend to be very plastic, and Flavobacterium strains are unique species with a tiny core genome and a large non-core region. The average nucleotide identity and phylogenomic analysis of the 27 Flavobacterium-type strains showed that F. faecale WV33T was positioned in a unique clade in the evolutionary tree.

Psychrobacillus glaciei sp. nov., a psychrotolerant species isolated from an Antarctic iceberg.

We performed taxonomic studies on a psychrotolerant strain, designated PB01T, isolated from an Antarctic iceberg. The cells of strain PB01T were Gram-stain-positive, strictly aerobic, white-yellow and rod-shaped. The results of 16S rRNA gene sequence analysis revealed that strain PB01T was closely related to Psychrobacillus psychrodurans DSM 11713T (99.19 % similarity), Psychrobacillus psychrotolerans DSM 11706T (98.91 %) and Psychrobacillus insolitus DSM 5T (98.85 %). Despite high 16S rRNA gene sequence similarity, the degrees of DNA-DNA relatedness between strain PB01T and its three closest phylogenetic neighbours were 62.4±7.3 % for P. psychrodurans DSM 11713T, 61.1±5.4 % for P. psychrotolerans DSM 11706T and 56.1±6.9 % for P. insolitus DSM 5T. The predominant cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and C16 : 1ω7с-OH. Menaquinone-8 was the major respiratory quinone, and phosphatidylethanolamine was the major polar lipid. The DNA G+C content of strain PB01T calculated from the complete genome sequence was 36.0 mol%. Based on the phenotypic, chemotaxonomic, genomic and phylogenetic data obtained in the present study, we conclude that strain PB01T represents a novel species of the genus Psychrobacillus, for which we propose the name Psychrobacillus glaciei sp. nov. The type strain is PB01T (=CECT 9792T=KCTC 43041T).

Redesign and reconstruction of a steviol-biosynthetic pathway for enhanced production of steviol in Escherichia coli.

BACKGROUND: Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry. Recently, efforts were made to produce these natural sweeteners in microorganisms using metabolic engineering. Nonetheless, the steviol titer is relatively low in metabolically engineered microorganisms, and therefore a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The purpose of this study was to redesign and reconstruct a steviol-biosynthetic pathway via synthetic-biology approaches in order to overproduce steviol in Escherichia coli. RESULTS: A genome-engineered E. coli strain, which coexpressed 5' untranslated region (UTR)-engineered geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, and kaurene synthase, produced 623.6 ± 3.0 mg/L ent-kaurene in batch fermentation. Overexpression of 5'-UTR-engineered, N-terminally modified kaurene oxidase of Arabidopsis thaliana yielded 41.4 ± 5 mg/L ent-kaurenoic acid. Enhanced ent-kaurenoic acid production (50.7 ± 9.8 mg/L) was achieved by increasing the cellular NADPH/NADP+ ratio. The expression of a fusion protein, UtrCYP714A2-AtCPR2 derived from A. thaliana, where trCYP714A2 was 5'-UTR-engineered and N-terminally modified, gave 38.4 ± 1.7 mg/L steviol in batch fermentation. CONCLUSIONS: 5'-UTR engineering, the fusion protein approach, and redox balancing improved the steviol titer in flask fermentation and bioreactor fermentation. The expression engineering of steviol-biosynthetic enzymes and the genome engineering described here can serve as the basis for producing terpenoids-including steviol glycosides and carotenoids-in microorganisms.

Genome Mining Reveals Two Missing CrtP and AldH Enzymes in the C30 Carotenoid Biosynthesis Pathway in Planococcus faecalis AJ003T.

Planococcus faecalis AJ003T produces glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid as its main carotenoid. Five carotenoid pathway genes were presumed to be present in the genome of P. faecalis AJ003T; however, 4,4-diaponeurosporene oxidase (CrtP) was non-functional, and a gene encoding aldehyde dehydrogenase (AldH) was not identified. In the present study, a genome mining approach identified two missing enzymes, CrtP2 and AldH2454, in the glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid biosynthetic pathway. Moreover, CrtP2 and AldH enzymes were functional in heterologous Escherichia coli and generated two carotenoid aldehydes (4,4'-diapolycopene-dial and 4,4'-diaponeurosporene-4-al) and two carotenoid carboxylic acids (4,4'-diaponeurosporenoic acid and 4,4'-diapolycopenoic acid). Furthermore, the genes encoding CrtP2 and AldH2454 were located at a distance the carotenoid gene cluster of P. faecalis.

Characterization of Carotenoid Biosynthesis in Newly Isolated Deinococcus sp. AJ005 and Investigation of the Effects of Environmental Conditions on Cell Growth and Carotenoid Biosynthesis.

Our purpose was to characterize the structures of deinoxanthin from Deinococcus sp. AJ005. The latter is a novel reddish strain and was found to synthesize two main acyclic carotenoids: deinoxanthin and its derivative. The derivative (2-keto-deinoxanthin) contains a 2-keto functional group instead of a 2-hydroxyl group on a ß-ionone ring. A deinoxanthin biosynthesis pathway of Deinococcus sp. AJ005 involving eight putative enzymes was proposed according to genome annotation analysis and chemical identification of deinoxanthin. Optimal culture pH and temperature for Deinococcus sp. AJ005 growth were pH 7.4 and 20 °C. Sucrose as a carbon source significantly enhanced the cell growth in comparison with glucose, glycerol, maltose, lactose, and galactose. When batch fermentation was performed in a bioreactor containing 40g/L sucrose, total carotenoid production was 650% higher than that in a medium without sucrose supplementation. The culture conditions found in this study should provide the basis for the development of fermentation strategies for the production of deinoxanthin and of its derivative by means of Deinococcus sp. AJ005.

Flavobacterium kingsejongi sp. nov., a carotenoid-producing species isolated from Antarctic penguin faeces.

Taxonomic studies were carried out on a carotenoid-producing strain, designated WV39T, isolated from the faeces of Antarctic penguins. Cells of strain WV39T were Gram-stain-negative, strictly aerobic, yellow and rod-shaped. 16S rRNA gene sequence analysis revealed that strain WV39T was closely related to Flavobacterium qiangtangense JCM 19739T (96.3 % similarity), Flavobacterium noncentrifugens NBRC 108844T (95.5 %) and Flavobacterium aquatile LMG 4008T (94.9 %). The predominant cellular fatty acids were iso-C15 : 0, iso-C15 : 0 3-OH and summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c). Menaquinone-6 was the sole quinone identified, and the major pigment was zeaxanthin. The major polar lipid was phosphatidylethanolamine. DNA-DNA relatedness of strain WV39T with respect to its closest phylogenetic neighbours was 41.8 % for F. qiangtangense JCM 19739T, 25.5 % for F. aquatile LMG 4008T and 25.2 % for F. noncentrifugens NBRC 108844T. The DNA G+C content of strain WV39T was 39.8 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain WV39T is concluded to represent a novel species of the genus Flavobacterium, for which the name Flavobacteriumkingsejongi sp. nov. is proposed. The type strain is WV39T (=KCTC 42908T=CECT 9085T).

Sphingomonas lacus sp. nov., an astaxanthin-dideoxyglycoside-producing species isolated from soil near a pond.

Taxonomic studies were performed on an astaxanthin-dideoxyglycoside-producing strain, designated PB304(T), isolated from soil near a pond in Daejeon city, South Korea. Cells of strain PB304(T) were Gram-staining-negative, strictly aerobic, orange-coloured and motile, and occurred as single or paired short chains. PB304(T) did not contain bacteriochlorophyll a. 16S rRNA gene sequence analysis revealed that strain PB304(T) was closely related to 'Sphingomonas humi' KCTC 12341 (98.7%), Sphingomonas kaistensis KCTC 12344(T)(97.9%), Sphingomonas astaxanthinifaciens DSM 22298(T) (97.6%) and Sphingomonas ginsengisoli KCTC 12630(T) (97.5%). Analysis of pufLM gene sequences revealed strain PB304(T) to be closely related to 'S. humi' KCTC 12341 (88.1%). The major cellular fatty acids were C16 : 0, summed feature 4 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c), and summed feature 7 (comprising C18  : 1ω7c/ω9t/ω12t). Ubiquinone 10 (Q-10) was the sole quinone identified, and the major pigment was astaxanthin dideoxyglycoside. The major polar lipids were sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The polyamine was spermidine. The DNA-DNA relatedness values of strain PB304(T) with respect to its closest phylogenetic neighbours were 57.1% for 'S. humi' KCTC 12341, 51.2% for Sphingomonas kaistensis KCTC 12334T, 50.6% for Sphingomonas astaxanthinifaciens DSM 22298(T) and 50.2% for Sphingomonas ginsengisoli KCTC 12630(T). The DNA G+C content of strain PB304(T) was 66.6 mol%. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain PB304T is concluded to represent a novel species of the genus Sphingomonas, for which the name Sphingomonas lacus is proposed. The type strain is PB304(T) ( = KCTC 32458(T) = CECT 8383(T)).

Planococcus faecalis sp. nov., a carotenoid-producing species isolated from stools of Antarctic penguins.

Taxonomic studies were performed on a novel carotenoid-producing strain, designated AJ003T, isolated from faeces of Antarctic penguins. Cells of strain AJ003T were aerobic, Gram-stain-positive, cocci-shaped and orange. Strain AJ003T was capable of growing in a broad temperature range, including sub-zero growth (below − 20 to 30 °C). 16S rRNA gene sequence analysis revealed that strain AJ003T was closely related to Planococcus halocryophilus Or1T (97.4 % similarity), Planococcus antarcticus DSM 14505T (97.3 %), Planococcus kocurii NCIMB 629T (97.3 %), and Planococcus donghaensis JH1T (97.1 %). The predominant cellular fatty acids were anteiso-C15 : 0, and iso-C16 : 0.MK-7 and MK-8 were the quinones identified, and the major pigment was glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid. The major polar lipid was phosphatidylglycerol. DNA­DNA relatedness of strain AJ003T with respect to its closest phylogenetic neighbours was 38.2 ± 0.5 % for Planococcus halocryophilus DSM 24743T, 32.2 ± 0.2 % for Planococcus antarcticus DSM 14505T, 21.0 ± 0.3 % for Planococcus kocurii DSM 20747T and 18.6 ± 1.4 % for Planococcus donghaensis KCTC 13050T. The DNA G+C content of strain AJ003T was 40.0 ± 0.6 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain AJ003T is concluded to represent a novel species of the genus Planococcus, for which the name Planococcus faecalis sp. nov. is proposed. The type strain is AJ003T ( = KCTC 33580T = CECT 8759T).

Proposed cytotoxic mechanisms of the saffron carotenoids crocin and crocetin on cancer cell lines.

We investigated the cytotoxic activities of crocin and crocetin, 2 major carotenoids isolated from the stigma of Crocus sativus (saffron), on 5 human cancer cell lines and proposed their possible anticancer mechanisms. Crocetin, a glycosylated carotenoid, showed approximately 5- to 18-fold higher cytotoxicity than crocin, a carboxylic carotenoid (IC50 of 0.16-0.61 mmol/L for crocetin vs. 2.0-5.5 mmol/L for crocin). This suggests that structural differences account for the different efficacies between them. Fluorescence-activated cell sorting (FACS) analysis showed that crocetin induced a significant level of cellular reactive oxygen species (ROS) in HeLa cells, whereas crocin did not. This ROS induction supported the cytotoxicity of crocetin, but not of crocin. A significant activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was observed in both HeLa cells treated with crocin and crocetin: a 3.0-fold increase by 1 mmol/L crocetin and a 1.6-fold increase by 0.8 mmol/L crocin compared to the control. Furthermore, both crocetin and crocin reduced the protein expression of lactate dehydrogenase A (LDHA), one of the targets for chemoprevention in cancer cells, by 34.2% and 10.5%, respectively, compared to the control in HeLa cells. These findings suggest that crocetin and crocin have different mechanisms for their observed cytotoxicity in cancer cell lines.

Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4-butanediol pathway in Escherichia coli.

1,4-Butanediol (1,4-BDO) is currently produced from succinate via six enzymatic reactions in an engineered Escherichia coli strain. Butyraldehyde dehydrogenase (Bld) and butanol dehydrogenase of Clostridium saccharoperbutylacetonicum were selected based on their activities of catalyzing the final two reactions in the 1,4-BDO pathway. To fit Bld into the non-natural 1,4-BDO pathway, we engineered it through random mutagenesis. Five Bld mutants were then isolated using a colorimetric Schiff's reagent-based method. Subsequent site-directed mutagenesis of Bld generated the two best Bld mutants, L273I and L273T, which produced 1,4-BDO titers fourfold greater than those of wild-type Bld. The enhanced 1,4-BDO titers obtained using L273I and L273T clearly correlated with their enhanced activities, which were caused by amino acid mutations at position 273 of Bld. The highest titer of 1,4-BDO (660 ± 40 mg/L) was obtained in a knock-out E. coli strain [ΔldhA ΔpflB ΔadhE ΔlpdA::K. lpd(E354K) Δmdh ΔarcA gltA(R164L)] coexpressing Bld273T+Bdh.

Flavobacterium faecale sp. nov., an agarase-producing species isolated from stools of Antarctic penguins.

Taxonomic studies were performed on an agarase-producing strain, designated WV33(T), isolated from faeces of Antarctic penguins. Cells of strain WV33(T) were Gram-staining-negative, strictly aerobic, orange and rod-shaped. Strain WV33(T) displayed agarase activity and was able to utilize galactose as a sole carbon source. 16S rRNA gene sequence analysis revealed that strain WV33(T) was closely related to Flavobacterium algicola TC2(T) (98.0% similarity), F. frigidarium ATCC 700810(T) (96.9%) and F. frigoris LMG 21922(T) (96.1%). The predominant cellular fatty acids were iso-C(15 : 1) G, iso-C(15 : 0), C(15 : 0), C(16 : 0) and summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c). Menaquinone 6 (MK-6) was the sole quinone identified, and the major pigment was zeaxanthin. The major polar lipid was phosphatidylethanolamine. DNA-DNA relatedness of strain WV33(T) with respect to its closest phylogenetic neighbours was 25% for F. algicola NBRC 102673(T), 23% for F. frigidarium DSM 17623(T) and 21% for F. frigoris DSM 15719(T). The DNA G+C content of strain WV33(T) was 37±0.6 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain WV33(T) is concluded to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium faecale sp. nov. is proposed. The type strain is WV33(T) ( = KCTC 32457(T) = CECT 8384(T)).

The astaxanthin dideoxyglycoside biosynthesis pathway in Sphingomonas sp. PB304.

A major carotenoid in Sphingomonas sp. PB304, originally isolated from a river in Daejon City, South Korea, was identified as astaxanthin dideoxyglycoside. Gene clusters encoding the astaxanthin dideoxyglycoside biosynthetic enzymes were identified by screening Sphingomonas sp. PB304 fosmid libraries using degenerate probes that harbor highly conserved sequences from the Sphigomonas elodea-derived crtI and Nostoc sp. PCC 7120-dervied crtW genes. Selected positive gene clusters were fully sequenced and annotated, revealing genes encoding six putative carotenogenic enzymes: phytoene synthase (CrtB), phytoene desaturase (CrtI), lycopene cyclase (CrtY), carotene hydroxylase (CrtZ), carotene ketolase (CrtW), and glycosyltransferase (CrtX). All of the carotenogenic enzymes, except for CrtX, were functional in the recombinant host Escherichia coli expressing synthetic carotenogenic modules from Pantoea agglomerans. CrtX did not take up UDP-glucose or GDP-fucose as sugar substrates during the in vitro reaction. Although no direct experimental evidence was obtained for the function of Sphingomonas sp. PB304 CrtX, it can be categorized as a putative deoxyglycosyltransferase based on the presence of astaxanthin dideoxyglycoside in Sphingomonas sp. PB304, a putative corresponding gene in the carotenoid biosynthetic gene cluster, and high amino acid sequence homology to the existing glycosyltransferases. Therefore, we propose that astaxanthin dideoxyglycoside can be synthesized in Sphingomonas sp. PB304 via sequential reactions of six pathway enzymes, including CrtX on the phytoene intermediate.

Carotenoid production from n-alkanes with a broad range of chain lengths by the novel species Gordonia ajoucoccus A2(T).

A novel diesel-degrading bacterial strain, A2(T), was isolated from soil that was heavily contaminated with oil. Based on phenotypic, phylogenetic, and DNA analyses, strain A2(T) was identified as a novel species of the genus Gordonia and named Gordonia ajoucoccus A2(T) (KCTC 11900BP and CECT8382). G. ajoucoccus A2(T) is able to synthesize carotenoids and produces mainly γ-carotene and keto-γ-carotene. G. ajoucoccus A2(T) is also capable of assimilating n-alkanes with a broad range of chain lengths (C6, C8-C25). Batch culture of G. ajoucoccus A2(T) in a bioreactor containing 1 % (v/v) hexadecane or 1 % (v/v) commercial diesel yielded 25 mg L⁻¹ and 2.6 mg L⁻¹ of carotenoids, respectively. Gas chromatography/mass spectrometry (GC-MS) analysis of hexadecane and hexane degradation metabolites suggested that G. ajoucoccus A2(T) may possess a terminal oxidation pathway that allows it to utilize n-alkanes and hexane as carbon and energy sources. G. ajoucoccus A2(T) could therefore serve as a good model system for understanding microbial n-alkane degradation pathways. Additionally, the metabolic capabilities of G. ajoucoccus A2(T) suggest potential biotechnological applications, such as the bioproduction of carotenoids from industrial discharge or other sources of n-alkanes.

Development of microalga Scenedesmus dimorphus mutant with higher lipid content by radiation breeding.

In this study, a high lipid-accumulating mutant strain of the microalgae Scenedesmus dimorphus was developed via radiation breeding. To induce mutant strain, S. dimorphus was gamma-irradiated at doses from 100 to 800 Gy, and then a mutant (Sd-Pm210) with 25 % increased lipid content was selected using Nile red staining methodology. Sd-Pm210 showed morphological changes and had higher growth rate compared to the wild type. From random amplified polymorphic DNA analysis, partial genetic modifications were also observed in Sd-Pm210. In comparisons of lipid content between wild type and Sd-Pm210 using thin-layer chromatography, the content of triacylglycerol was markedly higher in the Sd-Pm210 strain. The total peak area of fatty acid methyl ester was shown to have about 1.4-fold increase in Sd-Pm210, and major fatty acids were identified as palmitic acid, oleic acid, linoleic acid, and linolenic acid. To define the metabolic changes in the mutant strain, 2-dimensional electrophoresis was conducted. Several proteins related to lipid synthesis and energy metabolisms were overexpressed in the mutant strain. These results showed that radiation breeding can be utilized for the development of efficient microalgae strains for biofuel production.

Metagenomic Analysis of Antarctic Ocean near the King Sejong Station Reveals the Diversity of Carotenoid Biosynthetic Genes.

Carotenoids, biotechnologically significant pigments, play crucial biological roles in marine microorganisms. While various environments have been explored to understand the diversity of carotenoids and their biosynthesis, the Antarctic Ocean remains relatively under-investigated. This study conducted a metagenomic analysis of seawater from two depths (16 and 25 m) near the King Sejong Station in the Antarctic Ocean. The analysis revealed a rich genetic diversity underlying C40 (astaxanthin, myxol, okenone, spheroidene, and spirilloxanthin), C30 (diaponeurosporene, diapolycopene, and staphyloxanthin), and C50 (C.p. 450) carotenoid biosynthesis in marine microorganisms, with notable differential gene abundances between depth locations. Exploring carotenoid pathway genes offers the potential for discovering diverse carotenoid structures of biotechnological value and better understanding their roles in individual microorganisms and broader ecosystems.

Ductile Copolyesters Prepared Using Succinic Acid, 1,4-Butanediol, and Bis(2-hydroxyethyl) Terephthalate with Minimizing Generation of Tetrahydrofuran.

Poly(1,4-butylene succinate) (PBS) is a promising sustainable and biodegradable synthetic polyester. In this study, we synthesized PBS-based copolyesters by incorporating 5-20 mol% of -O2CC6H4CO2- and -OCH2CH2O- units through the polycondensation of succinic acid (SA) with 1,4-butanediol (BD) and bis(2-hydroxyethyl) terephthalate (BHET). Two different catalysts, H3PO4 and the conventional catalyst (nBuO)4Ti, were used comparatively in the synthesis process. The copolyesters produced using the former were treated with M(2-ethylhexanoate)2 (M = Mg, Zn, Mn) to connect the chains through ionic interactions between M2+ ions and either -CH2OP(O)(OH)O- or (-CH2O)2P(O)O- groups. By incorporating BHET units (i.e., -O2CC6H4CO2- and -OCH2CH2O-), the resulting copolyesters exhibited improved ductile properties with enhanced elongation at break, albeit with reduced tensile strength. The copolyesters prepared with H3PO4/M(2-ethylhexanoate)2 displayed a less random distribution of -O2CC6H4CO2- and -OCH2CH2O- units, leading to a faster crystallization rate, higher Tm value, and higher yield strength compared to those prepared with (nBuO)4Ti using the same amount of BHET. Furthermore, they displayed substantial shear-thinning behavior in their rheological properties due to the presence of long-chain branches of (-CH2O)3P=O units. Unfortunately, the copolyesters prepared with H3PO4/M(2-ethylhexanoate)2, and hence containing M2+, -CH2OP(O)(OH)O-, (-CH2O)2P(O)O- groups, did not exhibit enhanced biodegradability under ambient soil conditions.

Glycerol-derived organic carbonates: environmentally friendly plasticizers for PLA.

Polylactic acid (PLA) stands as a promising material, sourced from renewables and exhibiting biodegradability-albeit under stringent industrial composting settings. A primary challenge impeding PLA's broad applications is its inherent brittleness, as it fractures with minimal elongation despite its commendable tensile strength. A well-established remedy involves blending PLA with plasticizers. In this study, a range of organic carbonates-namely, 4-ethoxycarbonyloximethyl-[1,3]dioxolan-2-one (1), 4-methoxycarbonyloximethyl-[1,3]dioxolan-2-one (2), glycerol carbonate (3), and glycerol 1-acetate 2,3-carbonate (4)-were synthesized on a preparative scale (∼100 g), using renewable glycerol and CO2-derived diethyl carbonate (DEC) or dimethyl carbonate (DMC). Significantly, 1-4 exhibited biodegradability under ambient conditions within a week, ascertained through soil exposure at 25 °C-outpacing the degradation of comparative cellulose. Further investigations revealed 1's efficacy as a PLA plasticizer. Compatibility with PLA, up to 30 phr (parts per hundred resin), was verified using an array of techniques, including DSC, DMA, SEM, and rotational rheometry. The resulting blends showcased enhanced ductility, evident from tensile property measurements. Notably, the novel plasticizer 1 displayed an advantage over conventional acetyltributylcitrate (ATBC) in terms of morphological stability. Slow crystallization, observed in PLA/ATBC blends over time at room temperature, was absent in PLA/1 blends, preserving amorphous domain dimensions and mitigating plasticizer migration-confirmed through DMA assessments of aged and unaged specimens. Nevertheless, biodegradation assessments of the blends revealed that the biodegradable organic carbonate plasticizers did not augment PLA's biodegradation. The PLA in the blends remained mostly unchanged under ambient soil conditions of 25 °C over a 6 month period. This work underscores the potential of organic carbonates as both eco-friendly plasticizers for PLA and as biodegradable compounds, contributing to the development of environmentally conscious polymer systems.