Encoding acetyl-CoA Carboxylase Research Articles

Engineering Streptomyces albus B4 for Enhanced Production of (R)-Mellein: A High-Titer Heterologous Biosynthesis Approach.

The natural compound (R)-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 (pks8) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous (R)-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in β-oxidation (fadD, fadE, fadB, and fadA). Cell growth inhibition by (R)-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced (R)-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous (R)-(-)-mellein synthesis in microorganism with a high titer. (R)-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica.

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

Mechanisms of metabolic regulation and enhanced methane production by hydrolytic nanozyme in sludge anaerobic digestion

Artificial nanozymes have demonstrated potential as natural enzyme substitutes due to their efficient biomacromolecule hydrolysis. However, the metabolic and catalytic mechanisms of nanozyme during the biodegradation of complex organics remain inadequately understood. This study evaluated the effect of the hydrolytic mechanism of cerium-based metal–organic frameworks (Ce-MOFs) hydrolytic nanozyme on shaping microbiome and metabolic functions. Biochemical methane potential test revealed that Ce-MOF-based hydrolytic nanozyme increased methane production by 7.8 − 22.2% as the hydrolytic nanozyme dosage increased from 50 to 150 mg/g VS. The highest methane production observed in 150 mg/g VS dosage of Ce-MOF (Ce-MOF-150) could be attributed to the enhanced sludge hydrolysis, acidogenesis, and methanogenesis processes, proven by the increased relative abundance of dominant acidogens and methanogens. Furthermore, 150 mg/g VS hydrolytic nanozyme bolstered the relative abundance of genes encoding acetyl-CoA carboxylase (accABCD) and involving in glycolysis (hxk and pfkA). These quantities were higher (8.31 − 34.14%) than that without nanozyme addition. The enhanced relative abundance of ackA, pta, cdhCDE, mvhAD, fpoABCDHIJKLMNO, vhoC, rnfABCDEG, mtrABCDEFGH, and hdrABC in the Ce-MOF-150 test indicated that the high proton conductivity of nanozyme facilitated acetoclastic methanogenesis and membrane-bound electron transport system, thereby enhancing methane production. These findings contribute to the expanding understanding of the application of artificial nanozymes as biocatalysts for solid waste resource recovery, providing a promising approach for methane production from sewage sludge.

Standing genetic variation fuels rapid evolution of herbicide resistance in blackgrass

Repeated herbicide applications in agricultural fields exert strong selection on weeds such as blackgrass (Alopecurus myosuroides), which is a major threat for temperate climate cereal crops. This inadvertent selection pressure provides an opportunity for investigating the underlying genetic mechanisms and evolutionary processes of rapid adaptation, which can occur both through mutations in the direct targets of herbicides and through changes in other, often metabolic, pathways, known as non-target-site resistance. How much target-site resistance (TSR) relies on de novo mutations vs. standing variation is important for developing strategies to manage herbicide resistance. We first generated a chromosome-level reference genome for A. myosuroides for population genomic studies of herbicide resistance and genome-wide diversity across Europe in this species. Next, through empirical data in the form of highly accurate long-read amplicons of alleles encoding acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) variants, we showed that most populations with resistance due to TSR mutations-23 out of 27 and six out of nine populations for ACCase and ALS, respectively-contained at least two TSR haplotypes, indicating that soft sweeps are the norm. Finally, through forward-in-time simulations, we inferred that TSR is likely to mainly result from standing genetic variation, with only a minor role for de novo mutations.

Metabolomics and Transcriptomics Provide Insights into Lipid Biosynthesis in the Embryos of Walnut (Juglans regia L.).

Walnut (Juglans regia L.) is an important woody oilseed tree species due to its commercial value. However, the regulation mechanism of walnut oil accumulation is still poorly understood, which restricted the breeding and genetic improvement of high-quality oil-bearing walnuts. In order to explore the metabolic mechanism that regulates the synthesis of walnut oil, we used transcriptome sequencing technology and metabolome technology to comprehensively analyze the key genes and metabolites involved in oil synthesis of the walnut embryo at 60, 90, and 120 days after pollination (DAP). The results showed that the oil and protein contents increased gradually during fruit development, comprising 69.61% and 18.32% of the fruit, respectively, during ripening. Conversely, the contents of soluble sugar and starch decreased gradually during fruit development, comprising 2.14% and 0.84%, respectively, during ripening. Transcriptome sequencing generated 40,631 unigenes across 9 cDNA libraries. We identified 51 and 25 candidate unigenes related to the biosynthesis of fatty acid and the biosynthesis of triacylglycerol (TAG), respectively. The expression levels of the genes encoding Acetyl-CoA carboxylase (ACCase), long-chain acyl-CoA synthetases (LACS), 3-oxoacyl-ACP synthase II (KASII), and glycerol-3-phosphate acyl transfer (GPAT) were upregulated at 60 DAP relative to the levels at 90 and 120 DAP, while the stearoyl-ACP-desaturase (SAD) and fatty acid desaturase 2 (FAD2) genes were highly abundantly expressed during all walnut developmental periods. We found that ABSCISIC ACID INSENSEITIVE3 (ABI3), WRINKLEDl (WRI1), LEAFY COTYLEDON1 (LEC1), and FUSCA3 (FUS3) may be key transcription factors involved in lipid synthesis. Additionally, the metabolomics analysis detected 706 metabolites derived from 18 samples, among which, 4 are implicated in the TAG synthesis, 2 in the glycolysis pathway, and 5 in the tricarboxylic acid cycle (TCA cycle) pathway. The combined analysis of the related genes and metabolites in TAG synthesis showed that phospholipid:diacylglycerol acyltransferase (PDAT) genes were highly abundantly expressed across walnut fruit developmental periods, and their downstream metabolite TAG gradually accumulated with the progression of fruit development. The FAD2 gene showed consistently higher expression during fruit development, and its downstream metabolites 18:2-PC and 18:3-PC gradually accumulated. The ACCase, LACS, SAD, FAD2, and PDAT genes may be crucial genes required for walnut oil synthesis. Our data will enrich public databases and provide new insights into functional genes related to lipid metabolism in walnut.

Fatty Acid Production by Enhanced Malonyl-CoA Supply in Escherichia coli

The expression of exogenous genes encoding acetyl-CoA carboxylase (Acc) and pantothenate kinase (CoaA) in Escherichia coli enable highly effective fatty acid production. Acc-only strains grown at 37°C or 23°C produced an approximately twofold increase in fatty acid content, and additional expression of CoaA achieved a further twofold accumulation. In the presence of pantothenate, which is the starting material for the CoA biosynthetic pathway, the size of the intracellular CoA pool at 23°C was comparable to that at 30°C during cultivation, and more than 500mg/L of culture containing cellular fatty acids was produced, even at 23°C. However, the highest yield of cellular fatty acids (1100mg/L of culture) was produced in cells possessing the gene encoding type I bacterial fatty acid synthase (FasA) along with the acc and coaA, when the transformant was cultivated at 30°C in M9 minimal salt medium without pantothenate or IPTG. This E. coli transformant contained 141mg/L of oleic acid attributed to FasA under noninducible conditions. The increased fatty acid content was brought about by a greatly improved specific productivity of 289mg/g of dry cell weight. Thus, the effectiveness of the foreign acc and coaA in fatty acid production was unambiguously confirmed at culture temperatures of 23°C to 37°C. Cofactor engineering in E. coli using the exogenous coaA and acc genes resulted in fatty acid production over 1g/L of culture and could effectively function at 23°C.

Synthesis of Dolichols in Candida albicans Is Co-Regulated with Elongation of Fatty Acids

Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl reductase Dfg10 together with some other unknown enzymes. The aim of this study was to identify such enzymes in Candida. The Dfg10 polyprenyl reductase from S. cerevisiae comprises a C-terminal 3-oxo-5-alpha-steroid 4-dehydrogenase domain. Alignment analysis revealed such a domain in two ORFs (orf19.209 and orf19.3293) from C. albicans, which were similar, respectively, to Dfg10 polyprenyl reductase and Tsc13 enoyl-transferase from S. cerevisiae. Deletion of orf19.209 in Candida impaired saturation of polyprenols. The Tsc13 homologue turned out not to be capable of saturating polyprenols, but limiting its expression reduce the cellular level of dolichols and polyprenols. This reduction was not due to a decreased expression of genes encoding cis-prenyltransferases from the dolichol branch but to a lower expression of genes encoding enzymes of the early stages of the mevalonate pathway. Despite the resulting lower consumption of acetyl-CoA, the sole precursor of the mevalonate pathway, it was not redirected towards fatty acid synthesis or elongation. Lowering the expression of TSC13 decreased the expression of the ACC1 gene encoding acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid synthesis and elongation.

Metabolic engineering of Bacillus subtilis based on genome-scale metabolic model to promote fengycin production.

The online version contains supplementary material available at 10.1007/s13205-021-02990-7.

Global Transcriptome Analyses Provide Into Several Fatty Acid Biosynthesis-related Genes in Peanut (Arachis hypogaea L.)

Peanut (Arachis hypogaea L.) is one of the important oil crops worldwide. Revealing the molecular basis of seed oil synthesis and analyzing key candidate genes may have important implications for enhancing peanut production. In this study, we investigated the dynamic changes in the oil accumulation rate and gene expression levels in developing peanut seeds. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the up-regulated differentially expressed genes (DEGs) among three varieties at 25–35 days after flowering (DAF) were involved in fatty acid synthesis, carbon metabolism, and pyruvate metabolism. Between 25 and 35 DAF, the up-regulated DEGs in the high-oil varieties, but not in the low-oil variety, were related to acetyl-CoA synthesis. We examined the differences in the varieties at the same stages. We analyzed the lipid metabolism-related genes by protein–protein interaction (PPI), and finally identified 57 DEGs that may influence the peanut oil content. These candidate genes included three genes encoding acetyl-CoA carboxylase (ACCase), one gene encoding ketoacyl-ACP synthase III (KASIII), one gene encoding hydroxyacyl-ACP dehydrase (HAD), and one gene encoding enoyl-ACP reductase (EAR), which form the Type II fatty acid synthase complex. Additionally, we also found that 11 transcription factors may affect oil content. These identified candidate genes may help to further clarify the molecular basis of the oil accumulation in peanut seeds.

Recombinant overexpression of the Escherichia coli acetyl-CoA carboxylase gene in Synechocystis sp. boosts lipid production.

Microalgae have received continued attention as a potential source for biofuel production. However, the lack of suitable strains that provide a lipid-rich biomass and tolerate harsh condition inhibits their industrial application. This report describes an effort to transform Synechocystis sp. with genes encoding acetyl-CoA carboxylase (ACC), a key regulatory enzyme in the lipogenesis pathway, from the white mustard plant (Sinapis alba) and the bacterium Escherichia coli DH5α using chitosan nanoparticles. Although a recombinant plasmid encoding S. alba ACC failed to express, successful transformation was achieved with a recombinant plasmid encoding E. coli DH5α ACC. The successful transformant, Synechocystis sp. PAK13, exhibited increased ACC expression compared with its wild-type parent (11.8 vs. 7.2 ng), which significantly increased its lipid content (by 3.6-fold). Synechocystis sp. PAK13 also exhibited a significant (20%) reduction in photosynthetic pigments, a 1.52-fold higher glucose content and a 3.5-fold lower sucrose content than the wild-type. In conclusion, this report introduces a useful strategy to overexpress the ACC gene in microalgae, creating strains with improved lipid production that are suited to industrial applications.

UV-Induced Reduction of ACVR1C Decreases SREBP1 and ACC Expression by the Suppression of SMAD2 Phosphorylation in Normal Human Epidermal Keratinocytes.

Activin A receptor type 1C (ACVR1C), a type I transforming growth factor-β (TGF-β) receptor, has been implicated in sensitive skin and psoriasis and is involved in the regulation of metabolic homeostasis as well as cell proliferation and differentiation. In this study, we identified a novel role of ACVR1C in the ultraviolet (UV)-irradiation-induced reduction of epidermal lipogenesis in human skin. UV irradiation decreased ACVR1C expression and epidermal triglyceride (TG) synthesis in human skin in vivo and in primary normal human epidermal keratinocytes (NHEK) in vitro. Lipogenic genes, including genes encoding acetyl-CoA carboxylase (ACC) and sterol regulatory element binding protein-1 (SREBP1), were significantly downregulated in UV-irradiated NHEK. ACVR1C knockdown by shRNA resulted in greater decreases in SREBP1 and ACC in response to UV irradiation. Conversely, the overexpression of ACVR1C attenuated the UV-induced decreases in SREBP1 and ACC. Further mechanistic study revealed that SMAD2 phosphorylation mediated the ACVR1C-induced lipogenic gene modulation. Taken together, a decrease in ACVR1C may cause UV-induced reductions in SREBP1 and ACC as well as epidermal TG synthesis via the suppression of SMAD2 phosphorylation. ACVR1C may be a target for preventing or treating UV-induced disruptions in lipid metabolism and associated skin disorders.

Metabolic Engineering of Oleaginous Yeast Yarrowia lipolytica for Overproduction of Fatty Acids.

The oleaginous yeast Yarrowia lipolytica has attracted much attention due to its ability to utilize a wide range of substrates to accumulate high lipid content and its flexibility for genetic manipulation. In this study, intracellular lipid metabolism in Y. lipolytica was tailored to produce fatty acid, a renewable oleochemical and precursor for production of advanced biofuels. Two main strategies, including blocking activation and peroxisomal uptake of fatty acids and elimination of biosynthesis of lipids, were employed to reduce fatty acid consumption by the native pathways in Y. lipolytica. Both genetic modifications improved fatty acid production. However, disruption of the genes responsible for assembly of nonpolar lipid molecules including triacylglycerols (TAGs) and steryl esters resulted in the deleterious effects on the cell growth. The gene tesA encoding thioesterase from Escherichia coli was expressed in the strain with disrupted faa genes encoding fatty acyl-CoA synthetases and pxa1 encoding peroxisomal acyl-CoA transporter, and the titer of fatty acids resulted in 2.3 g/L in shake flask culture, representing 11-fold improvement compared with the parent strain. Expressing the native genes encoding acetyl-CoA carboxylase (ACC) and hexokinase also increased fatty acid production, although the improvement was not as significant as that with tesA expression. Saturated fatty acids including palmitic acid (C16:0) and stearic acid (C18:0) increased remarkably in the fatty acid composition of the recombinant bearing tesA compared with the parent strain. The recombinant expressing tesA gene resulted in high lipid content, indicating the great fatty acid producing potential of Y. lipolytica. The results highlight the achievement of fatty acid overproduction without adverse effect on growth of the strain. Results of this study provided insight into the relationship between fatty acid and lipid metabolism in Y. lipolytica, confirming the avenue to reprogram lipid metabolism of this host for overproduction of renewable fatty acids.

Use of acetate for the production of 3-hydroxypropionic acid by metabolically-engineered Pseudomonas denitrificans

The use of acetate as carbon feedstock can enhance sustainability and economics of the current bio-productions. This study explored the potential of acetate for the production of 3-hydroxypropionic acid by engineered Pseudomonas denitrificans. Heterologous mcr (encoding malonyl-CoA reductase) from Chloroflexus aurantiacus and endogenous accABCD (encoding acetyl-CoA carboxylase) were overexpressed in P. denitrificans. Carbon flux to 3-HP synthesis at the malonyl-CoA node was promoted by suppressing fatty acid synthesis through addition of cerulenin or deletion of fabF gene. In addition, stimulation of glyoxylate shunt and/or TCA cycle were attempted. Recombinant P. denitrificans overexpressing mcr and accABCD produced 19.3 mM 3-HP with cerulenin addition, and 14.2 mM with fabF deletion, respectively. Furthermore, the non-growing cells devoid of fabF could continuously produce 3-HP up to 40.4 mM without losing its production activity for 22 h. This study demonstrates that acetate is a good substrate for 3-HP production by recombinant P. denitrificans.

Genomic insights into the lifestyles, functional capacities and oleagenicity of members of the fungal family Trichosporonaceae

Trichosporonaceae incorporates six genera of physiologically and ecologically diverse fungi including both human pathogenic taxa as well as yeasts of biotechnological interest, especially those oleagenic taxa that accumulate large amounts of single cell oils (SCOs). Here, we have undertaken comparative genomic analysis of thirty-three members of the family with a view to gain insight into the molecular determinants underlying their lifestyles and niche specializations. Phylogenomic analysis revealed potential misidentification of three strains which could impact subsequent analyses. Evaluation of the predicted proteins coding sequences showed that the free-living members of the family harbour greater numbers of carbohydrate active enzymes (CAZYmes), metallo- and serine peptidases compared to their host-associated counterparts. Phylogenies of selected lipid biosynthetic enzymes encoded in the genomes of the studied strains revealed disparate evolutionary histories for some proteins inconsistent with the core genome phylogeny. However, the documented oleagenic members distinctly cluster based on the constitution of the upstream regulatory regions of genes encoding acetyl-CoA carboxylase (ACC), ATP-citrate synthase (ACS) and isocitrate dehydrogenase [NADP] (ICDH), which are among the major proteins in the lipid biosynthetic pathway of these yeasts, suggesting a possible pattern in the regulation of these genes.

Development and Quality of Barley Husk Adhesion Correlates With Changes in Caryopsis Cuticle Biosynthesis and Composition.

The caryopses of barley become firmly adhered to the husk during grain development through a cuticular cementing layer on the caryopsis surface. The degree of this attachment varies among cultivars, with poor quality adhesion causing “skinning”, an economically significant grain quality defect for the malting industry. Malting cultivars encompassing a range of husk adhesion qualities were grown under a misting treatment known to induce skinning. Development of the cementing layer was examined by electron microscopy and compositional changes of the cementing layer were investigated with gas-chromatography followed by mass spectroscopy. Changes in gene expression during adhesion development were examined with a custom barley microarray. The abundance of transcripts involved early in cuticular lipid biosynthesis, including those encoding acetyl-CoA carboxylase, and all four members of the fatty acid elongase complex of enzymes, was significantly higher earlier in caryopsis development than later. Genes associated with subsequent cuticular lipid biosynthetic pathways were also expressed higher early in development, including the decarbonylation and reductive pathways, and sterol biosynthesis. Changes in cuticular composition indicate that lowered proportions of alkanes and higher proportions of fatty acids are associated with development of good quality husk adhesion, in addition to higher proportions of sterols.

Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 6803

BackgroundCyanobacteria are potential sources for third generation biofuels. Their capacity for biofuel production has been widely improved using metabolically engineered strains. In this study, we employed metabolic engineering design with target genes involved in selected processes including the fatty acid synthesis (a cassette of accD, accA, accC and accB encoding acetyl-CoA carboxylase, ACC), phospholipid hydrolysis (lipA encoding lipase A), alkane synthesis (aar encoding acyl-ACP reductase, AAR), and recycling of free fatty acid (FFA) (aas encoding acyl–acyl carrier protein synthetase, AAS) in the unicellular cyanobacterium Synechocystis sp. PCC 6803.ResultsTo enhance lipid production, engineered strains were successfully obtained including an aas-overexpressing strain (OXAas), an aas-overexpressing strain with aar knockout (OXAas/KOAar), and an accDACB-overexpressing strain with lipA knockout (OXAccDACB/KOLipA). All engineered strains grew slightly slower than wild-type (WT), as well as with reduced levels of intracellular pigment levels of chlorophyll a and carotenoids. A higher lipid content was noted in all the engineered strains compared to WT cells, especially in OXAas, with maximal content and production rate of 34.5% w/DCW and 41.4 mg/L/day, respectively, during growth phase at day 4. The OXAccDACB/KOLipA strain, with an impediment of phospholipid hydrolysis to FFA, also showed a similarly high content of total lipid of about 32.5% w/DCW but a lower production rate of 31.5 mg/L/day due to a reduced cell growth. The knockout interruptions generated, upon a downstream flow from intermediate fatty acyl-ACP, an induced unsaturated lipid production as observed in OXAas/KOAar and OXAccDACB/KOLipA strains with 5.4% and 3.1% w/DCW, respectively.ConclusionsAmong the three metabolically engineered Synechocystis strains, the OXAas with enhanced free fatty acid recycling had the highest efficiency to increase lipid production.

Reduced skin lipid content in obese Japanese women mediated by decreased expression of rate-limiting lipogenic enzymes.

Skin barrier function is often deficient in obese individuals, but the underlying molecular mechanisms remain unclear. This study investigated how skin structure and lipid metabolism, factors strongly associated with barrier function, differed among 50 Japanese women of greatly varying body mass index (BMI). Subjects receiving breast reconstruction surgery were chosen for analysis to obtain skin samples from the same site. The subjects were classified into two groups, control (BMI < 25 kg/m2) and obese (25 kg/m2 ≤ BMI < 35 kg/m2), according to standards in Japan. Hematoxylin and eosin staining was used to assess skin thickness, Ki-67 immunostaining to examine keratinocyte proliferation, and real-time polymerase chain reaction to measure skin expression levels of genes associated with lipid metabolism. Total lipids, cholesterol, and fatty acids were also measured from these same skin samples. In the obese group, structural changes included epidermal thickening and an increase in the number of Ki-67-positive (proliferating) cells. Both skin cholesterol and fatty acid levels exhibited an “inverted-U” relationship with BMI, suggesting that there is an optimal BMI for peak lipid content and barrier function. Decreased lipid levels at higher BMI were accompanied by downregulated expression of PPARδ and other genes related to lipid metabolism, including those encoding acetyl-CoA carboxylase and HMG-CoA reductase, the rate-limiting enzymes for fatty acid and cholesterol synthesis, respectively. Thus, elevated BMI may lead to deficient skin barrier function by suppressing local lipid synthesis.

Driving Pest Insect Populations: Agricultural Chemicals Lead to an Adaptive Syndrome in Nilaparvata Lugens Stål (Hemiptera: Delphacidae).

The brown planthopper (BPH) is a devastating pest of rice throughout Asia. In this paper we document the BPH biogeographic range expansion in China over the 20-year period, 1992 to 2012. We posed the hypothesis that the range expansion is due to a syndrome of adaptations to the continuous presence of agricultural chemicals (insecticides and a fungicide) over the last 40 years. With respect to biogeography, BPH ranges have expanded by 13% from 1992 to 1997 and by another 3% from 1997 to 2012. In our view, such expansions may follow primarily from the enhancing effects of JGM, among other agricultural chemicals, and from global warming. JGM treatments led to increased thermotolerance, recorded as decreased mortality under heat stress at 40 ± 1 °C (down from 80% to 55%) and increased fecundity (by 49%) at 34 °C. At the molecular level, JGM treatments led to increased abundances of mRNA encoding Acetyl Co-A carboxylase (Acc) (up 25%) and Hsp70 (up 32%) in experimental BPH. RNAi silencing of Hsp70 and Acc eliminated the JGM effects on fecundity and silencing Hsp70 reduced JGM-induced thermotolerance. Integrated with global climate change scenarios, such syndromes in pest insect species have potential for regional- and global-scale agricultural disasters.

Acute toxicity and gene responses induced by endosulfan in zebrafish (Danio rerio) embryos

Endosulfan has been listed as a persistent organic pollutant, and is frequently found in agricultural environments during monitoring processes owing to its heavy use and persistent characteristics. This study was conducted to understand the effects of endosulfan on the development of zebrafish (Danio rerio) embryos by exposing them to a specific range of endosulfan concentrations. Exposing zebrafish embryos to endosulfan for 96 h yielded no acute toxicity until the concentration reached 1500 μg L−1, whereas malformed zebrafish larvae developed severely curved spines and shortened tails. About 50% of zebrafish larvae were malformed when exposed to 600 μg L−1 of endosulfan. Comparative gene expression using real-time quantitative polymerase chain reaction was assessed using endosulfan-exposed zebrafish embryos. CYP1A and CYP3A were significantly enhanced in response to endosulfan treatment. Two genes, acacb and fasn, encoding acetyl-CoA carboxylase b and fatty acid synthase proteins, respectively, were also up-regulated after treating zebrafish embryos with endosulfan. These genes are also involved in fatty acid biosynthesis. The genes encoding vitellogenin and Hsp70 increased in a concentration-dependent manner in embryos. Finally, biochemical studies showed that acetylcholinesterase activity was reduced, whereas glutathione S-transferase and carboxylesterase activities were enhanced in zebrafish embryos after endosulfan treatment. These biochemical and molecular biological differences might be used for tools to determine contamination of endosulfan in the aquatic environment.

Improvement in Oil Production by Increasing Malonyl-CoA and Glycerol-3-Phosphate Pools in Scenedesmus quadricauda.

In recent years, microalgae have attracted considerable interest as a biofuel resource owing to their rapid growth, tolerance to harsh conditions, and ability to accumulate a large amount of triacylglycerols (TAGs). However, the economic effectiveness of algal biofuel is still low. In this study, we attempted to increase oil production of the microalga Scenedesmus quadricauda by elevating intracellular malonyl-CoA and glycerol-3-phosphate (G3P) pools. To increase intracellular oil content, yeast-derived genes encoding acetyl-CoA carboxylase (ACC1), glycerol kinase (GPD1), and glycerol-3-phosphate dehydrogenase (GUT1) were overexpressed under the control of CaMV 35S and NOS promoters with SV40 large T antigen components. Fatty acid profiling, G3P content, and the number of cells with high oil content were analyzed by gas chromatography-mass spectrometry, G3P assay kit, and flow cytometry, respectively. Overexpression of ACC1 increased the total fatty acid content by 1.6-fold. Overexpression of GPD1 and GUT1 increased intracellular G3P content by 1.6- and 1.9-fold, respectively. Multi-gene expression of ACC1, GPD1, and GUT1 increased the number of cells with high oil content by 1.45-fold compared with that observed with the wild-type. This study is the first to report increased oil production by overexpression of the key genes (ACC1, GPD1, and GUT1) for TAG biosynthesis in microalgae.