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1.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1650-1661, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559747

RESUMO

Para-aminobenzoate (PABA) is an important chemical for organic synthesis and extensively used in pharmaceutical and dye industry. In recent years, PABA has received increasing attention as a potential component of high-strength polymer. In Escherichia coli, three genes of pabA, pabB and pabC are responsible for PABA production from chorismate in folate synthetic pathway. However, E. coli does not accumulate or accumulates very few amounts of PABA under normal growth condition. In this study, the tyrosine-producing E. coli TYR002 constructed previously was used as the starting strain for developing PABA-producing strain. First, the activity of bifunctional chorismate mutase/prephenate dehydrogenase TyrA in E. coli TYR002 was weakened to reduce the production of tyrosine. Then, three different constitutive promoters were used to regulate the expression of pabA, pabB and pabC in recombinant plasmid which was transformed into E. coli for improving PABA production. The shake-flask fermentation showed that the different combination of constitutive promoters significantly affected the production of PABA, and the highest shake-flask fermentation titer was 0.67 g/L. After further condition optimization, the engineered E. coli produced 6.4 g/L PABA under 5 L fed-batch fermentation. This study could be a good reference for improving microbial production of PABA.


Assuntos
Escherichia coli , Ácido 4-Aminobenzoico , Plasmídeos
2.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1317-1325, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328488

RESUMO

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 µL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.


Assuntos
Microfluídica , Pichia , Mutagênese , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes
3.
Metab Eng ; 55: 23-32, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31220662

RESUMO

Bioplastics produced from microbial source are promising green alternatives to traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids, especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential monomers for the synthesis of polymeric bioplastics as their primary amine and carboxylic acid are ideal functional groups for polymerization. Previous pathways for 5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from L-lysine catabolism and the citric acid cycle, respectively. Here, we show the construction of an artificial iterative carbon-chain-extension cycle in Escherichia coli for simultaneous production of a series of nonnatural amino acids with varying chain length. Overexpression of L-lysine α-oxidase in E. coli yields 2-keto-6-aminocaproate (2K6AC) as a non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-extended α-ketoacid products are decarboxylated and oxidized by an α-ketoacid decarboxylase and an aldehyde dehydrogenase, respectively, to yield their corresponding nonnatural straight-chain amino acids. The engineered system demonstrated simultaneous in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate and 4.78 mg/L of 7-aminoheptanoate after 8 h of enzyme catalysis starting from 2K6AC as the substrate. Furthermore, simultaneous production of 2.15 g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising metabolic-engineering strategy to access other medium-chain organic acids with -NH2, -SCH3, -SOCH3, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-elongation chemistry.

4.
5.
FEMS Yeast Res ; 19(3)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30776066

RESUMO

Enhanced capability of co-fermenting glucose and xylose at high temperature is highly desirable for yeast application in second-generation bioethanol production. Here, we obtained hybrid strains with improved glucose-xylose co-fermentation properties at high temperature by combining genome shuffling and adaptive evolution. Genome resequencing of these strains suggested predominantly inherited genetic information from one parental strain Spathaspora passalidarum SP rather than the other parental strain Saccharomyces cerevisiae ScY01, possibly due to that the CUG codon system of S. passalidarum might have systematically eliminated most of the functional proteins from S. cerevisiae through misfolding. Compared to SP, one-copy loss of a 146-kb fragment was found in the hybrid strain and regained after being evolved for a while, whereas one-copy loss of an 11-kb fragment was only found after being evolved for a longer time. Besides, the genes affected by nonsynonymous variants were also identified, especially the mutation S540F in the endoplasmic reticulum chaperon Kar2. Structural prediction indicated that S540F might change the substrate binding activity of Kar2, and thus play a role in preventing protein aggregation in yeast at high temperature. Our results illustrated genomic alterations during this process and revealed some genomic factors that might be involved to determine yeast thermotolerance.

6.
ACS Synth Biol ; 8(2): 297-306, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30609888

RESUMO

Biosensors for target metabolites provide powerful high-throughput screening tools to obtain high-performing strains. However, well-characterized metabolite-sensing modules are often unavailable and limit rapid access to the robust biosensors with successful applications. In this study, we developed a strategy of transcriptome-assisted metabolite-sensing (TAMES) to identify the target metabolite-sensing module based on selectively comparative transcriptome analysis between the target metabolite producing and nonproducing strains and a subsequent quantative reverse transcription (RT-qPCR) evaluation. The strategy was applied to identify the sensing module cusR that responds positively to the metabolite 3-dehydroshikimate (DHS) and proved it was effective to narrow down the candidates. We further constructed the cusR-based synthetic biosensor and established the DHS biosensor-based high-throughput screening (HTS) platform to screen higher DHS-producing strains and successfully increased DHS production by more than 90%. This study demonstrated that the TAMES strategy was effective at exploiting the metabolite-sensing transcriptional regulator, and this could likely be extended to develop the biosensor-based HTS platforms for other molecules.

8.
Anal Chim Acta ; 1029: 50-57, 2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29907290

RESUMO

Data analysis represents a key challenge for untargeted metabolomics studies and it commonly requires extensive processing of more than thousands of metabolite peaks included in raw high-resolution MS data. Although a number of software packages have been developed to facilitate untargeted data processing, they have not been comprehensively scrutinized in the capability of feature detection, quantification and marker selection using a well-defined benchmark sample set. In this study, we acquired a benchmark dataset from standard mixtures consisting of 1100 compounds with specified concentration ratios including 130 compounds with significant variation of concentrations. Five software evaluated here (MS-Dial, MZmine 2, XCMS, MarkerView, and Compound Discoverer) showed similar performance in detection of true features derived from compounds in the mixtures. However, significant differences between untargeted metabolomics software were observed in relative quantification of true features in the benchmark dataset. MZmine 2 outperformed the other software in terms of quantification accuracy and it reported the most true discriminating markers together with the fewest false markers. Furthermore, we assessed selection of discriminating markers by different software using both the benchmark dataset and a real-case metabolomics dataset to propose combined usage of two software for increasing confidence of biomarker identification. Our findings from comprehensive evaluation of untargeted metabolomics software would help guide future improvements of these widely used bioinformatics tools and enable users to properly interpret their metabolomics results.


Assuntos
Metabolômica/métodos , Software , Benchmarking , Biomarcadores/metabolismo , Piper nigrum/metabolismo
9.
J Ind Microbiol Biotechnol ; 45(6): 405-415, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29749580

RESUMO

Deficiency in petroleum resources and increasing environmental concerns have pushed a bio-based economy to be built, employing a highly reproducible, metal contaminant free, sustainable and green biomanufacturing method. Here, a chiral drug intermediate L-pipecolic acid has been synthesized from biomass-derived lysine. This artificial bioconversion system involves the coexpression of four functional genes, which encode L-lysine α-oxidase from Scomber japonicus, glucose dehydrogenase from Bacillus subtilis, Δ1-piperideine-2-carboxylase reductase from Pseudomonas putida, and lysine permease from Escherichia coli. Besides, a lysine degradation enzyme has been knocked out to strengthen the process in this microbe. The overexpression of LysP improved the L-pipecolic acid titer about 1.6-folds compared to the control. This engineered microbial factory showed the highest L-pipecolic acid production of 46.7 g/L reported to date and a higher productivity of 2.41 g/L h and a yield of 0.89 g/g. This biotechnological L-pipecolic acid production is a simple, economic, and green technology to replace the presently used chemical synthesis.

10.
J Proteome Res ; 17(5): 1812-1825, 2018 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-29611422

RESUMO

To gain a deep understanding of yeast-cell response to heat stress, multiple laboratory strains have been intensively studied via genome-wide expression analysis for the mechanistic dissection of classical heat-shock response (HSR). However, robust industrial strains of Saccharomyces cerevisiae have hardly been explored in global analysis for elucidation of the mechanism of thermotolerant response (TR) during fermentation. Herein, we employed data-independent acquisition and sequential window acquisition of all theoretical mass spectra based proteomic workflows to characterize proteome remodeling of an industrial strain, ScY01, responding to prolonged thermal stress or transient heat shock. By comparing the proteomic signatures of ScY01 in TR versus HSR as well as the HSR of the industrial strain versus a laboratory strain, our study revealed disparate response mechanisms of ScY01 during thermotolerant growth or under heat shock. In addition, through proteomics data-mining for decoding transcription factor interaction networks followed by validation experiments, we uncovered the functions of two novel transcription factors, Mig1 and Srb2, in enhancing the thermotolerance of the industrial strain. This study has demonstrated that accurate and high-throughput quantitative proteomics not only provides new insights into the molecular basis for complex microbial phenotypes but also pinpoints upstream regulators that can be targeted for improving the desired traits of industrial microorganisms.

11.
J Ind Microbiol Biotechnol ; 45(8): 719-734, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29654382

RESUMO

L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.

12.
FEMS Yeast Res ; 18(5)2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29672693

RESUMO

TALENs-assisted multiplex editing (TAME) toolbox was previously established and used to successfully enhance ethanol stress tolerance of Saccharomyces cerevisiae laboratory strain. Here, the TAME toolbox was harnessed to improve and elucidate stress tolerances of S. cerevisiae industrial strain. One osmotolerant strain and one thermotolerant strain were selected from the mutant library generated by TAME at corresponding stress conditions, and exhibited 1.2-fold to 1.3-fold increases of fermentation capacities, respectively. Genome resequencing uncovered genomic alterations in the selected stress-tolerant strains, suggesting that cell wall and membrane-related proteins might be major factors behind improved tolerances of yeast to different stresses. Furthermore, amplified mitochondrial DNA might also have an important impact on increased stress tolerance. Unexpectedly, none of predesigned target potential TALENs modification sites showed any genomic variants in sequenced genomes of the selected strains, implicating that the improved stress tolerances might be due to indirect impacts of genome editing via TALENs rather than introducing genomic variants at potential target sites. Our findings not only confirmed TAME could be a useful tool to accelerate the breeding of industrial strain with multiple stress tolerance, but also supported the previous understandings of the complicated mechanisms of multiple stress tolerance in yeast.

14.
Sheng Wu Gong Cheng Xue Bao ; 34(1): 54-67, 2018 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-29380571

RESUMO

Mig1 and Snf1 are two key regulatory factors involved in glucose repression of Saccharomyces cerevisiae. To enhance simultaneous utilization of glucose and xylose by engineered S. cerevisiae, single and double deletion strains of MIG1 and SNF1 were constructed. Combining shake flask fermentations and transcriptome analysis by RNA-Seq, the mechanism of Mig1 and Snf1 hierarchically regulating differentially expressed genes that might affect simultaneous utilization of glucose and xylose were elucidated. MIG1 deletion did not show any significant effect on co-utilization of mixed sugars. SNF1 deletion facilitated xylose consumption in mixed sugars as well as co-utilization of glucose and xylose, which might be due to that the SNF1 deletion resulted in the de-repression of some genes under nitrogen catabolite repression, thereby favorable to the utilization of nitrogen nutrient. Further deletion of MIG1 gene in the SNF1 deletion strain resulted in the de-repression of more genes under nitrogen catabolite repression and up-regulation of genes involved in carbon central metabolism. Compared with wild type strain, the MIG1 and SNF1 double deletion strain could co-utilize glucose and xylose, and accelerate ethanol accumulation, although this strain consumed glucose faster and xylose slower. Taken together, the MIG1 and SNF1 deletions resulted in up-regulation of genes under nitrogen catabolite repression, which could be beneficial to simultaneous utilization of glucose and xylose. Mig1 and Snf1 might be involved in the hierarchical regulatory network of genes under nitrogen catabolite repression. Dissection of this regulatory network could provide further insights to new targets for improving co-utilization of glucose and xylose.

15.
Int J Biol Macromol ; 109: 1302-1310, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29175162

RESUMO

Pullulanase with high catalytic efficiency has attracted great attention from both the academic and industrial communities for its wide application in cold starch hydrolysis. A novel pullulanase gene pul703 was cloned from a mesophilic bacteria Bacillus pseudofirmus 703. Pul703 was characterized to be a type I pullulanase with maximal activity at 45°C and good low-temperature stability, more than 70% of activity was detected after incubation at 25-35°C for 72h. Pul703 obtained the maximal activity around pH 7.0-8.0, and was highly active and stable over a wide pH range of 5.5-9.5, more than 80% of activity was retained after 12h incubation in these pHs. Pul703 was EDTA-resistant and detergent-tolerant, with a relative activity of 100, 99, and 114.8% at the presence of 10mM EDTA, 10% of Triton X-100 and Tween 20, respectively. Pul703 can efficiently hydrolyze pullulan with a specific activity of 270U/mg, which was higher than all reported type I pullulanases. In addition, Pul703 can act synergistically with α-amylase BLA to efficiently hydrolyze amylopectin. These results suggested that Pul703 was a good candidate for cold starch hydrolysis.


Assuntos
Bacillus/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Bacillus/genética , Catálise , Quelantes/química , Quelantes/farmacologia , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Sequência Conservada , Detergentes/química , Detergentes/farmacologia , Ativação Enzimática , Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Hidrólise , Cinética , Mutação , Proteínas Recombinantes , Análise de Sequência de DNA , Especificidade por Substrato , Temperatura Ambiente
16.
Lab Chip ; 18(1): 190-196, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29227495

RESUMO

Lipases are ubiquitous enzymes of great physiological significance that have been used extensively in multiple industries. Environmental microorganisms are a major source for the discovery of novel lipases with high catalytic efficiency and selectivity. However, current plate-based screening of lipase-producing strains is time consuming, labour intensive and inefficient. In this study, we developed an ultra-high throughput screening pipeline for lipase-producing strains based on fluorescence-activated droplet sorting (FADS) using a compact optical system that could be easily set up in an alignment-free manner. The pipeline includes droplet generation, droplet incubation, picoinjection of the fluorescence probe, and sorting of droplets with a throughput of 2 × 106 drops per h. We applied the pipeline to screen samples collected from different locations, including sediments from a hot spring in Tibet, soils from the Zoige wetland, contaminated soils from an abandoned oilfield, and a Chinese Daqu starter. In total, we obtained 47 lipase-producing bacterial strains belonging to seven genera, including Staphylococcus, Bacillus, Enterobacter, Serratia, Prolinoborus, Acinetobacter, and Leclercia. We believe that this FADS-based pipeline could be extended to screen various enzymes from the environment, and may find wide applications in breeding of industrial microorganisms.


Assuntos
Bactérias , Técnicas Bacteriológicas/métodos , Citometria de Fluxo/métodos , Lipase/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Bactérias/enzimologia , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/instrumentação , Desenho de Equipamento , Citometria de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação
17.
Sheng Wu Gong Cheng Xue Bao ; 33(12): 1913-1922, 2017 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-29271169

RESUMO

To develop a high-yield pyruvate strain, we first engineered a pyruvate-producing Escherichia coli KLPP from wild-type E. coli MG1655 by blocking the pathways for byproduct formation via gene knockout. Then, we built a library of mutant containing 7 197 monoclones by using the pUT Mini-Tn5 transposon vector for random mutagenesis with E. coli KLPP. We developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader. After two-round screening we successfully obtained six mutants with increased pyruvate titer using this method, the titer of pyruvate was increased by 38%, 31%, 19%, 28%, 44% and 14%, respectively. The position of transposon insertion was determined by whole genome re-sequencing, and the gene locus possibly influencing pyruvate production was analyzed, which laid the foundation for subsequent strain improvement by metabolic engineering.

18.
Stem Cell Res Ther ; 8(1): 263, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29141658

RESUMO

BACKGROUND: Radiation exposure poses a significant threat to public health. Hematopoietic injury is one of the major manifestations of acute radiation sickness. Protection and/or mitigation of hematopoietic stem cells (HSCs) from radiation injury is an important goal in the development of medical countermeasure agents (MCM). We recently identified thioredoxin (TXN) as a novel molecule that has marked protective and proliferative effects on HSCs. In the current study, we investigated the effectiveness of TXN in rescuing mice from a lethal dose of total body radiation (TBI) and in enhancing hematopoietic reconstitution following a lethal dose of irradiation. METHODS: We used in-vivo and in-vitro methods to understand the biological and molecular mechanisms of TXN on radiation mitigation. BABL/c mice were used for the survival study and a flow cytometer was used to quantify the HSC population and cell senescence. A hematology analyzer was used for the peripheral blood cell count, including white blood cells (WBCs), red blood cells (RBCs), hemoglobin, and platelets. Colony forming unit (CFU) assay was used to study the colongenic function of HSCs. Hematoxylin and eosin staining was used to determine the bone marrow cellularity. Senescence-associated ß-galactosidase assay was used for cell senescence. Western blot analysis was used to evaluate the DNA damage and senescence protein expression. Immunofluorescence staining was used to measure the expression of γ-H2AX foci for DNA damage. RESULTS: We found that administration of TXN 24 h following irradiation significantly mitigates BALB/c mice from TBI-induced death: 70% of TXN-treated mice survived, whereas only 25% of saline-treated mice survived. TXN administration led to enhanced recovery of peripheral blood cell counts, bone marrow cellularity, and HSC population as measured by c-Kit+Sca-1+Lin- (KSL) cells, SLAM + KSL cells and CFUs. TXN treatment reduced cell senescence and radiation-induced double-strand DNA breaks in both murine bone marrow lineage-negative (Lin-) cells and primary fibroblasts. Furthermore, TXN decreased the expression of p16 and phosphorylated p38. Our data suggest that TXN modulates diverse cellular processes of HSCs. CONCLUSIONS: Administration of TXN 24 h following irradiation mitigates radiation-induced lethality. To the best of our knowledge, this is the first report demonstrating that TXN reduces radiation-induced lethality. TXN shows potential utility in the mitigation of radiation-induced hematopoietic injury.


Assuntos
Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/farmacologia , Tiorredoxinas/farmacologia , Irradiação Corporal Total , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Biomarcadores/metabolismo , Contagem de Células Sanguíneas , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Medula Óssea/efeitos da radiação , Linhagem Celular , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Expressão Gênica , Hematócrito , Hematopoese/genética , Hematopoese/efeitos da radiação , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Lesões Experimentais por Radiação/genética , Lesões Experimentais por Radiação/mortalidade , Lesões Experimentais por Radiação/patologia , Proteínas Recombinantes/farmacologia , Análise de Sobrevida , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Front Microbiol ; 8: 2184, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163455

RESUMO

Medium-chain (C8-C14) α, ω-dicarboxylic acids (α, ω-DCAs), which have numerous applications as raw materials for producing various commodities and polymers in chemical industry, are mainly produced from chemical or microbial conversion of petroleum-derived alkanes or plant-derived fatty acids at present. Recently, significant attention has been gained to microbial production of medium-chain α, ω-DCAs from simple renewable sugars. Here, we designed and created a synthetic omega oxidation pathway in Saccharomyces cerevisiae to produce C10 and C12 α, ω-DCAs from renewable sugars and fatty acids by introducing a heterogeneous cytochrome P450 CYP94C1 and cytochrome reductase ATR1. Furthermore, the deletion of fatty acyl-CoA synthetase genes FAA1 and FAA4 increased the production of medium-chain α, ω-DCAs from 4.690 ± 0.088 mg/L to 12.177 ± 0.420 mg/L and enabled the production of C14 and C16 α, ω-DCAs at low percentage. But blocking ß-oxidation pathway by deleting fatty-acyl coenzyme A oxidase gene POX1 and overexpressing different thioesterase genes had no significant impact on the production and the composition of α, ω-dicarboxylic acids. Overall, our study indicated the potential of microbial production of medium-chain α, ω-DCAs from renewable feedstocks using engineered yeast.

20.
J Biol Chem ; 292(50): 20707-20719, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29038295

RESUMO

An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within Saccharomyces cerevisiae cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention.


Assuntos
Aminoácidos Aromáticos/química , Retículo Endoplasmático/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Retículo Endoplasmático/enzimologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peso Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie , Técnicas do Sistema de Duplo-Híbrido
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