An Integrated Approach to Produce a Recombinant Yeast Pichia Pastoris For GLA Production

γ-Linolenic acid (GLA) is an important n-6 polyunsaturated fatty acid (PUFA) used in many nutritional and medicinal applications such as the treatment of cancer, inflammatory disorders, and diabetes. However, GLA level of the total fatty acids in plant seeds and nuts as prominent sources of GLA is not enough to utilize on an industrial scale. The study aimed to improve the expression of delta-6 desaturase, which is one of the important enzymes in GLA production pathway. The expression vector pPICZC was selected for clone M.rouxii delta-6 desaturase. The engineered vector was first cloned into E. coli DH5α and after plasmid extraction and confirmation of sequencing was transformed by electroporation into Pichia pastoris GS115. The results indicated that the recombinant yeast strain expressed the gene in the presence of methanol 0.5%. The lipids and essential fatty acids especially GLA were extracted to confirm the expression. The results of studies of lipid and fatty acid production by Sudan black and Nile red staining, GC, and flow cytometry revealed that recombinant strain can produce GLA levels up to 19.2% of total fatty acids. The present study may provide an opportunity for the development of an alternative host for manufacturing GLA on an industrial scale.

Fractionation, Chemical Analysis, and In Vitro Testing Identify Bioactive Components in MC252 Crude Oil

Crude oil is a complex mixture that includes polycyclic aromatic hydrocarbons (PAHs) as one of its major components. The toxicity of some chemically substituted PAHs found in oil, such as the methylated species, are relatively understudied. A combination of chemical fractionation and analysis coupled with a bioassay was used to identify a subset of oil PAHs that activated aryl hydrocarbon receptor (AHR). Silica gel chromatography was used for primary and secondary oil fractionation, and standard and reverse phase high performance liquid chromatography (HPLC) were used for the final fractionation steps. Both gas chromatography (GC-) and HPLC-coupled with mass spectrometry (MS) were used to separate and identify compounds present in the petroleum fractions. Bioactivity of the individual fractions was identified and measured using a recombinant yeast strain that expressed the human aryl hydrocarbon receptor complex (AHRC) transcription factor that is composed of human AHR and the ARNT proteins. AHRC activation by oil components results in expression of β-galactosidase, and readout from this enzymatic activity is proportional to the amount and potency of the compounds that activated the system. Silica gel separations produced 25–29 fractions that were assessed for bioactivity using the AHRC reporter system. Bioactivity peaked with the fractions that contained larger PAHs that included four ring compounds such as the triphenylenes, benzanthracenes, and chrysenes (MW 228 + additional methyl groups). When tested as individual compounds, the triphenylenes and benzanthracenes were less potent than the chrysenes, so the latter constituted more of the AHRC signaling activity in the oil fractions. The chrysenes in bioactive fractions were mixtures of the parent compound along with mono-, di-, tri-, and tetra-methyl derivatives and other PAHs. The six possible mono-methylchrysenes were obtained and tested for AHRC activity and for their concentrations in oil. Chrysene, 1-, 2-, 3-, and 6-methylchrysene were present, but 4- and 5-methylchrysene were not detected in the bioactive fractions of oil that were resolved by HPLC. When tested individually in the AHRC bioassay, 4-methylchrysene was the most potent ligand, and 5-methylchrysene was the least potent. Synthetic mixtures of PAHs were reconstructed based upon the chemical composition of one fraction with the high AHRC activity. Collectively, these data show that: 1) the six methylchrysene isomers are within an order of magnitude of chrysene in their ability to activate the AHRC bioassay; 2) although they are a minor group, the chrysene compounds in oil potently activate AHRC signaling; 3) chrysenes diminish as oil weathers, while triphenylenes of identical molecular weight persist, 4) this methodology can be useful for identification and characterization of the bioactivity of sub-fractions and individual compounds found in oil.

Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

ABSTRACTSeveral yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases inSaccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.

Methylamine-Sensitive Amperometric Biosensor Based on (His)6-TaggedHansenula polymorphaMethylamine Oxidase Immobilized on the Gold Nanoparticles

A novel methylamine-selective amperometric bienzyme biosensor based on recombinant primary amine oxidase isolated from the recombinant yeast strainSaccharomyces cerevisiaeand commercial horseradish peroxidase is described. Two amine oxidase preparations were used: free enzyme (AMO) and covalently immobilized on the surface of gold nanoparticles (AMO-nAu). Some bioanalytical parameters (sensitivity, selectivity, and storage stability) of the developed biosensors were investigated. The sensitivity for both sensors is high:1450 ± 113and700 ± 30 A−1·M−1·m−2for AMO-nAu biosensor, respectively. The biosensors exhibit the linear range from 15 μM to 150 μM (AMO-nAu) and from 15 μM to 60 μM (AMO). The developed biosensor demonstrated a good selectivity toward methylamine (MA) (signal for dimethylamine and trimethylamine is less than 5% and for ethylamine 15% compared to MA output) and reveals a satisfactory storage stability. The constructed amperometric biosensor was used for MA assay in real samples of fish products in comparison with chemical method. The values obtained with both approaches different methods demonstrated a high correlation.

Fuel Ethanol Production from Lignocellulosic Biomass Using a Recombinant Yeast Strain

Fuel ethanol production from lignocellulosic biomass corn stover was investigated. Compared with acid pretreatment and ammonia steeping pretreatment, alkali pretreatment with 2% NaOH markedly enhanced lignin removal and thereby improved the enzymatic hydrolysis yield to 81.2% by 48 h. Fed-batch hydrolysis was started with a batch hydrolysis containing 80 g/l substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 110 g/l. After 72 h of hydrolysis, the reducing sugar concentration reached 89.5 g/l with a hydrolysis yield of 83.3%. Further fermentation of the cellulosic hydrolysate containing 56.7 g/l glucose and 23.6 g/l xylose was performed using a recombinant yeast strain Saccharomyces cerevisiae ZU-10, and 36.3 g/l ethanol was produced within 72 h. The research results are meaningful in fuel ethanol production from renewable lignocellulosic biomass.