Biocatalytic, one-pot diterminal oxidation and esterification of n-alkanes for production of α,ω-diol and α,ω-dicarboxylic acid esters

Direct and selective terminal oxidation of medium-chain n-alkanes is a major challenge in chemistry. Efforts to achieve this have so far resulted in low specificity and overoxidized products. Biocatalytic oxidation of medium-chain n-alkanes – with for example the alkane monooxygenase AlkB from P. putida GPo1- on the other hand is highly selective. However, it also results in overoxidation. Moreover, diterminal oxidation of medium-chain n-alkanes is inefficient. Hence, α,ω-bifunctional monomers are mostly produced from olefins using energy intensive, multi-step processes. By combining biocatalytic oxidation with esterification we drastically increased diterminal oxidation upto 92 mol% and reduced overoxidation to 3% for n-hexane. This methodology allowed us to convert medium-chain n-alkanes into α,ω-diacetoxyalkanes and esterified α,ω-dicarboxylic acids. We achieved this in a one-pot reaction with resting-cell suspensions of genetically engineered Escherichia coli. The combination of terminal oxidation and esterification constitutes a versatile toolbox to produce α,ω-bifunctional monomers from n-alkanes

Purification and characterization of an NAD+-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotroph Arthrobacter P1

When Arthrobacter P1 is grown on choline, betaine, dimethylglycine or sarcosine, an NAD+-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa ± 10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa ± 3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the Km values were 0.10 and 0.80 mM for formaldehyde and NAD+, respectively. The enzyme was heat-stable at 50°C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity

Metabolic engineering of Pseudomonas putida KT2440 for medium-chain-length fatty alcohol and ester production from fatty acids

publishedVersio

Pilot-scale conversion of lime-treated wheat straw into bioethanol: quality assessment of bioethanol and valorization of side streams by anaerobic digestion and combustion

The limited availability of fossil fuel sources, worldwide rising energy demands and anticipated climate changes attributed to an increase of greenhouse gasses are important driving forces for finding alternative energy sources. One approach to meeting the increasing energy demands and reduction of greenhouse gas emissions is by large-scale substitution of petrochemically derived transport fuels by the use of carbon dioxide-neutral biofuels, such as ethanol derived from lignocellulosic material. Results This paper describes an integrated pilot-scale process where lime-treated wheat straw with a high dry-matter content (around 35% by weight) is converted to ethanol via simultaneous saccharification and fermentation by commercial hydrolytic enzymes and bakers' yeast (Saccharomyces cerevisiae). After 53 hours of incubation, an ethanol concentration of 21.4 g/liter was detected, corresponding to a 48% glucan-to-ethanol conversion of the theoretical maximum. The xylan fraction remained mostly in the soluble oligomeric form (52%) in the fermentation broth, probably due to the inability of this yeast to convert pentoses. A preliminary assessment of the distilled ethanol quality showed that it meets transportation ethanol fuel specifications. The distillation residue, which contained non-hydrolysable and non-fermentable (in)organic compounds, was divided into a liquid and solid fraction. The liquid fraction served as substrate for the production of biogas (methane), whereas the solid fraction functioned as fuel for thermal conversion (combustion), yielding thermal energy, which can be used for heat and power generation. Conclusion Based on the achieved experimental values, 16.7 kg of pretreated wheat straw could be converted to 1.7 kg of ethanol, 1.1 kg of methane, 4.1 kg of carbon dioxide, around 3.4 kg of compost and 6.6 kg of lignin-rich residue. The higher heating value of the lignin-rich residue was 13.4 MJ thermal energy per kilogram (dry basis)

From Eat to trEat : engineering the mitochondrial Eat1 enzyme for enhanced ethyl acetate production in Escherichia coli

Genetic engineering of microorganisms has become a common practice to establish microbial cell factories for a wide range of compounds. Ethyl acetate is an industrial solvent that is used in several applications, mainly as a biodegradable organic solvent with low toxicity. While ethyl acetate is produced by several natural yeast species, the main mechanism of production has remained elusive until the discovery of Eat1 in Wickerhamomyces anomalus. Unlike other yeast alcohol acetyl transferases (AATs), Eat1 is located in the yeast mitochondria, suggesting that the coding sequence contains a mitochondrial pre-sequence. For expression in prokaryotic hosts such as E. coli, expression of heterologous proteins with eukaryotic signal sequences may not be optimal. Results Unprocessed and synthetically truncated eat1 variants of Kluyveromyces marxianus and Wickerhamomyces anomalus have been compared in vitro regarding enzyme activity and stability. While the specific activity remained unaffected, half-life improved for several truncated variants. The same variants showed better performance regarding ethyl acetate production when expressed in E. coli. Conclusion By analysing and predicting the N-terminal pre-sequences of different Eat1 proteins and systematically trimming them, the stability of the enzymes in vitro could be improved, leading to an overall improvement of in vivo ethyl acetate production in E. coli. Truncated variants of eat1 could therefore benefit future engineering approaches towards efficient ethyl acetate production.publishedVersio

Lactic acid production from lime-treated wheat straw by Bacillus coagulans: neutralization of acid by fed-batch addition of alkaline substrate

Conventional processes for lignocellulose-to-organic acid conversion requires pretreatment, enzymatic hydrolysis, and microbial fermentation. In this study, lime-treated wheat straw was hydrolyzed and fermented simultaneously to lactic acid by an enzyme preparation and Bacillus coagulans DSM 2314. Decrease in pH because of lactic acid formation was partially adjusted by automatic addition of the alkaline substrate. After 55 h of incubation, the polymeric glucan, xylan, and arabinan present in the lime-treated straw were hydrolyzed for 55%, 75%, and 80%, respectively. Lactic acid (40.7 g/l) indicated a fermentation efficiency of 81% and a chiral l(+)-lactic acid purity of 97.2%. In total, 711 g lactic acid was produced out of 2,706 g lime-treated straw, representing 43% of the overall theoretical maximum yield. Approximately half of the lactic acid produced was neutralized by fed-batch feeding of lime-treated straw, whereas the remaining half was neutralized during the batch phase with a Ca(OH)2 suspension. Of the lime added during the pretreatment of straw, 61% was used for the neutralization of lactic acid. This is the first demonstration of a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime

Enhancing Jatropha oil extraction yield from the kernels assisted by a xylan-degrading bacterium to preserve protein structure

We investigated the use of bacterial cells isolated from paddy crab for the extraction of oil from Jatropha seed kernels in aqueous media while simultaneously preserving the protein structures of this protein-rich endosperm. A bacterial strain—which was marked as MB4 and identified by means of 16S rDNA sequencing and physiological characterization as either Bacillus pumilus or Bacillus altitudinis—enhanced the extraction yield of Jatropha oil. The incubation of an MB4 starter culture with preheated kernel slurry in aqueous media with the initial pH of 5.5 at 37 °C for 6 h liberated 73% w/w of the Jatropha oil. Since MB4 produces xylanases, it is suggested that strain MB4 facilitates oil liberation via degradation of hemicelluloses which form the oil-containing cell wall structure of the kernel. After MB4 assisted oil extraction, SDS-PAGE analysis showed that the majority of Jatropha proteins were preserved in the solid phase of the extraction residues. The advantages offered by this process are: protein in the residue can be further processed for other applications, no purified enzyme preparation is needed, and the resulting oil can be used for biodiesel production

Production of cyanophycin in Rhizopus oryzae through the expression of a cyanophycin synthetase encoding gene

Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid

Metabolic engineering of Rhizopus oryzae for the production of platform chemicals

Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes. It is among others known for its ability to produce the sustainable platform chemicals l-(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for l-(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products