Lipoxygenase : a game-changing enzyme

Many challenges lie ahead in using LOXs as tools in industrial oleochemistry. One of these challenges is the supply of PUFAs. Although we are moving towards a biobased economy where second and third generation biomass is taking a leading role, it is still faster and cheaper to use first generation biomass. Industrialization of microbial oils is a good alternative to supply the demand of PUFAs. Another challenge is the production of heterologous LOX in sufficient quantities. Since the last decade this problem is being tackled and more research is being done in heterologous expression of LOXs. The LOX with the highest potential so far is the secreted Pseudomonas aeruginosa LOX produced in Escherichia coli. During this thesis research different lox genes were tried for heterologous production of LOX using different Aspergillus niger and Aspergillus nidulans strains as expression hosts. These LOXs were identified as discussed in Chapter 3 and Chapter 6. Unfortunately, heterologous production in sufficient quantities was unsuccessful using these expression hosts as discussed in Chapter 5 and Chapter 6. Since production of Gaeumannomyces graminis LOX was successful in Trichoderma reesei, as discussed in Chapter 4, the production of polymers used for bioplastics could be demonstrated in this ERA-NOEL project anyway. Therefore this thesis shifted its focus on resolving the question of the difficulties in the heterologous expression of LOX in different Aspergillus species. Chapter 5 is the result of a systematic approach to analyze different aspects of G. graminis LOX expression in A. nidulans. Chapter 2 shows that heterologous expression of extracellular fungal LOX can be performed using T. reesei and Pichia pastoris as production hosts, and E. coli can be used for the production of intracellular LOXs of plant, mammal, bacterial, and fungal origin. As shown in Chapter 2, E. coli is not very efficient in the production of heterologous LOX due to the formation of inclusion bodies and low induction temperature necessary for production. The use of Aspergillus oryzae can be exploited further in the heterologous production of LOXs. Due to the choice of using A. niger and A. nidulans as expression hosts, this expression host was not exploited for its potential. The last challenge is to synthetically engineer LOX to broaden its use in industry. In this way more building blocks for chemicals can be synthetically produced and more products based on LOX origin can be made. Therefore, LOX can be a world-wide game-changing enzyme in a biobased economy as its use can decrease the demand for petroleum-based products. </p

Large-scale cultivation of Caenorhabditis elegans in a bioreactor using a labor-friendly fed-batch approach.

Caenorhabditis elegans is an invertebrate model organism used in many areas of biology including developmental biology and the identification of molecular mechanisms and pathways. However, several experimental approaches require large quantities of worms, which is limiting and time-consuming. We present a protocol that uses modern fermentation methodology to effectively produce large numbers of C. elegans using a 7-l bioreactor in a fed-batch cultivation procedure. The production is modular and flexible as well as being a self-controlled system, thus not much labor is required until harvesting C. elegans. The high-yield worm cultivation is flexible and simple to amend, and now allows for the extended application of C. elegans as a model organism and expression system, including large-scale protein production

Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans

Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans

A novel class of fungal lipoxygenases

Lipoxygenases (LOXs) are well-studied enzymes in plants and mammals. However, fungal LOXs are less studied. In this study, we have compared fungal LOX protein sequences to all known characterized LOXs. For this, a script was written using Shell commands to extract sequences from the NCBI database and to align the sequences obtained using Multiple Sequence Comparison by Log-Expectation. We constructed a phylogenetic tree with the use of Quicktree to visualize the relation of fungal LOXs towards other LOXs. These sequences were analyzed with respect to the signal sequence, C-terminal amino acid, the stereochemistry of the formed oxylipin, and the metal ion cofactor usage. This study shows fungal LOXs are divided into two groups, the Ile- and the Val-groups. The Ile-group has a conserved WRYAK sequence that appears to be characteristic for fungal LOXs and has as a C-terminal amino acid Ile. The Val-group has a highly conserved WL-L/F-AK sequence that is also found in LOXs of plant and animal origin. We found that fungal LOXs with this conserved sequence have a Val at the C-terminus in contrast to other LOXs of fungal origin. Also, these LOXs have signal sequences implying these LOXs will be expressed extracellularly. Our results show that in this group, in addition to the Gaeumannomyces graminis and the Magnaporthe salvinii LOXs, the Aspergillus fumigatus LOX uses manganese as a cofacto

Production of tailor-made enzymes to facilitate lipid extraction from the oleaginous yeast Schwanniomyces occidentalis

Due to the depletion of fossil fuel resources and concern about increasing atmospheric CO2 levels, the production of microbial oil as source for energy and chemicals is considered as a sustainable alternative. A promising candidate strain for the production of microbial oil is the oleaginous yeast Schwanniomyces occidentalis CBS 2864. To compete with fossil resources, cultivation and processing of S. occidentalis requires improvement. Currently, different cell wall disruption techniques based on mechanical, chemical, physiological, and biological methods are being investigated using a variety of oil producing yeasts and microalgae. Most of these techniques are not suitable for upscaling because they are technically or energetically unfavorable. Therefore, new techniques have to be developed to overcome this challenge. Here, we demonstrate an effective mild enzymatic approach for cell disruption to facilitate lipid extraction from the oleaginous yeast S. occidentalis. Most oil was released by applying 187 mg L−1 tailor-made enzymes from Trichoderma harzianum CBS 146429 against the yeast cell wall of S. occidentalis at pH 5.0 and 40 °C with 4 h of incubation time after applying 1 M NaOH as a pretreatment step