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Abstract Objectives The ascomycotic yeast-like fungus Aureobasidium exhibits the natural ability to synthesize several secondary metabolites, like polymalic acid, pullulan, or polyol lipids, with potential biotechnological applications. Combined with its polyextremotolerance, these properties make Aureobasidium a promising production host candidate. Hence, plenty of genomes of Aureobasidia have been sequenced recently. Here, we provide the annotated draft genome sequence of the polyol lipid-producing strain A. pullulans NRRL 62042. Data description The genome of A. pullulans NRRL 62042 was sequenced using Illumina NovaSeq 6000. Genome assembly revealed a genome size of 24.2 Mb divided into 39 scaffolds with a GC content of 50.1%. Genome annotation using Genemark v4.68 and GenDBE yielded 9,596 genes.

Abstract Background To contribute to the discovery of new microbial strains with metabolic and physiological robustness and develop them into successful chasses, Paracoccus pantotrophus DSM 2944, a Gram-negative bacterium from the phylum Alphaproteobacteria and the family Rhodobacteraceae, was chosen. The strain possesses an innate ability to tolerate high salt concentrations. It utilizes diverse substrates, including cheap and renewable feedstocks, such as C1 and C2 compounds. Also, it can consume short-chain alkanes, predominately found in hydrocarbon-rich environments, making it a potential bioremediation agent. The demonstrated metabolic versatility, coupled with the synthesis of the biodegradable polymer polyhydroxyalkanoate, positions this microbial strain as a noteworthy candidate for advancing the principles of a circular bioeconomy. Results The study aims to follow the chassis roadmap, as depicted by Calero and Nikel, and de Lorenzo, to transform wild-type P. pantotrophus DSM 2944 into a proficient SynBio (Synthetic Biology) chassis. The initial findings highlight the antibiotic resistance profile of this prospective SynBio chassis. Subsequently, the best origin of replication (ori) was identified as RK2. In contrast, the non-replicative ori R6K was selected for the development of a suicide plasmid necessary for genome integration or gene deletion. Moreover, when assessing the most effective method for gene transfer, it was observed that conjugation had superior efficiency compared to electroporation, while transformation by heat shock was ineffective. Robust host fitness was demonstrated by stable plasmid maintenance, while standardized gene expression using an array of synthetic promoters could be shown. pEMG-based scarless gene deletion was successfully adapted, allowing gene deletion and integration. The successful integration of a gene cassette for terephthalic acid degradation is showcased. The resulting strain can grow on both monomers of polyethylene terephthalate (PET), with an increased growth rate achieved through adaptive laboratory evolution. Conclusion The chassis roadmap for the development of P. pantotrophus DSM 2944 into a proficient SynBio chassis was implemented. The presented genetic toolkit allows genome editing and therewith the possibility to exploit Paracoccus for a myriad of applications.

Abstract Background Itaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable of directly converting recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. A method for process monitoring was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online during co-cultivation. Results The efficiency of the process was compared to a simultaneous saccharification and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. Thereby, substrate availability was again identified as key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation. Conclusion This study demonstrates the feasibility of consortium-based CBP for itaconic acid production and also lays the fundamentals for the development and improvement of similar microbial consortia for cellulose-based organic acid production.

Abstract Background Ustilago cynodontis ranks among the relatively unknown itaconate production organisms. In comparison to the well-known and established organisms like Aspergillus terreus and Ustilago maydis, genetic engineering and first optimizations for itaconate production were only recently developed for U. cynodontis, enabling metabolic and morphological engineering of this acid-tolerant organism for efficient itaconate production. These engineered strains were so far mostly characterized in small scale shaken cultures. Results In pH-controlled fed-batch experiments an optimum pH of 3.6 could be determined for itaconate production in the morphology-engineered U. cynodontis Δfuz7. With U. cynodontis ∆fuz7 r ∆cyp3 r P etef mttA P ria1 ria1, optimized for itaconate production through the deletion of an itaconate oxidase and overexpression of rate-limiting production steps, titers up to 82.9 ± 0.8 g L−1 were reached in a high-density pulsed fed-batch fermentation at this pH. The use of a constant glucose feed controlled by in-line glucose analysis increased the yield in the production phase to 0.61 gITA gGLC−1, which is 84% of the maximum theoretical pathway yield. Productivity could be improved to a maximum of 1.44 g L−1 h−1 and cell recycling was achieved by repeated-batch application. Conclusions Here, we characterize engineered U. cynodontis strains in controlled bioreactors and optimize the fermentation process for itaconate production. The results obtained are discussed in a biotechnological context and show the great potential of U. cynodontis as an itaconate producing host.

Abstract Background Itaconic acid is an unsaturated, dicarboxylic acid which finds a wide range of applications in the polymer industry and as a building block for fuels, solvents and pharmaceuticals. Currently, Aspergillus terreus is used for industrial production, with titers above 100 g L−1 depending on the conditions. Besides A. terreus, Ustilago maydis is also a promising itaconic acid production host due to its yeast-like morphology. Recent strain engineering efforts significantly increased the yield, titer and rate of production. Results In this study, itaconate production by U. maydis was further increased by integrated strain- and process engineering. Next-generation itaconate hyper-producing strains were generated using CRISPR/Cas9 and FLP/FRT genome editing tools for gene deletion, promoter replacement, and overexpression of genes. The handling and morphology of this engineered strain were improved by deletion of fuz7, which is part of a regulatory cascade that governs morphology and pathogenicity. These strain modifications enabled the development of an efficient fermentation process with in situ product crystallization with CaCO3. This integrated approach resulted in a maximum itaconate titer of 220 g L−1, with a total acid titer of 248 g L−1, which is a significant improvement compared to best published itaconate titers reached with U. maydis and with A. terreus. Conclusion In this study, itaconic acid production could be enhanced significantly by morphological- and metabolic engineering in combination with process development, yielding the highest titer reported with any microorganism.

Abstract Background Thermotolerance is a highly desirable trait of microbial cell factories and has been the focus of extensive research. Yeast usually tolerate only a narrow temperature range and just two species, Kluyveromyces marxianus and Ogataea polymorpha have been described to grow at reasonable rates above 40 °C. However, the complex mechanisms of thermotolerance in yeast impede its full comprehension and the rare physiological data at elevated temperatures has so far not been matched with corresponding metabolic analyses. Results To elaborate on the metabolic network response to increased fermentation temperatures of up to 49 °C, comprehensive physiological datasets of several Kluyveromyces and Ogataea strains were generated and used for 13C-metabolic flux analyses. While the maximum growth temperature was very similar in all investigated strains, the metabolic network response to elevated temperatures was not conserved among the different species. In fact, metabolic flux distributions were remarkably irresponsive to increasing temperatures in O. polymorpha, while the K. marxianus strains exhibited extensive flux rerouting at elevated temperatures. Conclusions While a clear mechanism of thermotolerance is not deducible from the fluxome level alone, the generated data can be valued as a knowledge repository for using temperature to modulate the metabolic activity towards engineering goals.

Abstract Background Microbial production of chemicals from renewable carbon sources enables a sustainable route to many bioproducts. Sugar streams, such as those derived from biomass pretreated with ionic liquids (IL), provide efficiently derived and cost-competitive starting materials. A limitation to this approach is that residual ILs in the pretreated sugar source can be inhibitory to microbial growth and impair expression of the desired biosynthetic pathway. Results We utilized laboratory evolution to select Escherichia coli strains capable of robust growth in the presence of the IL, 1-ethyl-3-methyl-imidizolium acetate ([EMIM]OAc). Whole genome sequencing of the evolved strain identified a point mutation in an essential gene, cydC, which confers tolerance to two different classes of ILs at concentrations that are otherwise growth inhibitory. This mutation, cydC-D86G, fully restores the specific production of the bio-jet fuel candidate d-limonene, as well as the biogasoline and platform chemical isopentenol, in growth medium containing ILs. Similar amino acids at this position in cydC, such as cydC-D86V, also confer tolerance to [EMIM]OAc. We show that this [EMIM]OAc tolerance phenotype of cydC-D86G strains is independent of its wild-type function in activating the cytochrome bd-I respiratory complex. Using shotgun proteomics, we characterized the underlying differential cellular responses altered in this mutant. While wild-type E. coli cannot produce detectable amounts of either product in the presence of ILs at levels expected to be residual in sugars from pretreated biomass, the engineered cydC-D86G strains produce over 200 mg/L d-limonene and 350 mg/L isopentenol, which are among the highest reported titers in the presence of [EMIM]OAc. Conclusions The optimized strains in this study produce high titers of two candidate biofuels and bioproducts under IL stress. Both sets of production strains surpass production titers from other IL tolerant mutants in the literature. Our application of laboratory evolution identified a gain of function mutation in an essential gene, which is unusual in comparison to other published IL tolerant mutants.

Abstract Renewable raw materials in sustainable biorefinery processes pose new challenges to the manufacturing routes of platform chemicals. Beside the investigations of individual unit operations, the research on process chains, leading from plant biomass to the final products like lactic acid, succinic acid, and itaconic acid is increasing. This article presents a complete process chain from wooden biomass to the platform chemical itaconic acid. The process starts with the mechanical pretreatment of beech wood, which subsequently is subjected to chemo-catalytic biomass fractionation (OrganoCat) into three phases, which comprise cellulose pulp, aqueous hydrolyzed hemicellulose, and organic lignin solutions. Lignin is transferred to further chemical valorization. The aqueous phase containing oxalic acid as well as hemi-cellulosic sugars is treated by nanofiltration to recycle the acid catalyst back to the chemo-catalytic pretreatment and to concentrate the sugar hydrolysate. In a parallel step, the cellulose pulp is enzymatically hydrolyzed to yield glucose, which—together with the pentose-rich stream—can be used as a carbon source in the fermentation. The fermentation of the sugar fraction into itaconic acid can either be performed with the established fungi Aspergillus terreus or with Ustilago maydis. Both fermentation concepts were realized and evaluated. For purification, (in situ) filtration, (in situ) extraction, and crystallization were investigated. The presented comprehensive examination and discussion of the itaconate synthesis process—as a case study—demonstrates the impact of realistic process conditions on product yield, choice of whole cell catalyst, chemocatalysts and organic solvent system, operation mode, and, finally, the selection of a downstream concept.

Abstract Background Itaconate is getting growing biotechnological significance, due to its use as a platform compound for the production of bio-based polymers, chemicals, and novel fuels. Currently, Aspergillus terreus is used for its industrial production. The Ustilaginaceae family of smut fungi, especially Ustilago maydis, has gained biotechnological interest, due to its ability to naturally produce this dicarboxylic acid. The unicellular, non-filamentous growth form makes these fungi promising alternative candidates for itaconate production. Itaconate production was also observed in other Ustilaginaceae species such as U. cynodontis, U. xerochloae, and U. vetiveriae. The investigated species and strains varied in a range of 0–8 g L−1 itaconate. The genes responsible for itaconate biosynthesis are not known for these strains and therefore not characterized to explain this variability. Results Itaconate production of 13 strains from 7 species known as itaconate producers among the family Ustilaginaceae were further characterized. The sequences of the gene cluster for itaconate synthesis were analyzed by a complete genome sequencing and comparison to the annotated itaconate cluster of U. maydis. Additionally, the phylogenetic relationship and inter-species transferability of the itaconate cluster transcription factor Ria1 was investigated in detail. Doing so, itaconate production could be activated or enhanced by overexpression of Ria1 originating from a related species, showing their narrow phylogenetic relatedness. Conclusion Itaconate production by Ustilaginaceae species can be considerably increased by changing gene cluster regulation by overexpression of the Ria1 protein, thus contributing to the industrial application of these fungi for the biotechnological production of this valuable biomass derived chemical.

Abstract Background Expanding the application of technical enzymes, e.g., in industry and agriculture, commands the acceleration and cost-reduction of bioprocess development. Microplates and shake flasks are massively employed during screenings and early phases of bioprocess development, although major drawbacks such as low oxygen transfer rates are well documented. In recent years, miniaturization and parallelization of stirred and shaken bioreactor concepts have led to the development of novel microbioreactor concepts. They combine high cultivation throughput with reproducibility and scalability, and represent promising tools for bioprocess development. Results Parallelized microplate cultivation of the eukaryotic protein production host Pichia pastoris was applied effectively to support miniaturized phenotyping of clonal libraries in batch as well as fed-batch mode. By tailoring a chemically defined growth medium, we show that growth conditions are scalable from microliter to 0.8 L lab-scale bioreactor batch cultivation with different carbon sources. Thus, the set-up allows for a rapid physiological comparison and preselection of promising clones based on online data and simple offline analytics. This is exemplified by screening a clonal library of P. pastoris constitutively expressing AppA phytase from Escherichia coli. The protocol was further modified to establish carbon-limited conditions by employing enzymatic substrate-release to achieve screening conditions relevant for later protein production processes in fed-batch mode. Conclusion The comparison of clonal rankings under batch and fed-batch-like conditions emphasizes the necessity to perform screenings under process-relevant conditions. Increased biomass and product concentrations achieved after fed-batch microscale cultivation facilitates the selection of top producers. By reducing the demand to conduct laborious and cost-intensive lab-scale bioreactor cultivations during process development, this study will contribute to an accelerated development of protein production processes.

Abstract Background Ustilago maydis is known for its natural potential to produce a broad range of valuable chemicals, such as itaconate, from both industrial carbon waste streams and renewable biomass. Production of itaconate, and many other secondary metabolites, is induced by nitrogen limitation in U. maydis. The clustered genes responsible for itaconate production have recently been identified, enabling the development of new expression tools that are compatible with biotechnological processes. Results Here we report on the investigation of two of the native promoters, P tad1 and P mtt1 , from the itaconate cluster of U. maydis MB215. For both promoters the specific activation upon nitrogen limitation, which is known to be the trigger for itaconate production in Ustilago, could be demonstrated by gfp expression. The promoters cover a broad range of expression levels, especially when combined with the possibility to create single- and multicopy construct integration events. In addition, these reporter constructs enable a functional characterization of gene induction patterns associated with itaconate production. Conclusions The promoters are well suited to induce gene expression in response to nitrogen limitation, coupled to the itaconate production phase, which contributes towards the further improvement of organic acid production with Ustilago.

Abstract Background The family of Ustilaginaceae is known for their capability to naturally produce industrially valuable chemicals from different carbon sources. Recently, several Ustilaginaceae were reported to produce organic acids from glycerol, which is the main side stream in biodiesel production. Results In this study, we present Ustilago vetiveriae as new production organism for itaconate synthesis from glycerol. In a screening of 126 Ustilaginaceae, this organism reached one of the highest titers for itaconate combined with a high-glycerol uptake rate. By adaptive laboratory evolution, the production characteristics of this strain could be improved. Further medium optimization with the best single colony, U. vetiveriae TZ1, in 24-deep well plates resulted in a maximal itaconate titer of 34.7 ± 2.5 g L−1 produced at a rate of 0.09 ± 0.01 g L−1 h−1 from 196 g L−1 glycerol. Simultaneously, this strain produced 46.2 ± 1.4 g L−1 malate at a rate of 0.12 ± 0.00 g L−1 h−1. Due to product inhibition, the itaconate titer in NaOH-titrated bioreactor cultivations was lower (24 g L−1). Notably, an acidic pH value of 5.5 resulted in decreased itaconate production, however, completely abolishing malate production. Overexpression of ria1 or mtt1, encoding a transcriptional regulator and mitochondrial transporter, respectively, from the itaconate cluster of U. maydis resulted in a 2.0-fold (ria1) and 1.5-fold (mtt1) higher itaconate titer in comparison to the wild-type strain, simultaneously reducing malate production by 75 and 41%, respectively. Conclusions The observed production properties of U. vetiveriae TZ1 make this strain a promising candidate for microbial itaconate production. The outcome of the overexpression experiments, which resulted in reduced malate production in favor of an increased itaconate titer, clearly strengthens its potential for industrial itaconate production from glycerol as major side stream of biodiesel production.

Abstract Background Ogataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. While previously mainly used for protein synthesis, it also holds promise for producing platform chemicals. O. polymorpha has the distinct advantage of using methanol as a substrate, which could be potentially derived from carbon capture and utilization streams. Full development of the organism into a production strain and estimation of the metabolic capabilities require additional strain design, guided by metabolic modeling with a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha. Results To overcome this limitation, we used a published reconstruction of the closely related yeast Komagataella phaffii as a reference and corrected reactions based on KEGG and MGOB annotation. Additionally, we conducted phenotype microarray experiments to test the suitability of 190 substrates as carbon sources. Over three-quarter of the substrate use was correctly reproduced by the model and 27 new substrates were added, that were not present in the K. phaffii reference model. Conclusion The developed genome-scale metabolic model of O. polymorpha will support the engineering of synthetic metabolic capabilities and enable the optimization of production processes, thereby supporting a sustainable future methanol economy.