Exploration of Issatchenkia orientalis as a host for the production of itaconic acid

Recent years have raised awareness towards bio-based production routes starting from raw material to commercial goods, as an alternative to petrochemical derivates chemical synthesis. Looking towards the achievement of ecological and economic sustainability it is crucial to take advantage of lignocellulosic feedstocks for biomass production, as these derive from what is commonly conceived as waste. In particular, organic acid production has taken the centre stage relative to the broad applicability of organic acids for usage as polymer building blocks, as well as for commodity chemical synthesis. In 2004, the United States Department of Energy (US-DOE) produced a report on the top twelve added value chemical platforms to be obtained from renewable biomass as the result of an extensive screening effort, eight of which are identified as organic acids []. In 2015, a more specific study was conducted relating to the synthesis and application of organic acids as chemical building blocks, listing the seven market-share wise most promising organic acids which may be obtained in a biorefinery setting []. Among these, the C5 itaconic acid is featured as a promising chemical platform due to the presence of two carboxylic acid functionalities and an α-β unsaturated double-bond which make it a relevant precursor for diverse chemical transformations [] Specifically, itaconic acid is of great interest for the bio-based polymer synthesis industry, namely polyester-based ones with added functionalities such as UV/thermal curing and shape memory polymers. Other applications include detergents, coatings and rubber such as nitrile latex. The most promising application relates to the conversion of IA to methyl methacrylate, also known as Plexiglas, with a potential market of up to 3.2 million tons/year []. Presently, itaconic acid synthesis is performed by the fungus Aspergillus terreus with highest reported concentration of 86.2 g L 1 [] 150 g L [] for the shake flask and 15 L bioreactor fermentation settings, respectively. In A. terreus, the itaconic acid pathway involves the transport of the TCA cycle intermediate cisaconitate from the TCA cycle to the cytosol, where the cis-aconitate decarboxylase enzyme (cadA) catalyses the conversion to itaconic acid. One other relevant itaconic acid producer is A.niger which due to its citrate accumulating phenotype has been extensively explored as cell factory for the production of itaconic acid []. Most recent efforts report a itaconic acid titer of 26.2 g L in a heavily engineered strain [] Itaconic acid production is presently established in fungi, however the process requires further optimization looking to achieve higher final concentrations, identify suitable non-filamentous microorganisms and also to allow the use of low cost sustainable raw materials as a carbon source. Other concerns relate to A.terreus associated mycotoxin production, fungal morphology growth related complications in broth rheology and nutrient distribution as well as poor genome tractability of fungi [] Looking to overcome these limitations we turned to the model yeast S.cerevisiae with its well described tolerance to low pH’s, simple growth morphology and associated available genetic edition tools [] the suitability of this organism to produce itaconic acid has been the target of different studies performed in the laboratory. Preliminary results revealed the metabolic background of S.cerevisiae relevant for itaconic acid production as well as identifying genes involved in determining itaconic acid resistance []. Posterior work performed by Vila-Santa et al., [] reports the successful establishment of S.cerevisiae via heterologous expression of AtCad and in silico guided genetic edition to titers of up to 25 mg L. This work also resulted in a list of OptKnock suggested genes for deletion to improve itaconic acid titer which was later explored by Santos et al.,[] resulting in an increase to 68 mg L. Production of organic acids subjects the microorganisms to acidic stress at characteristically low pH’s, which may hinder metabolism, in this sense, process-robust strains are required, this coupled with the need of moving to more sustainable substrates has been bringing non-conventional yeasts to the spotlight as these organisms are known for having developed extreme tolerance strategies and ability to consume pentose sugars [] . The novel yeast Issatchenkia orientalis has been reported to grow at low pH’s [] as well as displaying resistances to several fermentation inhibiting stress factors [] A work from 2014 reports the usage of this same yeast in the production of succinic acid, while simultaneously establishing the hardy nature of this potential microbial cell factory []. The present work reports the successful inclusion of the plasmid-borne Aspergilus terreus CadA gene in a uracil auxotroph of the nonconventional yeast Issatchenkia orientalis SD108 and assays were conducted to find the optimal MM medium formulation for itaconic acid production. A transporter overexpression strategy was also applied looking to increase production and gain insights on metabolic pathways and compartmentalization. Furthermore, the physiology and traits of this non-conventional yeast have been explored looking to understand the mechanisms underlying I.orientalis’ high organic acid tolerance. André Costa 1, 2