Evaluating the Mode of Antifungal Action of Heat-Stable Antifungal Factor (HSAF) in Neurospora crassa.

Heat-stable antifungal factor (HSAF) isolated from Lysobacter enzymogenes has shown a broad-spectrum of antifungal activities. However, little is known about its mode of action. In this study, we used the model filamentous fungus Neurospora crassa to investigate the antifungal mechanism of HSAF. We first used HSAF to treat the N. crassa strain at different time points. Spore germination, growth phenotype and differential gene expression analysis were conducted by utilizing global transcriptional profiling combined with genetic and physiological analyses. Our data showed that HSAF could significantly inhibit the germination and aerial hyphae growth of N. crassa. RNA-seq analysis showed that a group of genes, associated with cell wall formation and remodeling, were highly activated. Screening of N. crassa gene deletion mutants combined with scanning electron microscopic observation revealed that three fungal cell wall integrity-related genes played an important role in the interaction between N. crassa and L. enzymogens. In addition, Weighted Gene Co-Expression Network Analysis (WGCNA), accompanied by confocal microscopy observation revealed that HSAF could trigger autophagy-mediated degradation and eventually result in cell death in N. crassa. The findings of this work provided new insights into the interactions between the predatory Lysobacter and its fungal prey.

High-Level Production of Indole-3-acetic Acid in the Metabolically Engineered Escherichia coli.

Indole-3-acetic acid (IAA) is a critical plant hormone that regulates cell division, development, and metabolism. IAA synthesis in plants and plant-associated microorganisms cannot fulfill the requirement for large-scale agricultural production. Here, two novel IAA biosynthesis pathways, tryptamine (TAM) and indole-3-acetamide (IAM), were developed for IAA production by whole-cell catalysis and de novo biosynthesis in an engineered Escherichia coli MG1655. When 10 g/L l-tryptophan was used as a substrate, an MIA-6 strain containing a heterologous IAM pathway had the highest IAA titer of 7.10 g/L (1.34 × 103 mg/g DCW), which was 98.4 times more than MTAI-5 containing the TAM pathway by whole-cell catalysis. De novo IAA biosynthesis was optimized by improving NAD(P)H availability, resulting in an increased IAA titer of 906 mg/L obtained by the MGΔadhE::icd strain, which is 29.7% higher than the control. These strategies exhibit the potential for IAA production in engineered E. coli and possible industrial applications.

Improved Production of Majority Cellulases in Trichoderma reesei by Integration of cbh1 Gene From Chaetomium thermophilum.

Lignocellulose is an abundant waste resource and has been considered as a promising material for production of biofuels or other valuable bio-products. Currently, one of the major bottlenecks in the economic utilization of lignocellulosic materials is the cost-efficiency of converting lignocellulose into soluble sugars for fermentation. One way to address this problem is to seek superior lignocellulose degradation enzymes or further improve current production yields of lignocellulases. In the present study, the lignocellulose degradation capacity of a thermophilic fungus Chaetomium thermophilum was firstly evaluated and compared to that of the biotechnological workhorse Trichoderma reesei. The data demonstrated that compared to T. reesei, C. thermophilum displayed substantially higher cellulose-utilizing efficiency with relatively lower production of cellulases, indicating that better cellulases might exist in C. thermophilum. Comparison of the protein secretome between C. thermophilum and T. reesei showed that the secreted protein categories were quite different in these two species. In addition, to prove that cellulases in C. thermophilum had better enzymatic properties, the major cellulase cellobiohydrolase I (CBH1) from C. thermophilum and T. reesei were firstly characterized, respectively. The data showed that the specific activity of C. thermophilum CBH1 was about 4.5-fold higher than T. reesei CBH1 in a wide range of temperatures and pH. To explore whether increasing CBH1 activity in T. reesei could contribute to improving the overall cellulose-utilizing efficiency of T. reesei, T. reesei cbh1 gene was replaced with C. thermophilum cbh1 gene by integration of C. thermophilum cbh1 gene into T. reesei cbh1 gene locus. The data surprisingly showed that this gene replacement not only increased the cellobiohydrolase activities by around 4.1-fold, but also resulted in stronger induction of other cellulases genes, which caused the filter paper activities, Azo-CMC activities and ß-glucosidase activities increased by about 2.2, 1.9, and 2.3-fold, respectively. The study here not only provided new resources of superior cellulases genes and new strategy to improve the cellulase production in T. reesei, but also contribute to opening the path for fundamental research on C. thermophilum.

Carotenoids and lipid production from Rhodosporidium toruloides cultured in tea waste hydrolysate.

BACKGROUND: In this study, renewable tea waste hydrolysate was used as a sole carbon source for carotenoids and lipid production. A novel Rhodosporidium toruloides mutant strain, RM18, was isolated through atmospheric and room-temperature plasma mutagenesis and continuous domestication in tea waste hydrolysate from R. toruloides ACCC20341. RESULTS: RM18 produced a larger biomass and more carotenoids and α-linolenic acid compared with the control strain cultured in tea waste hydrolysate. The highest yields of torularhodin (481.92 µg/g DCW) and torulene (501 µg/g DCW) from RM18 cultured in tea waste hydrolysate were 12.86- and 1.5-fold higher, respectively, than that of the control strain. In addition, α-linolenic acid production from RM18 in TWH accounted for 5.5% of total lipids, which was 1.58 times more than that of the control strain. Transcriptomic profiling indicated that enhanced central metabolism and terpene biosynthesis led to improved carotenoids production, whereas aromatic amino acid synthesis and DNA damage checkpoint and sensing were probably relevant to tea waste hydrolysate tolerance. CONCLUSION: Tea waste is suitable for the hydrolysis of microbial cell culture mediums. The R. toruloides mutant RM18 showed considerable carotenoids and lipid production cultured in tea waste hydrolysate, which makes it viable for industrial applications.

[Reinforcement of Rhodobacter sphaeroides cofactor NADPH to increase the production of farnesol].

Farnesol (FOH) is produced by dephosphorylation of farnesyl diphosphate (FPP) derived from two universal building blocks, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). In Rhodobacter sphaeroides these building blocks are generated by MEP pathway, however, many of the biosynthetic reactions and biotransformations in the MEP pathway are limited by low availability of NADPH. Improvement of the amount of intracellular NADPH may enhance the synthesis of FOH. In this study, we utilized the strategies of increasing the production of NADPH and decreasing the consumption of NADPH. The expression of glucose 6-phosphate isomerase (pgi) and glutamate dehydrogenase (gdhA) were inhibited by RNA interference, respectively, and overexpression of 6-glucose phosphate dehydrogenase (zwf) and 6-glucose phosphate dehydrogenase (gnd) in the pentose phosphate pathway were carried out. The results showed that the content of NADPH in the recombinant strains increased significantly, the highest FOH production of RSpgii in the RNA interfered strain was 3.91 mg/g, and the FOH production increased to 3.43 mg/g after zwf gene and gnd gene has been overexpressed. In order to obtain strains with higher FOH production, we used RSpgii as the starting strain, and zwf, gnd and co-overexpressed zwf + gnd gene were overexpressed in RSpgii, respectively. The highest FOH production of the strain RSzgpi reached to 4.48 mg/g which was 2.24 times that of the starting strain RS-GY2.

Enhancement of NADPH availability for coproduction of coenzyme Q10 and farnesol from Rhodobacter sphaeroides.

Coenzyme Q10 (CoQ10)-an essential cofactor in the respiratory electron transport chain-has important pharmaceutical and healthcare applications. Farnesol (FOH)-an acyclic sesquiterpene alcohol-has garnered interest owing to its valuable clinical and medical benefits. Here, the coproduction of CoQ10 and FOH in Rhodobacter sphaeroides GY-2 was greatly improved through the enhancement of intracellular NADPH availability. Transcription of pgi, gdhA, and nuocd was, respectively, inhibited using RNA interference to reduce intracellular NAD(P)H consumption. Moreover, zwf, gnd, and zwf + gnd were overexpressed to enhance the pentose phosphate pathway, resulting in improved NADPH availability in most metabolically engineered R. sphaeroides strains. RSg-pgi with RNAi of pgi combined with overexpression of gnd produced 55.05 mg/L FOH that is twofold higher than the parental strain GY-2, and 185.5 mg/L CoQ10 can be coproduced at the same time. In conclusion, improved carbon flux can be redirected toward NADPH-dependent biosynthesis through the enhancement of NADPH availability.

Co-production of farnesol and coenzyme Q10 from metabolically engineered Rhodobacter sphaeroides.

BACKGROUND: Farnesol is an acyclic sesquiterpene alcohol present in the essential oils of various plants in nature. It has been reported to be valuable in medical applications, such as alleviation of allergic asthma, gliosis, and edema as well as anti-cancerous and anti-inflammatory effects. Coenzyme Q10 (CoQ10), an essential cofactor in the aerobic respiratory electron transport chain, has attracted growing interest owing to its clinical benefits and important applications in the pharmaceutical, food, and health industries. In this work, co-production of (E,E)-farnesol (FOH) and CoQ10 was achieved by combining 3 different exogenous terpenes or sesquiterpene synthase with the RNA interference of psy (responsible for phytoene synthesis in Rhodobacter sphaeroides GY-2). RESULTS: FOH production was significantly increased by overexpressing exogenous terpene synthase (TPS), phosphatidylglycerophosphatase B (PgpB), and sesquiterpene synthase (ATPS), as well as RNAi-mediated silencing of psy coding phytoene synthase (PSY) in R. sphaeroides strains. Rs-TPS, Rs-ATPS, and Rs-PgpB respectively produced 68.2%, 43.4%, and 21.9% higher FOH titers than that of the control strain. Interestingly, the CoQ10 production of these 3 recombinant R. sphaeroides strains was exactly opposite to that of FOH. However, CoQ10 production was almost unaffected in R. sphaeroides strains modified by psy RNA interference. The highest FOH production of 40.45 mg/L, which was twice as high as that of the control, was obtained from the TPS-PSYi strain, where the exogenous TPS was combined with the weakening of the phytoene synthesis pathway via psy RNA interference. CoQ10 production in TPS-PSYi, ATPS-PSYi, and PgpB-PSYi was decreased and lower than that of the control strain. CONCLUSIONS: The original flux that contributed to phytoene synthesis was effectively redirected to provide precursors toward FOH or CoQ10 synthesis via psy RNA interference, which led to weakened carotenoid synthesis. The improved flux that was originally involved in CoQ10 production and phytoene synthesis was redirected toward FOH synthesis via metabolic modification. This is the first reported instance of FOH and CoQ10 co-production in R. sphaeroides using a metabolic engineering strategy.

Identification of two integration sites in favor of transgene expression in Trichoderma reesei.

BACKGROUND: The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei. Currently, only the locus of exocellobiohydrolases I encoding gene (cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized. RESULTS: To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified. CONCLUSIONS: We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei. Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.

Mathematical modeling of fed-batch fermentation of Schizochytrium sp. FJU-512 growth and DHA production using a shift control strategy.

To obtain high-cell-density cultures of Schizochytrium sp. FJU-512 for DHA production, two stages of fermentation strategy were used and carbon/nitrogen ratio, DO and temperature were controlled at different levels. The final dry cell weight, total lipid production and DHA yield in 15 l bioreactor reached 103.9, 37.2 and 16.0 g/l, respectively. For the further study of microbial growth and DHA production dynamics, we established a set of kinetic models for the fed-batch production of DHA by Schizochytrium sp. FJU-512 in 15 and 100 l fermenters and a compensatory parameter n was integrated into the model in order to find the optimal mathematical equations. A modified Logistic model was proposed to fit the cell growth data and the following kinetic parameters were obtained: µm = 0.0525/h, Xm = 100 g/l and n = 4.1717 for the 15 l bioreactor, as well as µm = 0.0382/h, Xm = 107.4371 g/l and n = 10 for the 100 l bioreactor. The Luedeking-Piret equations were utilized to model DHA production, yielding values of α = 0.0648 g/g and ß = 0.0014 g/g/h for the 15 l bioreactor, while the values of α and ß obtained for the 100 l fermentation were 0.0209 g/g and 0.0030 g/g/h. The predicted results compared with experimental data showed that the established models had a good fitting precision and were able to exactly depict the dynamic features of the DHA production process.

Functional identification and regulatory analysis of Δ6-fatty acid desaturase from the oleaginous fungus Mucor sp. EIM-10.

OBJECTIVES: To enlarge the diversity of the desaturases associated with PUFA biosynthesis and to better understand the transcriptional regulation of desaturases, a Δ6-desaturase gene (Md6) from Mucor sp. and its 5'-upstream sequence was functionally identified in Saccharomyces cerevisiae. RESULTS: Expression of the Δ6-fatty acid desaturase (Md6) in S. cerevisiae showed that Md6 could convert linolenic acid to γ-linolenic acid. Computational analysis of the promoter of Md6 suggested it contains several eukaryotic fundamental transcription regulatory elements. In vivo functional analysis of the promoter showed the 5'-upstream sequence of Md6 could initiate expression of GFP and Md6 itself in S. cerevisiae. A series deletion analysis of the promoter suggested that sequence between -919 to -784 bp (relative to start site) named as eMd6 is the key factor for high activity of Δ6-desaturase. The activity of Δ6-desaturase was increased by 2.8-fold and 2.5-fold when the eMd6 sequence was placed upstream of -434 with forward or reverse orientations respectively. CONCLUSION: To our best knowledge, the native promoter of Md6 from Mucor is the strongest promoter for Δ6-desaturase reported so far and the sequence between -919 to -784 bp is an enhancer for Δ6-desaturase activity.

A lignocellulosic hydrolysate-tolerant Aurantiochytrium sp. mutant strain for docosahexaenoic acid production.

To utilize lignocellulosic hydrolysate for docosahexaenoic acid (DHA) production, a novel mutant Aurantiochytrium sp. FN21 with strong tolerance against inhibitory lignocellulosic hydrolysate was obtained through continuous domestication processes from the parent strain Aurantiochytrium sp. FJU-512. Aurantiochytrium sp. FN21 can accumulate 21.3% and 30.7% more DHA compared to its parent strain cultured in fermentation medium and a medium with 50% (v/v) sugarcane bagasse hydrolysate (SBH), respectively. After optimization with different nitrogen sources, the highest lipid (11.84g/L) and DHA (3.15g/L) production were achieved in SBH. The results demonstrated that Aurantiochytrium sp. FN21 has the commercial applications for DHA production using lignocellulosic hydrolysate. In order to elucidate the tolerance mechanism, transcriptomic profiling of the two strains was studied. The highly up-regulated genes and corresponding cellular pathways (TCA cycle, amino acid biosynthesis, fatty acid metabolism and degradation of aromatic compounds) are considered to be associated with the hydrolysate-tolerance of Aurantiochytrium sp. FN21.

[High-level production of glucose oxidase by recombinant Pichia pastoris using a combined strategy].

To enhance the production of glucose oxidase by recombinant Pichia pastoris, two strategies were developed, which were namely co-feeding of methanol and sorbitol and co-expressing of the protein disulfide isomerase (PDI) and Vitreoscialla hemoglobin (VHb). The volumetric activity reached 456 U/mL by using the strain X33/pPIC9k-GOD, in 5 liter fermentator, with the co-feeding of methanol and sorbitol, it was 0.2 fold higher than that only feeding by methanol. The improved strain was obtained by co-expressing PDI-VHb with GOD. While fermented in a 5 liter fermentator by feeding methanol and sorbitol, the activity of the improved strain reached 716 U/mL with a yield of 7 400 mg/L total soluble protein concentration. These results indicated that heterologous protein expression level can be enhanced by optimizing fermentation condition and co-expression molecular chaperon in Pichia pastoris.

[Cloning, expression and characterization of a lipase gene, lipC24, from Burkholderia sp. ZYB002].

OBJECTIVE: We cloned a lipase gene, lipC24, from Burkholderia sp. ZYB002 and characterized the recombinant lipase LipC24. METHOD: Based on the known genomic DNA sequence from Burkholderia cecapia JK321, we designed a pair of specific primers for the lipC24 gene and then obtained the full length of lipC24 gene. The lipC24 gene fragment enconding the mature peptide LipC24 was then subcloned into expression plasmid, pACYC-Duet-lipB, and expressed in E. coli. The recombinant protein, LipC24, was purified to homogeneity by HisTrap HP chromatography column and HiTrap DEAE FF chromatography column. RESULTS: We expressed the lipC24 gene from Burkholderia sp. ZYB002 in E. coli Origami 2(DE3). Nucleotide sequencing revealed that the lipC24 gene had an open reading frame of 1317 bp, and the deduced amino acid sequence of LipC24 corresponded to 438 amino acid residues, including a conserved -G-X1-S-X2-G- motif. The relative molecular weight of the purified LipC24 was about 45 kDa. The purified LipC24 displayed hydrolysis activity to various 4-nitrophenyl esters and substrate preference for the medium chain length 4-nitrophenyl-esters. The optimal temperature was 40°C and the optimal pH was 7.5. The lipase was stable between pH 7.0 and 8.0 for 24 hours. However, the half-life was only 16 min at 40°C. CONCLUSION: The LipC24 was a 45 kDa protein, a mesotherm and neutral lipase.

[Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1].

The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.

Cloning and functional identification of delta5 fatty acid desaturase gene and its 5'-upstream region from marine fungus Thraustochytrium sp. FJN-10.

A gene encoding delta5 fatty acid desaturase (fad5) was cloned from marine fungus Thraustochytrium sp. FJN-10, a species capable of producing docosahexaenoic acid. The open reading frame of fad5 was 1,320 bp and encoded a protein comprising 439 amino acids. Expression of the fad5 in Saccharomyces cerevisiae INVSC1 revealed that FAD5 is able to introduce a double bond at position 5 of the dihomo-γ-linolenic acid (20:3 Δ(8,11,14)), resulting in arachidonic acid (20:4 Δ(5,8,11,14)) with a conversion rate of 56.40% which is the highest among engineering yeasts reported so far. The 5'-upstream region of fad5 was cloned by LA-PCR and analyzed. Phylogenetic analysis of this sequence with the 5'-upstream region of other delta5 desaturases showed that the 5'-upstream region of fad5 from Thraustochytrium share the smallest evolution distance with human and rhesus. Computational analysis of the nucleotide sequence of the 5'-upstream region of fad5 has revealed several basic transcriptional elements including five TATA boxes, three CCAAT boxes, 12 GC boxes, and several putative target-binding sites for transcription factors such as HSF, CAP, and ADR1. Preliminary functional analysis of this promoter in S. cerevisiae shows that the 5'-upstream region of fad5 could drive the expression of green fluorescent protein.

Investigation of protease-mediated cuticle-degradation of nematodes by using an improved immunofluorescence-localization method.

In order to facilitate the understanding of the actual process of enzyme-based degradation of nematodes, we visualized the localization of BLG4, a cuticle-degrading protease from the nematophagous bacterium Brevibacillus laterosporus G4, on nematode cuticle by using an improved immuno-labeled fluorescent method. Live nematodes, heat-killed nematodes and extracted nematode cuticles were exposed to the protease, and the localization of the protease and the resulting tissue degradation and destruction were observed microscopically. The bioassay findings showed that live nematodes were significantly more resistant to the protease than the dead nematodes and the extracted cuticles were. The observation of the immuno-labeling fluorescence for BLG4 revealed that the protease localized first in the tail region of the live target; and then spread over the entire target and ultimately destroyed it, including the cuticle. The results indicated the resistance of nematode cuticles to enzymatic attacks and the differences in protease susceptibilities at different regions on the nematode cuticles.

Expressed sequence tag analysis of marine fungus Schizochytrium producing docosahexaenoic acid.

To investigate docosahexaenoic acid (DHA, C22:6n-3) biosynthesis pathway in marine fungus Schizochytrium sp. FJU-512, a cDNA library of the fungus was constructed and analyzed. The titers of primary library were up to 5.0 x 10(6). A total of 4005 ESTs were assembled into 1947 unigenes. Sequences annotation and function analysis were carried out by using Blast, GO and KEGG programs. Compared with other eukaryote genomes, Schizochytrium sp. FJU-512 ESTs shared at least 26.6% genes with Arabidopsis thaliana (E < or = 10(-10)). The cDNA (Contig46, assembled by EH401977 and EH404532) and EH40321 were found to encode serine/threonine protein phosphatase type 1 and cell division control protein 2 which were involved in successive binary cell division. Notably, the key enzymes involved in biosynthesis of fatty acid via polyketide synthases (PKS) such as beta-ketoacyl synthase, beta-ketoacyl reductase, hydroxyacyl dehydrogenase, enoyl-CoA hydratase/isomerase, and enoyl reductase were found in the cDNA library. The results indicated that DHA synthesis in Schizochytrium sp. FJU-512 had undergone PKS pathway.

[Cloning and expression of two elongase genes involved in the biosynthesis of docosahexaenoic acid in Thraustochytrium sp. FJN-10].

Docosahexaenoic acid (DHA C22:6n-3), a typical long chain polyunsaturated fatty acids (PUFAs) has many positive effects on diseases such as artherosclerosis, hypertriglyceridemia, hypertension and cancers. Marine fungi, especially Thraustochytrium spp. producing much DHA can serve as model organisms for explaining the mechanism on the biosynthesis of PUFA. We described two elongase genes (TFD6 and TFD5) involved in the biosynthesis of DHA in Thraustochytrium sp. FJN-10 was cloned by using reverse transcription PCR and rapid amplification of cDNA ends. TFD6 cDNA was 816 bp in length and encoded a protein of 271 amino acids. TFD5 cDNA was 831 bp in length and encoded a protein of 276 amino acids. Transmembrane analysis revealed that TFD6 contained five transmembrane domains while TFD5 contained seven. Tertiary structures of TFD6, TFD5 elongases were predicted by HHMMSTR (Hidden markov model for local sequence-structure) model and Rosetta program. Alignment of TFD6, TFD5 with other elongases showed that both of them shared an HXXHH conserved histidine-rich motif. Phylogenetic analysis showed that TFD6 was the closest to Thraustochytrium 66 elongase, while TFD5 was the closest to Thraustochytrium sp. delta5 elongase. TFD6 and TFD5 were subcloned into the Hind III/Xba I restriction site of pYES2 vector respectively. Recombined plasmids were transformed into Saccharomyces cerevisiae using lithium acetate method. Gas chromatography analysis showed that TFD6 could elongate C18:3n-3 to C20:3n-3 while TFD5 could elongate C20:5n-3 to C22:5n-3.