Yeast Expression Research Articles

Characterization of recombinant Bacillus halodurans CM1 xylanase produced by Pichia pastoris KM71 and its potential application in bleaching process of bagasse pulp

Thermoalkalophilic xylanases promise potential application in pulp biobleaching to reduce the use of toxic chlorinated chemical agents, which are harmful to the environment. In this study, a thermoalkalophilic endoxylanase gene (bhxyn3) originating from Indonesian indigenous Bacillus halodurans CM1 was cloned into yeast expression vector pPICZα A and expressed in Pichia pastoris KM71 under the control of AOX1 promoter. Recombinant P. pastoris expressed the highest final level of xylanase (146 U/mL) on BMGY medium after five days of cultivation. Optimization of xylanase production on a small scale was carried out by varying the methanol concentrations and the optimal xylanase production by the recombinant P. pastoris was observed in the culture with 2% (v/v) methanol after four days of the induction phase. The recombinant xylanase (BHxyn3E) was thermotolerant and alkalophilic, with an optimal temperature at around 55‐65 °C and under pH 8.0. The enzyme activity was slightly induced by K+, Fe2+, and MoO42‐. Enzymatic bleaching of bagasse pulp with no prior pH adjustment (pH 9) using BHxyn3E at 200 U/g oven dried pulp increased the lightness index (L*) and changed substantially the color a index (a*); however, the treatments did not change the whiteness index in a significant way. Therefore, further optimization and assessment such as adjustment of incubation temperature and pH in biobleaching were needed to reduce the use of harmful chemical agents in industrial applications.

Genome-wide identification and functional characterization of the PheE2F/DP gene family in Moso bamboo

BackgroundE2F/DP proteins have been shown to regulate genes implicated in cell cycle control and DNA repair. However, to date, research into the potential role of the Moso bamboo E2F/DP family has been limited.ResultsHere, we identified 23 E2F/DPs in the Moso bamboo genome, including nine E2F genes, six DP genes, eight DEL genes and one gene with a partial E2F domain. An estimation of the divergence time of the paralogous gene pairs suggested that the E2F/DP family expansion primarily occurred through a whole-genome duplication event. A regulatory element and coexpression network analysis indicated that E2F/DP regulated the expression of cell cycle-related genes. A yeast two-hybrid assay and expression analysis based on transcriptome data and in situ hybridization indicated that the PheE2F-PheDP complex played important roles in winter Moso bamboo shoot growth. The qRT-PCR results showed that the PheE2F/DPs exhibited diverse expression patterns in response to drought and salt treatment and diurnal cycles.ConclusionOur findings provide novel insights into the Moso bamboo E2F/DP family and partial experimental evidence for further functional verification of the PheE2F/DPs.

Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters.

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

The quest for selective Cs+ transport in plants

The quest for selective Cs+ transport in plants

A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

Biosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

Identification of KCS gene family and functional analysis of FAE-like genes from Malania oleifera

KCS (3-ketoacyl-CoA synthase) is the key enzyme catalyzing the first step of very long chain fatty acid (VLCFA) biosynthesis. Studies showed that different KCSs possessed different substrate preference. Malania oleifera are abundance of VLCFAs in its mature seeds, especially the nervonic acid, which is essential for human health. In this study, we identified and characterized 18 KCS genes in M. oleifera genome. Phylogenetic analysis showed that these KCS genes were classified into four subfamilies, including two FAE-like, six KCS-like, eight FDH-like and two CER6. We concentrated on the functional role of two FAE-like genes, Maole003085.T1 and Maole004215.T1 which encoded predicted amino acid residues of 516 and 518 in protein, respectively. Multiple sequence alignment showed that their two proteins contained the known and conserved active sites among FAE-like subfamily. Upon heterologous expression in wild type yeast (Saccharomyces cerevisiae) INVSc1, we found that Maole004215.T1 could produce four new fatty acids including C22:0/C22:1 and C24:0/C24:1, but Maole003085.T1 only produced C22:1. Besides, upon heterologous expression in mutant yeast BY4741-△elo3, we found the Maole003085.T1 could produce C24:0 and C26:0, while the Maole004215.T1 could catalyze the formation of fatty acids C24:0, C26:0 and C28:0. These results showed Maole003085.T1 and Maole004215.T1 had fatty acid elongation activity in yeast, and possessed different substrate preference in the production of different VLCFAs. Interestingly, we found Maole004215.T1 could produce nervonic acid in yeast, which provides molecular basis on the genetic improvement and genic engineering for producing nervonic acid resources by using biotechnological methods.

Biosynthesis of the Sex Pheromone Component (E,Z)-7,9-Dodecadienyl Acetate in the European Grapevine Moth, Lobesia botrana, Involving \u220611 Desaturation and an Elusive \u22067 Desaturase

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.

Transient Gene Expression: an Approach for Recombinant Vaccine Production

The production of recombinant vaccines in green plants is an attractive and promising topic in genetic engineering. However, the stable transformation of green plants is a time-consuming, costly, and labor-intensive practice. Moreover, public concerns about genetically modified plants put another limitation on the development and release of transgenic plant-based recombinant vaccines. These shortcomings were addressed by developing transient gene expression systems that allow researchers to investigate candidate recombinant vaccines quickly without tedious work and high costs. A comprehensive literature review was used to gather relevant information. This approach has received much attention in various recombinant vaccine production platforms, including mammalian cell culture, insect cell culture, yeast expression systems, and, more importantly, in plant hosts. Due to their simplicity and efficiency, transient gene expression systems are now widely used to validate gene constructs and transgene expression within plant tissues. This paper describes the concept of transient gene expression and discusses the significant advantages of this approach for producing recombinant vaccines. Notably, the major types of transient gene expression viz. agroinfiltration, viral-based systems, and application of naked plasmid in plant cell culture are introduced, and some examples illustrate the pros and cons of each system. Our literature review also discusses some practical notes on the successful application of this system to provide a more comprehensive image of transient gene expression applicability in green plants. As a whole, this review contributes to the existing literature by shedding more light on various aspects of transient gene expression that have not been addressed thoroughly yet.

Identification of Fungal Limonene-3-Hydroxylase for Biotechnological Menthol Production.

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively.IMPORTANCE (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.

Enhanced in vitro anticancer activity of yeast expressed recombinant glucose oxidase versus commercial enzyme.

Cancer treatments continue to have many disadvantages. Reactive oxygen species, such as H2O2, in high concentrations, can cause cytotoxicity to cells, being even greater in cancer cells. One of the H2O2-producing enzymes is glucose oxidase; its application in cancer treatment should be explored. In this work, the extracellular expression of the mutated recombinant enzyme glucose oxidase was carried out in the eukaryotic expression system Pichia pastoris SMD1168, through the modification and optimization of the gox gene of Aspergillus niger to improve its expression in yeast and its purification. Also, the secretion signal of the alpha-mating factor from Saccharomyces cerevisiae was added to the gene for extracellular expression, and it was inserted into the expression vector pPIC3.5k. The extracellular expression of the enzyme facilitated purification by anion exchange chromatography; the purification was corroborated by SDS-PAGE, with a molecular weight of its subunit between 63 kDa and 100 kDa. The mutated recombinant enzyme glucose oxidase showed greater anticancer activity compared to the commercial glucose oxidase and could have potential for cancer treatment. KEY POINTS: • Pichia pastoris is an excellent eukaryotic expression system for proteins that need post-translational modifications. • Extracellular expression facilitates protein purification. • Glucose oxidase has potential application in cancer treatment.

Futile cycling by human microsomal cytochrome P450 enzymes within intact fission yeast cells

Human cytochrome P450 enzymes (CYPs or P450s) are known to be reduced by their electron transfer partners in the absence of substrate and in turn to reduce other acceptor molecules such as molecular oxygen, thereby creating superoxide anions (O2−•). This process is known as futile cycling. Using our previously established fission yeast expression system we have monitored cells expressing each one of the 50 human microsomal CYPs in the absence of substrate for oxidation of dihydroethidium in living cells by flow cytometry. It was found that 38 of these display a statistically significant increase in O2−• production. More specifically, cells expressing some CYPs were found to be intermediate strength O2−• producers, which means that their effect was comparable to that of treatment with 3 mM H2O2. Cells expressing other CYPs had an even stronger effect, with those expressing CYP2B6, CYP5A1, CYP2A13, CYP51A1, or CYP1A2, respectively, being the strongest producers of O2−•.

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation.

Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.

Preliminary investigations on the pathogenesis-related protein expression profile of the medicinal herb Macleaya cordata and anti-bacterial properties of recombinant proteins

The plant pathogenesis-related (PR) proteins play a crucial role in the defense of plants against pathogens and orchestrate the innate immune system of plants. In this paper, a non-normalized cDNA library of the leaf was constructed to obtain a comprehensive view of PR proteins of Macleaya cordata. Specifically, 511 expressed sequence tags (ESTs) were generated using Sanger sequencing. All ESTs were assembled into 364 non-redundancy sequences, including 78 clusters and 286 singlets. The PR protein expression profile of the medicinal herb M. cordata has been investigated and is represented by defensin, lipid-transfer protein, (S)-norcoclaurine synthase, and major allergen protein, suggesting that the herb contains rich active proteins against pathogens. Furthermore, two defensins were selected for recombinant expression in yeast, and the antimicrobial activities were explored. Since they both present a broad antimicrobial spectrum, they are of particular importance for agricultural and medicinal applications. Our study describes defensins in Papaveraceae for the first time and provides novel insights into the effective components. In addition to the alkaloids, PR proteins (such as defensins, lipid transfer proteins, (S) - norcoclaurine synthase, major allergen protein, and Class IV chitinases) are involved in the antibacterial and anti-inflammatory activities of M. cordata.

Expression and functional characterization in yeast of an endoglucanase from Bacillus sonorensis BD92 and its impact as feed additive in commercial broilers

Some ingredients used in poultry feed formulation contain carbohydrate polymers which are difficult to digest and thus hinder nutritional feed value. Toward overcoming this limitation, exogenous enzymes have been added to poultry feed to improve its nutritive value. The present study was designed to provide first enzymatic characterization of endoglucanase (BsEgl) from the genome of B. sonorensis BD92 expressed in Pichia pastoris. Further, we tested its impact alone and in combination with a β-glucosidase (Bteqβgluc) on growth in commercial broilers as feed additive. The expressed enzyme displayed features of GH5 family and had optimum activity against carboxymethyl cellulose at pH 5 and 50 °C. The BsEgl was stable at a range of pH from 4 to 8 for 60 min and at 50 °C for 180 min. Supplementing broilers diet with BsEgl alone or in combination with Bteqβgluc resulted in better feed conversion ratio among treatments during a five weeks testing period. Moreover, meat percentage was also highest for this treatment, and all treatments with recombinant enzymes increased intestinal length in birds compared to treatment control group. Blood parameters and serum biochemistry profile showed non-significant difference among groups. These results support that recombinant cellulolytic enzymes supplement high fiber diets improve their nutritional performance.

Two plastidic glycolate/glycerate translocator 1 isoforms function together to transport photorespiratory glycolate and glycerate in rice chloroplasts.

The photorespiratory pathway is highly compartmentalized. As such, metabolite shuttles between organelles are critical to ensure efficient photorespiratory carbon flux. Arabidopsis plastidic glycolate/glycerate translocator 1 (PLGG1) has been reported as a key chloroplastic glycolate/glycerate transporter. Two homologous genes, OsPLGG1a and OsPLGG1b, have been identified in the rice genome, although their distinct functions and relationships remain unknown. Herein, our analysis of exogenous expression in oocytes and yeast shows that both OsPLGG1a and OsPLGG1b have the ability to transport glycolate and glycerate. Furthermore, we demonstrate in planta that the perturbation of OsPLGG1a or OsPLGG1b expression leads to extensive accumulation of photorespiratory metabolites, especially glycolate and glycerate. Under ambient CO2 conditions, loss-of-function osplgg1a or osplgg1b mutant plants exhibited significant decreases in photosynthesis efficiency, starch accumulation, plant height, and crop productivity. These morphological defects were almost entirely recovered when the mutant plants were grown under elevated CO2 conditions. In contrast to osplgg1a, osplgg1b mutant alleles produced a mild photorespiratory phenotype and had reduced accumulation of photorespiratory metabolites. Subcellular localization analysis showed that OsPLGG1a and OsPLGG1b are located in the inner and outer membranes of the chloroplast envelope, respectively. In vitro and in vivo experiments revealed that OsPLGG1a and OsPLGG1b have a direct interaction. Our results indicate that both OsPLGG1a and OsPLGG1b are chloroplastic glycolate/glycerate transporters required for photorespiratory metabolism and plant growth, and that they may function as a singular complex.

Wheat 2-Cys peroxiredoxin plays a dual role in chlorophyll biosynthesis and adaptation to high temperature.

The molecular mechanism of high-temperature stress (HTS) response, in plants, has so far been investigated using transcriptomics, while the dynamics of HTS-responsive proteome remain unexplored. We examined the adaptive responses of the resilient wheat cultivar 'Unnat Halna' and dissected the HTS-responsive proteome landscape. This led to the identification of 55 HTS-responsive proteins (HRPs), which are predominantly involved in metabolism and defense pathways. Interestingly, HRPs included a 2-cysteine peroxiredoxin (2CP), designated Ta2CP, presumably involved in stress perception and adaptation. Complementation of Ta2CP in yeast and heterologous expression in Arabidopsis demonstrated its role in thermotolerance. Both Ta2CP silencing and overexpression inferred the involvement of Ta2CP in plant growth and chlorophyll biosynthesis. We demonstrated that Ta2CP interacts with protochlorophyllide reductase b, TaPORB. Reduced TaPORB expression was found in Ta2cp-silenced plants, while upregulation was observed in Ta2CP-overexpressed plants. Furthermore, the downregulation of Ta2CP in Taporb-silenced plants and reduction of protochlorophyllide in Ta2cp-silenced plants suggested the key role of Ta2CP in chlorophyll metabolism. Additionally, the transcript levels of AGPase1 and starch were increased in Ta2cp-silenced plants. More significantly, HTS-treated Ta2cp-silenced plants showed adaptive responses despite increased reactive oxygen species and peroxide concentrations, which might help in rapid induction of high-temperature acclimation.

Piper nigrum CYP719A37 Catalyzes the Decisive Methylenedioxy Bridge Formation in Piperine Biosynthesis.

Black pepper (Piper nigrum) is among the world’s most popular spices. Its pungent principle, piperine, has already been identified 200 years ago, yet the biosynthesis of piperine in black pepper remains largely enigmatic. In this report we analyzed the characteristic methylenedioxy bridge formation of the aromatic part of piperine by a combination of RNA-sequencing, functional expression in yeast, and LC-MS based analysis of substrate and product profiles. We identified a single cytochrome P450 transcript, specifically expressed in black pepper immature fruits. The corresponding gene was functionally expressed in yeast (Saccharomyces cerevisiae) and characterized for substrate specificity with a series of putative aromatic precursors with an aromatic vanilloid structure. Methylenedioxy bridge formation was only detected when feruperic acid (5-(4-hydroxy-3-methoxyphenyl)-2,4-pentadienoic acid) was used as a substrate, and the corresponding product was identified as piperic acid. Two alternative precursors, ferulic acid and feruperine, were not accepted. Our data provide experimental evidence that formation of the piperine methylenedioxy bridge takes place in young black pepper fruits after a currently hypothetical chain elongation of ferulic acid and before the formation of the amide bond. The partially characterized enzyme was classified as CYP719A37 and is discussed in terms of specificity, storage, and phylogenetic origin of CYP719 catalyzed reactions in magnoliids and eudicots.

Ectopic Expression of CrPIP2;3, a Plasma Membrane Intrinsic Protein Gene from the Halophyte Canavalia rosea, Enhances Drought and Salt-Alkali Stress Tolerance in Arabidopsis.

Aquaporins are channel proteins that facilitate the transmembrane transport of water and other small neutral molecules, thereby playing vital roles in maintaining water and nutrition homeostasis in the life activities of all organisms. Canavalia rosea, a seashore and mangrove-accompanied halophyte with strong adaptability to adversity in tropical and subtropical regions, is a good model for studying the molecular mechanisms underlying extreme saline-alkaline and drought stress tolerance in leguminous plants. In this study, a PIP2 gene (CrPIP2;3) was cloned from C. rosea, and its expression patterns and physiological roles in yeast and Arabidopsis thaliana heterologous expression systems under high salt-alkali and high osmotic stress conditions were examined. The expression of CrPIP2;3 at the transcriptional level in C. rosea was affected by high salinity and alkali, high osmotic stress, and abscisic acid treatment. In yeast, the expression of CrPIP2;3 enhanced salt/osmotic and oxidative sensitivity under high salt/osmotic and H2O2 stress. The overexpression of CrPIP2;3 in A. thaliana could enhance the survival and recovery of transgenic plants under drought stress, and the seed germination and seedling growth of the CrPIP2;3 OX (over-expression) lines showed slightly stronger tolerance to high salt/alkali than the wild-type. The transgenic plants also showed a higher response level to high-salinity and dehydration than the wild-type, mostly based on the up-regulated expression of salt/dehydration marker genes in A. thaliana plants. The reactive oxygen species (ROS) staining results indicated that the transgenic lines did not possess stronger ROS scavenging ability and stress tolerance than the wild-type under multiple stresses. The results confirmed that CrPIP2;3 is involved in the response of C. rosea to salt and drought, and primarily acts by mediating water homeostasis rather than by acting as an ROS transporter, thereby influencing physiological processes under various abiotic stresses in plants.

Production of 10-methyl branched fatty acids in yeast

BackgroundDespite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation.ResultsWe cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids.ConclusionsWe report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis

BackgroundHaematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis remains ambiguous.ResultsFive putative DGAT2 genes (HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of the HpDGAT2A, HpDGAT2D, and HpDGAT2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated that HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E had the capacity to restore TAG synthesis in a TAG-deficient yeast strain (H1246) showing a large difference in enzymatic activity. Fatty acid (FA) profile assays revealed that HpDGAT2A, HpDGAT2D, and HpDGAT2E, but not HpDGAT2B, preferred monounsaturated fatty acyl-CoAs (MUFAs) for TAG synthesis in yeast cells, and showed a preference for polyunsaturated fatty acyl-CoAs (PUFAs) based on their feeding strategy. The heterologous expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFAs and PUFAs contents.ConclusionsOur study represents systematic work on the characterization of HpDGAT2s by integrating expression patterns, AST/TAG accumulation, functional complementation, and heterologous expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove the TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFAs and PUFAs, and (4) offer target genes to modulate TAG biosynthesis by using genetic engineering methods.