EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.     

Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens

xynB is one of at least four genes from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that encode xylanase activity. The xynB gene is predicted to encode a 781-amino acid product starting with a signal peptide, followed by an amino-terminal xylanase domain which is identical at 89% and 78% of residues, respectively, to the amino-terminal xylanase domains of the bifunctional XynD and XynA enzymes from the same organism. Two separate regions within the carboxy-terminal 537 amino acids of XynB also show close similarities with domain B of XynD. These regions show no significant homology with cellulose- or xylan-binding domains from other species, or with any other sequences, and their functions are unknown. In addition a 30 to 32-residue threonine-rich region is present in both XynD and XynB. Codon usage shows a consistent pattern of bias in the three xylanase genes from R. flavefaciens that have been sequenced.     

Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens

xynB is one of at least four genes from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that encode xylanase activity. The xynB gene is predicted to encode a 781-amino acid product starting with a signal peptide, followed by an amino-terminal xylanase domain which is identical at 89% and 78% of residues, respectively, to the amino-terminal xylanase domains of the bifunctional XynD and XynA enzymes from the same organism. Two separate regions within the carboxy-terminal 537 amino acids of XynB also show close similarities with domain B of XynD. These regions show no significant homology with cellulose- or xylan-binding domains from other species, or with any other sequences, and their functions are unknown. In addition a 30 to 32-residue threonine-rich region is present in both XynD and XynB. Codon usage shows a consistent pattern of bias in the three xylanase genes from R. flavefaciens that have been sequenced.     

Sequence of a cellulase gene from the rumen anaerobe Ruminococcus flavefaciens 17

Summary A cellulase gene (endA) was isolated from a library of Ruminococcus flavefaciens strain 17 DNA fragments inserted in pUC13. The endA product showed activity against acid-swollen cellulose, carboxymethyl-cellulose, lichenan, cellopentaose and cellotetraose, but showed no activity against cellotriose or binding to avicel. Nucleotide sequencing indicated an encoded product of 455 amino acids which showed significant sequence similarity (ranging from 56% to 61%) with three endoglucanases from Ruminococcus albus, and with Clostridium thermocellum endoglucanase E. Little relatedness was found with a cellodextrinase previously isolated from R. flavefaciens FD1.     

Influence of Plant Phenolic Acids on Growth and Cellulolytic Activity of Rumen Bacteria

Isolated rumen bacteria were examined for growth and, where appropriate, for their ability to degrade cellulose in the presence of the hydroxycinnamic acids trans-p-coumaric acid and trans-ferulic acid and the hydroxybenzoic acids vanillic acid and 4-hydroxybenzoic acid. Ferulic and p-coumaric acids proved to be the most toxic of the acids examined and suppressed the growth of the cellulolytic strains Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes when included in a simple sugars medium at concentrations of >5 mM. The extent of cellulose digestion by R. flavefaciens and B. succinogenes but not R. albus was also substantially reduced. Examination of rumen fluid from sheep maintained on dried grass containing 0.51% phenolic acids showed the presence of phloretic acid (0.1 mM) and 3-methoxyphloretic acid (trace) produced by hydrogenation of the 2-propenoic side chain of p-coumaric and ferulic acids, respectively. The parent acids were found in trace amounts only, although they represented the major phenolic acids ingested. Phloretic and 3-methoxyphloretic acids proved to be considerably less toxic than their parent acids. All of the cellulolytic strains (and Streptococcus bovis) showed at least a limited ability to hydrogenate hydroxycinnamic acids, with Ruminococcus spp. proving the most effective. No further modification of hydroxycinnamic acids was produced by the single strains of bacteria examined. However, a considerable shortfall in the recovery of added phenolic acids was noted in media inoculated with rumen fluid. It is suggested that hydrogenation may serve to protect cellulolytic strains from hydroxycinnamic acids.     

Isolation and characterisation of anaerobic cellulose-degrading bacteria from East African porcupine (Hystrix cristata)

Two strains of obligately anaerobic cellulolytic bacteria designated as PS7 and PS8 (PS for porcupine species) were isolated from hindgut fluid of a crested porcupine (Hystrix cristata). The rates of cellulose degradation, total volatile fatty acids, and gas production from cellulose by the isolates were determined in comparison with Ruminococcus flavefaciens FD-1. Ruminococcus flavefaciens FD-1 degraded acid swollen cellulose and produced total volatile fatty acid at a faster rate (0.03145 mg/d; 3.8350 μmol/mL) than PS7 (0.03113 mg/d; 2.5278 μmol/mL) and PS8 (0.0125 mg/d; 2.1080 μm/mL). However, PS7 degraded cellulose strips (untreated) faster (1.5 weeks) than R. flavefaciens FD-1 (2 weeks). Furthermore, PS7 produced gas at a higher rate (0.1055 ml/d) than R. flavefaciens FD-1 (0.03145 ml/d) more produced butyric, isovaleric acids and almost twice the amounts of total volatile fatty acids from acid swollen cellulose. Both PS7 and PS8 were Gram variable, rod shaped and motile. On cellobiose medium, PS7 grew at temperature ranges from 25 to 45°C while PS8 did not grow at 25 and 45°C. Both isolates grew at pH levels between 6.2 and 11. Characterisation based on carbohydrate fermentation and morphology indicated that these two isolates were similar. Characterisation by RAPD-PCR suggested that PS7 and PS8 were genotypically similar but distinct. Phylogenetic analysis using the nucleotide sequence (1450 bp) of the 16S rRNA gene suggested that PS7 clustered with Clostridium sub-phylum and exhibited the highest similarity (95%) withClostridium lentocellum . The phylogenetic results suggest that PS7 might represent a new taxon.     

A conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity

In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge-helix-bulge motif RNAs as substrates.     

The modular cellulase CelZ of the thermophilic bacterium Clostridium stercorarium contains a thermostabilizing domain

The non-catalytic region of the Clostridium stercorarium cellulase CelZ (Avicelase I) comprises two protein segments (C and C′) grouped into different subfamilies of cellulose-binding domain (CBD) family III. The C-terminally located family IIIb domain C was identified as a true cellulose-binding domain responsible for anchoring the CelZ enzyme to cellulose. The family IIIc domain C′ immediately adjacent to the catalytic domain was unable to mediate binding to cellulose. A deletion study revealed a lack of independence of this pair of domains: almost the entire C′ domain was required to maintain the catalytic activity and the thermostability of the enzyme.     

Butyric acid production from red algae by a newly isolated <Emphasis Type="Italic">Clostridium</Emphasis> sp. S1

Objective

To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required.

Results

A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5–2 g levulinic acid/l and recovered from HMF inhibition at 0.6–2.5 g/l, resulting in 85–92 % butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l.

Conclusion

Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

     

Functional importance of Crenarchaea-specific extra-loop revealed by an X-ray structure of a heterotetrameric crenarchaeal splicing endonuclease

Archaeal splicing endonucleases (EndAs) are currently classified into three groups. Two groups require a single subunit protein to form a homodimer or homotetramer. The third group requires two nonidentical protein components for the activity. To elucidate the molecular architecture of the two-subunit EndA system, we studied a crenarchaeal splicing endonuclease from Pyrobaculum aerophilum. In the present study, we solved a crystal structure of the enzyme at 1.7-Å resolution. The enzyme adopts a heterotetrameric form composed of two catalytic and two structural subunits. By connecting the structural and the catalytic subunits of the heterotetrameric EndA, we could convert the enzyme to a homodimer that maintains the broad substrate specificity that is one of the characteristics of heterotetrameric EndA. Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif. These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems. Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself.     

Production of H2 from cellulose by rumen microorganisms: effects of inocula pre-treatment and enzymatic hydrolysis

H₂ production from cellulose, using rumen fluid as the inoculum, has been investigated in batch experiments. Methanogenic archaea were inhibited by acid pre-treatment, which also inhibited cellulolytic microorganisms, and in consequence, the conversion of cellulose to H₂. Positive results were observed only with the addition of cellulase. H₂ yields were 18.5 and 9.6 mmol H₂ g cellulose^?1 for reactors with 2 and 4 g cellulose l^?1 and cellulase, respectively. H₂ was primarily generated by the butyric acid pathway and this was followed by formation of acetic acid, ethanol and n-butanol. In reactors using 4 g cellulose l^?1 and cellulase, the accumulation of alcohols negatively affected the H₂ yield, which changed the fermentation pathways to solventogenesis. PCR–DGGE analysis showed changes in the microbial communities. The phylogenetic affiliations of the bands of DGGE were 99 % similar to Clostridium sp.     

Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library

This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-β-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20–50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20–40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.     

Effect of Whole-Grain Barley on the Human Fecal Microbiota and Metabolome

In this study, we compared the fecal microbiota and metabolomes of 26 healthy subjects before (HS) and after (HSB) 2 months of diet intervention based on the administration of durum wheat flour and whole-grain barley pasta containing the minimum recommended daily intake (3 g) of barley β-glucans. Metabolically active bacteria were analyzed through pyrosequencing of the 16S rRNA gene and community-level catabolic profiles. Pyrosequencing data showed that levels of Clostridiaceae (Clostridium orbiscindens and Clostridium sp.), Roseburia hominis, and Ruminococcus sp. increased, while levels of other Firmicutes and Fusobacteria decreased, from the HSB samples to the HS fecal samples. Community-level catabolic profiles were lower in HSB samples. Compared to the results for HS samples, cultivable lactobacilli increased in HSB fecal samples, while the numbers of Enterobacteriaceae, total coliforms, and Bacteroides, Porphyromonas, Prevotella, Pseudomonas, Alcaligenes, and Aeromonas bacteria decreased. Metabolome analyses were performed using an amino acid analyzer and gas chromatography-mass spectrometry solid-phase microextraction. A marked increase in short-chain fatty acids (SCFA), such as 2-methyl-propanoic, acetic, butyric, and propionic acids, was found in HSB samples with respect to the HS fecal samples. Durum wheat flour and whole-grain barley pasta containing 3% barley β-glucans appeared to be effective in modulating the composition and metabolic pathways of the intestinal microbiota, leading to an increased level of SCFA in the HSB samples.     

Characterization, cloning and functional expression of novel xylanase from Thermomyces lanuginosus SS-8 isolated from self-heating plant wreckage material

Extracellular cellulase free xylanase from Thermomyces lanuginosus sp. SS-8, isolated from self heating plant wreckage material was identified as β-1,4-endo-xylanase precursor, a monomer of 21.3 kDa with no carbohydrate residue. This xylanase retained 80 % activity at 60 °C for 96 h, was active at a wide pH range of 3–11 and uniquely hydrolyzed xylan to xylose without production of xylo-oligosaccharides. Gene xynSS8 encoding xylanase from T. lanuginosus SS-8 was cloned and functionally expressed in Escherichia coli XL1 Blue using pTZ57R/T plasmid and xynSS8/pQE-9 expression vector construct respectively. Gene xynSS8 was of 777 bp and deduced amino acid sequence was a mature xylanase of 258 amino acids. XynSS8 has extra 33 amino acids compared to its nearest homolog and was thermo-alkali tolerant as that of native protein. The xylanase could degrade pulp and release substantial chromophoric materials and lignin derived compounds indicating its effective utility in pulp bleaching. Novel characteristics of the enzyme may contribute to its wide industrial usage. This is first report of cloning and functional expression of the novel xylanase from T. lanuginosus SS-8.     

Hjc resolvase is a distantly related member of the type II restriction endonuclease family

Hjc resolvase is an archaeal enzyme involved in homologous DNA recombination at the Holliday junction intermediate. However, the structure and the catalytic mechanism of the enzyme have not yet been identified. We performed database searching using the amino acid sequence of the enzyme from Pyrococcus furiosus as a query. We detected 59 amino acid sequences showing weak but significant sequence similarity to the Hjc resolvase. The detected sequences included DpnII, HaeII and Vsr endonuclease, which belong to the type II restriction endonuclease family. In addition, a highly conserved region was identified from a multiple alignment of the detected sequences, which was similar to an active site of the type II restriction endonucleases. We substituted three conserved amino acid residues in the highly conserved region of the Hjc resolvase with Ala residues. The amino acid replacements inactivated the enzyme. The experimental study, together with the results of the database searching, suggests that the Hjc resolvase is a distantly related member of the type II restriction endonuclease family. In addition, the results of our database searches suggested that the members of the RecB domain superfamily are evolutionarily related to the type II restriction endonuclease family.     

Cloning and Expression of a Chitinase Gene from Sanguibacter sp. C4

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank dadabase. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.     

Triphenylmethane Reductase from Citrobacter sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in Escherichia coli

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.     

Metabolism of glycyrrhetic acid by rat liver microsomes: glycyrrhetinate dehydrogenase

Glycyrrhetic acid, derived from a main component of liquorice, was converted to 3-ketoglycyrrhetic acid reversibly by rat liver homogenates in the presence of NADPH or NADP⁺. Glycyrrhetic acid-oxidizing and 3-ketoglycyrrhetic acid-reducing activities were localized in microsomes among the subcellular fractions of rat liver. Glycyrrhetic acid-oxidizing activity and 3-ketoglycyrrhetic acid-reducing activities showed pH optima at 6.3 and 8.5, respectively, and required NADP⁺ or NAD⁺ and NADPH or NADH, respectively, indicating that these activities were due to glycyrrhetinate dehydrogenase. The dehydrogenase was not solubilized from the membranes by the treatment with 1 M NaCl or sonication, indicating that the enzyme is a membrane component. The dehydrogenase was solubilized with detergents such as Emalgen 913, Triton X-100 and sodium cholate, and then separated from 3β-hydroxysteroid dehydrogenase (5β-androstan-3β-ol-17-one-oxidizing activity) by butyl-Toyopearl 650 M column chromatography. Partially purified enzyme catalyzed the reversible reaction between glycyrrhetic acid and 3-ketoglycyrrhetic acid, but was inactive toward 3-epiglycyrrhetic acid and other steroids having the 3β-hydroxyl group. The enzyme required NADP⁺ and NADPH for the highest activities of oxidation and reduction, respectively, and NAD⁺ and NADH for considerable activities, similar to the results with microsomes. From these results the enzyme is defined as glycyrrhetinate dehydrogenase, being quite different from 3β-hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestine, which is active for both glycyrrhetic acid and steroids having the 3β-hydroxyl group.     

A metagenomic approach for the identification and cloning of an endoglucanase from rice straw compost

Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the “great plate count anomaly” phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12 kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7 kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.     

Cloning and functional analysis of putative malonyl-CoA:acyl-carrier protein transacylase gene from the docosahexaenoic acid-producer Schizochytrium sp. TIO1101

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), which transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), is a key enzyme in fatty acid biosynthesis. Schizochytrium sp. TIO1101 is a marine protist with high levels of docosahexaenoic acid accumulation. In this study, the putative fabD gene coding MCAT was isolated from Schizochytrium sp. TIO1101. The Schizochytrium MCAT gene (ScTIOfabD) contained an 1176 bp open reading frame encoding a protein of 391 amino acids. The ScTIOfabD gene exhibited high novelty in nucleotide and amino acid sequence. The highest amino acid identity was only 35 % between ScTIOMCAT and the reported MCATs. Further studies demonstrated that ScTIOMCAT could bind malonyl-CoA directly and transfer malonyl group from malonyl-CoA to the ACP domain in vitro. Phylogenetic analysis suggested that ScTIOMCAT was relative close to MCATs of yeast strains. Overexpression of ScTIOMCAT in Saccharomyces cereviseae significantly increased the MCAT activity, without negative effects on the growth rate of the host strain. In addition, ScTIOMCAT generated 16.8 and 62 % increase in biomass and fatty acid accumulation, respectively, and did not alter the profile of fatty acid. Our results indicated that the novel MCAT gene from Schizochytrium sp. TIO1101 was crucial for fatty acid synthesis and had potential applications for genetic modifications of oil-producing species.