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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 We here explore the potential of the fungal genus Aureobasidium as a prototype for a microbial chassis for industrial biotechnology in the context of a developing circular bioeconomy. The study emphasizes the physiological advantages of Aureobasidium, including its polyextremotolerance, broad substrate spectrum, and diverse product range, making it a promising candidate for cost‐effective and sustainable industrial processes. In the second part, recent advances in genetic tool development, as well as approaches for up‐scaled fermentation, are described. This review adds to the growing body of scientific literature on this remarkable fungus and reveals its potential for future use in the biotechnological industry.

Abstract Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever‐improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one‐plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I‐SceI expressing plasmid pSW‐2 as a counterselection marker to accelerated plasmid curing; (2) double‐strand break introducing gene I‐sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG‐RIS. The single plasmid of pEMG‐RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I‐SceI encoding gene. Here, the usability of the pEMG‐RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform.

Abstract To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.

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 Foam formation is a massive challenge in submerged aerated bioprocesses, e.g., in beer fermentation. While the use of antifoam may easily overcome foaming at laboratory scale, it is often an unattractive solution since the challenge remains in future upscaling, as reduced mass transfer and extra steps in product purification and analytics result in increased costs. Interestingly, the number of studies tackling this challenge is relatively low, although literature suggests a range of alternatives, from avoiding foaming to means of controlling or even using foaming as an in situ product removal. Here we give an overview of the topic in five subsections. (1) We argue that a sound understanding of the molecular origin of foaming can facilitate solutions for overcoming the challenge while introducing some long-known challenges (i.e., in beer fermentation). We then review in (2) the apparent avoidance of foam formation before we in (3) summarize possibilities to reduce and control foam after its formation. Subsequently, in (4), we discuss possible solutions that take advantage of foam formation, for example, via foam fractionation for in situ product removal. Finally, in (5), we provide an overview of microbial strain engineering approaches to cope with some aspects of foaming in fermentations. With this review, we would like to sensitize and inform the interested reader while offering an overview of the current literature for the expert, particularly with regard to the foam special issue in Discover Chemical Engineering.

Abstract To reduce carbon dioxide emissions, carbon-neutral fuels have recently gained renewed attention. Here we show the development and evaluation of process routes for the production of such a fuel, the cyclic acetal 4,5-dimethyl-1,3-dioxolane, from glucose via 2,3-butanediol. The selected process routes are based on the sequential use of microbes, enzymes and chemo-catalysts in order to exploit the full potential of the different catalyst systems through a tailor-made combination. The catalysts (microbes, enzymes, chemo-catalysts) and the reaction medium selected for each conversion step are key factors in the development of the respective production methods. The production of the intermediate 2,3-butanediol by combined microbial and enzyme catalysis is compared to the conventional microbial route from glucose in terms of specific energy demand and overall yield, with the conventional route remaining more efficient. In order to be competitive with current 2,3-butanediol production, the key performance indicator, enzyme stability to high aldehyde concentrations, needs to be increased. The target value for the enzyme stability is an acetaldehyde concentration of 600 mM, which is higher than the current maximum concentration (200 mM) by a factor of three.

Abstract Plastic usage by microbes as a carbon source is a promising strategy to increase the recycling quota. 1,4‐butanediol (BDO) is a common monomer derived from polyesters and polyurethanes. In this study, Ustilago trichophora was found to be an efficient cell‐factory to valorize BDO. To investigate product formation by U. trichophora, we refined the traditional ion exclusion liquid chromatography method by examining eluent, eluent concentrations, oven temperatures, and organic modifiers to make the chromatography compatible with mass spectrometry. An LC‐UV/RI‐MS2 method is presented here to identify and quantify extracellular metabolites in the cell cultures. With this method, we successfully identified that U. trichophora secreted malic acid, succinic acid, erythritol, and mannitol into the culture medium. Adaptive laboratory evolution followed by medium optimization significantly improved U. trichophora growth on BDO and especially malic acid production. Overall, the carbon yield on the BDO substrate was approximately 33% malic acid. This study marks the first report of a Ustilaginaceae fungus capable of converting BDO into versatile chemical building blocks. Since U. trichophora is not genetically engineered, it is a promising microbial host to produce malic acid from BDO, thereby contributing to the development of the envisaged sustainable bioeconomy.

Polyol lipids (a.k.a. liamocins) produced by the polyextremotolerant, yeast-like fungus Aureobasidium pullulans are amphiphilic molecules with high potential to serve as biosurfactants. So far, cultivations of A. pullulans have been performed in media with complex components, which complicates further process optimization due to their undefined composition. In this study, we developed and optimized a minimal medium, focusing on biosurfactant production. Firstly, we replaced yeast extract and peptone in the best-performing polyol lipid production medium to date with a vitamin solution, a trace-element solution, and a nitrogen source. We employed a design of experiments approach with a factor screening using a two-level-factorial design, followed by a central composite design. The polyol lipid titer was increased by 56% to 48 g L−1, and the space-time yield from 0.13 to 0.20 g L−1 h−1 in microtiter plate cultivations. This was followed by a successful transfer to a 1 L bioreactor, reaching a polyol lipid concentration of 41 g L−1. The final minimal medium allows the investigation of alternative carbon sources and the metabolic pathways involved, to pinpoint targets for genetic modifications. The results are discussed in the context of the industrial applicability of this robust and versatile fungus.

Abstract As one of the main precursors, acetyl‐CoA leads to the predominant production of even‐chain products. From an industrial biotechnology perspective, extending the acyl‐CoA portfolio of a cell factory is vital to producing industrial relevant odd‐chain alcohols, acids, ketones and polyketides. The bioproduction of odd‐chain molecules can be facilitated by incorporating propionyl‐CoA into the metabolic network. The shortest pathway for propionyl‐CoA production, which relies on succinyl‐CoA catabolism encoded by the sleeping beauty mutase operon, was evaluated in Pseudomonas taiwanensis VLB120. A single genomic copy of the sleeping beauty mutase genes scpA, argK and scpB combined with the deletion of the methylcitrate synthase PVLB_08385 was sufficient to observe propionyl‐CoA accumulation in this Pseudomonas. The chassis' capability for odd‐chain product synthesis was assessed by expressing an acyl‐CoA hydrolase, which enabled propionate synthesis. Three fed‐batch strategies during bioreactor fermentations were benchmarked for propionate production, in which a maximal propionate titre of 2.8 g L−1 was achieved. Considering that the fermentations were carried out in mineral salt medium under aerobic conditions and that a single genome copy drove propionyl‐CoA production, this result highlights the potential of Pseudomonas to produce propionyl‐CoA derived, odd‐chain products.

Biosurfactants are much-discussed alternatives to petro- and oleochemical surfactants. Alcanivorax borkumensis, a marine, Gram-negative γ-proteobacterium, produces a glycine-glucolipid biosurfactant from hydrocarbons, pyruvate, and acetate as carbon sources. Sustainable acetate production from lignocellulose or syngas adds to its relevance for the bioeconomy. This study investigated nitrogen sources and carbon-to-nitrogen ratios (C/N) to optimize fed-batch fermentation for biosurfactant production using A. borkumensis with acetate as the carbon source. Urea enabled high biosurfactant production, which was confirmed in DO-based fed-batch fermentation. Varying C/N ratios led to increased glycine-glucolipid production and decreased biomass production, with improvement plateauing at a C/N ratio of 26.7 Cmol Nmol−1. pH-stat fed-batch fermentation using glacial acetic acid as the carbon source and a pH-adjusting agent doubled the biosurfactant production. Finally, bubble-free membrane aeration was used to prevent extensive foam formation observed during conventional bubble aeration. The efficient production made it possible to investigate the bioactivity of glycine-glucolipid in combination with antibiotics against various microorganisms. Our findings allow for the leverage of glycine-glucolipid biosurfactant production using acetate as a carbon source.

Glycine-glucolipid, a glycolipid, is natively synthesized by the marine bacterium Alcanivorax borkumensis SK2. A. borkumensis is a Gram-negative, non-motile, aerobic, halophilic, rod-shaped γ-proteobacterium, classified as an obligate hydrocarbonoclastic bacterium. Naturally, this bacterium exists in low cell numbers in unpolluted marine environments, but during oil spills, the cell number significantly increases and can account for up to 90% of the microbial community responsible for oil degradation. This growth surge is attributed to two remarkable abilities: hydrocarbon degradation and membrane-associated biosurfactant production. This study aimed to characterize and enhance the growth and biosurfactant production of A. borkumensis, which initially exhibited poor growth in the previously published ONR7a, a defined salt medium. Various online analytic tools for monitoring growth were employed to optimize the published medium, leading to improved growth rates and elongated growth on pyruvate as a carbon source. The modified medium was supplemented with different carbon sources to stimulate glycine-glucolipid production. Pyruvate, acetate, and various hydrophobic carbon sources were utilized for glycolipid production. Growth was monitored via online determined oxygen transfer rate in shake flasks, while a recently published hyphenated HPLC-MS method was used for glycine-glucolipid analytics. To transfer into 3 L stirred-tank bioreactor, aerated batch fermentations were conducted using n-tetradecane and acetate as carbon sources. The challenge of foam formation was overcome using bubble-free membrane aeration with acetate as the carbon source. In conclusion, the growth kinetics of A. borkumensis and glycine-glucolipid production were significantly improved, while reaching product titers relevant for applications remains a challenge.

Rationale: Liver cirrhosis is known to affect drug pharmacokinetics, but the functional assessment of the underlying pathophysiological alterations in drug metabolism is difficult.Methods: Cirrhosis in mice was induced by repeated treatment with carbon tetrachloride for 12 months. A cocktail of six drugs was administered, and parent compounds as well as phase I and II metabolites were quantified in blood, bile, and urine in a time-dependent manner. Pharmacokinetics were modeled in relation to the altered expression of metabolizing enzymes. In discrepancy with computational predictions, a strong increase of glucuronides in blood was observed in cirrhotic mice compared to vehicle controls.Results: The deviation between experimental findings and computational simulations observed by analyzing different hypotheses could be explained by increased sinusoidal export and corresponded to increased expression of export carriers (Abcc3 and Abcc4). Formation of phase I metabolites and clearance of the parent compounds were surprisingly robust in cirrhosis, although the phase I enzymes critical for the metabolism of the administered drugs in healthy mice, Cyp1a2 and Cyp2c29, were downregulated in cirrhotic livers. RNA-sequencing revealed the upregulation of numerous other phase I metabolizing enzymes which may compensate for the lost CYP isoenzymes. Comparison of genome-wide data of cirrhotic mouse and human liver tissue revealed similar features of expression changes, including increased sinusoidal export and reduced uptake carriers.Conclusion: Liver cirrhosis leads to increased blood concentrations of glucuronides because of increased export from hepatocytes into the sinusoidal blood. Although individual metabolic pathways are massively altered in cirrhosis, the overall clearance of the parent compounds was relatively robust due to compensatory mechanisms.

Agricultural products, have a high phytate content that has a hidden potential as a renewable source of phosphate. We present the first biotechnological route for the production of food-grade, organic polyphosphate (polyP) from deoiled seeds or bran, included in a vision for a circular phosphorus economy. The three-step production process includes phosphorus mobilization (e.g., 37 mg PO43−/g bran) using phytase enzymes. Non-genetically modified phosphate starved Saccharomyces cerevisiae is then fed with soluble P-extracts. The yeast intracellularly polymerizes the phosphate to polyP (≥ 30% mol polyP in yeast per mol PO43− in medium) and polyP-rich yeast extract or pure polyP is purified from the biomass. We demonstrate that the obtained polyP-rich yeast extract is an excellent green surrogate for polyP from fossil P-sources in meat manufacturing. The valorization of phytate–P while producing P-depleted biomass as a demanded feed in livestock production is shown. Our sustainable process enables the production of food-grade polyP from renewable resources and is contributing to the sustainable management of the dwindling nutrient phosphorus.

Summary: Bile acid (BA) metabolism is a complex system that includes a wide variety of primary and secondary, as well as conjugated and unconjugated BAs that undergo continuous enterohepatic circulation (EHC). Alterations in both composition and dynamics of BAs have been associated with various diseases. However, a mechanistic understanding of the relationship between altered BA metabolism and related diseases is lacking. Computational modeling may support functional analyses of the physiological processes involved in the EHC of BAs along the gut-liver axis. In this study, we developed a physiologically based model of murine BA metabolism describing synthesis, hepatic and microbial transformations, systemic distribution, excretion, and EHC of BAs at the whole-body level. For model development, BA metabolism of specific pathogen-free (SPF) mice was characterized in vivo by measuring BA levels and composition in various organs, expression of transporters along the gut, and cecal microbiota composition. We found significantly different BA levels between male and female mice that could only be explained by adjusted expression of the hepatic enzymes and transporters in the model. Of note, this finding was in agreement with experimental observations. The model for SPF mice could also describe equivalent experimental data in germ-free mice by specifically switching off microbial activity in the intestine. The here presented model can therefore facilitate and guide functional analyses of BA metabolism in mice, e.g., the effect of pathophysiological alterations on BA metabolism and translation of results from mouse studies to a clinically relevant context through cross-species extrapolation.

Modern biotechnology benefits from the introduction of novel chassis organisms in remedying the limitations of already-established strains. For this, Paracoccus pantotrophus was chosen for in-depth assessment. Its unique broad metabolism and robustness against abiotic stressors make this strain a well-suited chassis candidate. This study set out to comprehensively overview abiotic influences on the growth performance of five P. pantotrophus strains. These data can aid in assessing the suitability of this genus for chassis development by using the type strain as a preliminary model organism. The five P. pantotrophus strains DSM 2944T, DSM 11072, DSM 11073, DSM 11104, and DSM 65 were investigated regarding their growth on various carbon sources and other nutrients. Our data show a high tolerance against osmotic pressure for the type strain with both salts and organic osmolytes. It was further observed that P. pantotrophus prefers organic acids over sugars. All of the tested strains were able to grow on short-chain alkanes, which would make P. pantotrophus a candidate for bioremediation and the upcycling of plastics. In conclusion, we were able to gain insights into several P. pantotrophus strains, which will aid in further introducing this species, or even another species from this genus, as a candidate for future biotechnological processes.

Pseudomonas putida has been engineered to produce medium chain length oleochemicals, including medium chain length free fatty acids and their ester forms, compounds which may be useful as biodiesel blending agents.

Abstract Microbial activity is the driving force of the carbon cycle, including the digestion of biomass in the soil, oceans, and oil deposits. This natural diversity of microbial carbon sources poses challenges for humans. Contamination monitoring can be difficult in oil tanks and similar settings. To assess microbial activity in such industrial settings, off‐gas analysis can be employed by considering growth and non‐growth‐associated metabolic activity. In this work, we describe the monitoring of CO2 as a method for measuring microbial activity. We revealed that the CO2 signal corresponds to classical growth curves, exemplified by Pseudomonas fluorescens, Yarrowia lipolytica, and Penicillium chrysogenum. Deviations of the CO2 signal from the growth curves occurred when the yield of biomass on the substrate changed (i.e., the non‐growth‐associated metabolic activities). We monitored CO2 to track the onset of microbial contamination in an oil tank. This experimental setup was applied to determine the susceptibility of heating oil and biodiesel to microbial contamination long before the formation of problematic biofilms. In summary, the measurement of CO2 production by bacteria, yeasts, and molds allowed the permanent monitoring of microbial activity under oil storage conditions without invasive sampling.

Abstract Volatile organic compounds play an essential role in every domain of life, with diverse functions. In this study, we use novel secondary electrospray ionisation high-resolution Orbitrap mass spectrometry (SESI-Orbitrap MS) to monitor the complete yeast volatilome every 2.3 s. Over 200 metabolites were identified during growth in shake flasks and bioreactor cultivations, all with their unique intensity profile. Special attention was paid to ethanol as biotech largest product and to acetaldehyde as an example of a low-abundance but highly-volatile metabolite. While HPLC and Orbitrap measurements show a high agreement for ethanol, acetaldehyde could be measured five hours earlier in the SESI-Orbitrap MS. Volatilome shifts are visible, e.g. after glucose depletion, fatty acids are converted to ethyl esters in a detoxification mechanism after stopped fatty acid biosynthesis. This work showcases the SESI-Orbitrap MS system for tracking microbial physiology without the need for sampling and for time-resolved discoveries during metabolic transitions.

Abstract Since 2008, European and German legislative initiatives for climate protection and reduced dependency on fossil resources led to the introduction of biofuels as CO2‐reduced alternatives in the heating oil sector. In the case of biodiesel, customers were confronted with accelerated microbial contaminations during storage. Since then, other fuel alternatives, like hydrogenated vegetable oils (HVOs), gas‐to‐liquid (GtL) products, or oxymethylene ether (OME) have been developed. In this study, we use online monitoring of microbial CO2 production and the simulation of onset of microbial contamination to investigate the contamination potential of fuel alternatives during storage. As references, fossil heating oil of German refineries are used. Biodiesel blends with fossil heating oils confirmed the promotion of microbial activity. In stark contrast, OMEs have an antimicrobial effect. The paraffinic Fischer–Tropsch products and biogenic hydrogenation products demonstrate to be at least as resistant to microbial contamination as fossil heating oils despite allowing a diversity of representative microbes. Through mass spectrometry, elemental analysis, and microbial sequencing, we can discuss fuel properties that affect microbial contaminations. In summary, novel, non‐fossil heating oils show clear differences in microbial resistance during long‐term storage. Designing blends with an intrinsic resistance against microbial contamination and hence reduced activity might be an option.

Plastic mulch films and biofertilizers (processed sewage sludge, compost or manure) have helped to increase crop yields. However, there is increasing evidence that these practices significantly contribute to microplastic contamination in agricultural soils, affecting biodiversity and soil health. Here, we draw attention to the use of hydrolase enzymes that depolymerize polyester-based plastics as a bioremediation technique for agricultural soils (in situ), biofertilizers and irrigation water (ex situ), and discuss the need for fully biodegradable plastic mulches. We also highlight the need for ecotoxicological assessment of the proposed approach and its effects on different soil organisms. Enzymes should be optimized to work effectively and efficiently under the conditions found in natural soils (typically, moist solids at an ambient temperature with low salinity). Such optimization is also necessary to ensure that already distressed ecosystems are not disrupted any further.

ABSTRACT Biotechnology has experienced innovations in analytics and data processing. As the volume of data and its complexity grow, new computational procedures for extracting information are being developed. However, the rate of change outpaces the adaptation of biotechnology curricula, necessitating new teaching methodologies to equip biotechnologists with data analysis abilities. To simulate experimental data, we created a virtual organism simulator (silvio) by combining diverse cellular and subcellular microbial models. With the silvio Python package, we constructed a computer-based instructional workflow to teach growth curve data analysis, promoter sequence design, and expression rate measurement. The instructional workflow is a Jupyter Notebook with background explanations and Python-based experiment simulations combined. The data analysis is conducted either within the Notebook in Python or externally with Excel. This instructional workflow was separately implemented in two distance courses for Master's students in biology and biotechnology with assessment of the pedagogic efficiency. The concept of using virtual organism simulations that generate coherent results across different experiments can be used to construct consistent and motivating case studies for biotechnological data literacy.

Poly-γ-glutamic acid (γ-PGA) is an emerging biopolymer produced by several Bacillus species. To improve γ-PGA synthesis, metabolic engineering of the production host B. subtilis poses great potential and is facilitated by the convenient genetical amenability of the organism. In this study, a 3.7-fold increase in γ-PGA production using a bdhA, alsSD, pta, yvmC, and cypX deletion mutant with blocked by-product synthesis pathways was obtained. A detailed analysis of intracellular metabolites for reference strains and the γ-PGA-producing deletion strain identified the accumulation of pyruvate and acetyl-CoA in deletion mutants, highlighting the citrate synthase activity as an important metabolic engineering target for further metabolic flux optimization towards γ-PGA synthesis. An in-depth analysis of growth and γ-PGA production with on-line measurement techniques revealed significant variations across cultivations with deletion mutants that are likely caused by culture acidification due to pyruvate accumulation. Despite the observed acidification, the by-product deletion mutants outperformed the reference strains independent of the promoter controlling the PGA synthetase expression. The constructed deletion strains exhibit high γ-PGA production in minimal medium with glucose as sole carbon source as well as in modified Medium E reaching γ-PGA concentrations of 0.57 gL-1 and 14.46 gL-1, respectively. The results presented in this work broaden the understanding of the microbial metabolism during γ-PGA production and will be useful to guide future metabolic engineering for improved γ-PGA production.

Introduction: Bioproduction of plant-derived triterpenoids in recombinant microbes is receiving great attention to make these biologically active compounds industrially accessible as nutraceuticals, pharmaceutics, and cosmetic ingredients. So far, there is no direct method for detecting triterpenoids under physiological conditions on a cellular level, information yet highly relevant to rationalizing microbial engineering.Methods: Here, we show in a proof-of-concept study, that triterpenoids can be detected and monitored in living yeast cells by combining coherent anti-Stokes Raman scattering (CARS) and second-harmonic-generation (SHG) microscopy techniques. We applied CARS and SHG microscopy measurements, and for comparison classical Nile Red staining, on immobilized and growing triterpenoid-producing, and non-producing reference Saccharomyces cerevisiae strains.Results and Discussion: We found that the SHG signal in triterpenoid-producing strains is significantly higher than in a non-producing reference strain, correlating with lipophile content as determined by Nile red staining. In growing cultures, both CARS and SHG signals showed changes over time, enabling new insights into the dynamics of triterpenoid production and storage inside cells.

Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of Pseudomonas taiwanensis VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was actP, coding for a cation/acetate symporter, and the other gene was paaA coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmolrhl/Cmol4-coumarate) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmolrhl/Cmolferulate), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.

The yeast Ogataea polymorpha is an upcoming host for bio-manufacturing due to its unique physiological properties, including its broad substrate spectrum, and particularly its ability to utilize methanol as the sole carbon and energy source. However, metabolic engineering tools for O. polymorpha are still rare. In this study we characterized the influence of 6 promoters and 15 terminators on gene expression throughout batch cultivations with glucose, glycerol, and methanol as carbon sources as well as mixes of these carbon sources. For this characterization, a short half-life Green Fluorescent Protein (GFP) variant was chosen, which allows a precise temporal resolution of gene expression. Our promoter studies revealed how different promoters do not only influence the expression strength but also the timepoint of maximal expression. For example, the expression strength of the catalase promoter (pCAT) and the methanol oxidase promoter (pMOX) are comparable on methanol, but the maximum expression level of the pCAT is reached more than 24 h earlier. By varying the terminators, a 6-fold difference in gene expression was achieved with the MOX terminator boosting gene expression on all carbon sources by around 50% compared to the second-strongest terminator. It was shown that this exceptional increase in gene expression is achieved by the MOX terminator stabilizing the mRNA, which results in an increased transcript level in the cells. We further found that different pairing of promoters and terminators or the expression of a different gene (β-galactosidase gene) did not influence the performance of the genetic parts. Consequently, it is possible to mix and match promoters and terminators as independent elements to tune gene expression in O. polymorpha.

Commercial carbon fiber (CF) fabrics are popular electrode materials for bioelectrochemical systems (BES), but are usually not optimized for the specific application. This study investigates BES-relevant material characteristics on fabric level, such as weave types and weave parameters. The two contrasting weave types plain and leno weave were characterized with respect to their envisaged application types: 1) BES with mainly advective flow regimes and 2) stirred systems, which could benefit from fluid flow through a fabric electrode. Experiments with batch and continuously fed pure cultures of Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1 reveal that µm-scale electrode topologies are of limited use for the thick biofilms of G. sulfurreducens, but can boost S. oneidensis’ current generation especially in batch and fed-batch reactors. For advective flow regimes, deeper layers of biofilm inside microporous electrodes are often mass transport limited, even with thin biofilms of S. oneidensis. Therefore, low porosity plain weave electrodes for advective flow operation as in wastewater treating BES should be thin and flat. A trade-off between maximized current density and electrode material utilization exists, which is optimized exemplarily for an advective flow operation. For stirred BES of biotechnological applications, a flow-through of electrolyte is desired. For this, leno weave fabrics with pores at cm-scale are produced from 100% CF for the first time. In a preliminary evaluation, they outperform plain weave fabrics. Mass transfer investigations in stirred BES demonstrate that the large pores enable efficient electrode utilization at lower power input in terms of stirring speed.

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.

With their ability of CO2 fixation using sunlight as an energy source, algae and especially microalgae are moving into the focus for the production of proteins and other valuable compounds. However, the valorization of algal biomass depends on the effective disruption of the recalcitrant microalgal cell wall. Especially cell walls of Chlorella species proved to be very robust. The wall structures that are responsible for this robustness have been studied less so far. Here, we evaluate different common methods to break up the algal cell wall effectively and measure the success by protein and carbohydrate release. Subsequently, we investigate algal cell wall features playing a role in the wall’s recalcitrance towards disruption. Using different mechanical and chemical technologies, alkali catalyzed hydrolysis of the Chlorella vulgaris cells proved to be especially effective in solubilizing up to 56 wt% protein and 14 wt% carbohydrates of the total biomass. The stepwise degradation of C. vulgaris cell walls using a series of chemicals with increasingly strong conditions revealed that each fraction released different ratios of proteins and carbohydrates. A detailed analysis of the monosaccharide composition of the cell wall extracted in each step identified possible factors for the robustness of the cell wall. In particular, the presence of chitin or chitin-like polymers was indicated by glucosamine found in strong alkali extracts. The presence of highly ordered starch or cellulose was indicated by glucose detected in strong acidic extracts. Our results might help to tailor more specific efforts to disrupt Chlorella cell walls and help to valorize microalgae biomass.

Summary Ustilago maydis is a promising yeast for the production of a range of valuable metabolites, including itaconate, malate, glycolipids and triacylglycerols. However, wild‐type strains generally produce a potpourri of all of these metabolites, which hinders efficient production of single target chemicals. In this study, the diverse by‐product spectrum of U. maydis was reduced through strain engineering using CRISPR/Cas9 and FLP/FRT, greatly increasing the metabolic flux into the targeted itaconate biosynthesis pathway. With this strategy, a marker‐free chassis strain could be engineered, which produces itaconate from glucose with significantly enhanced titre, rate and yield. The lack of by‐product formation not only benefited itaconate production, it also increases the efficiency of downstream processing improving cell handling and product purity.

Microbial co-cultures have been used in several biotechnological applications. Within these co-cultures, the microorganisms tend to interact with each other and perform complex actions. Investigating metabolic interactions in microbial co-cultures is crucial in designing microbial consortia. Here, we present a pipeline integrating modelling and experimental approaches to understand metabolic interactions between organisms in a community. We define a new index named “Metabolic Support Index (MSI)”, which quantifies the benefits derived by each organism in the presence of the other when grown as a co-culture. We computed MSI for several experimentally demonstrated co-cultures and showed that MSI, as a metric, accurately identifies the organism that derives the maximum benefit. We also computed MSI for a commonly used yeast co-culture consisting of Saccharomyces cerevisiae and Pichia stipitis and observed that the latter derives higher benefit from the interaction. Further, we designed two-stage experiments to study mutual interactions and showed that P. stipitis indeed derives the maximum benefit from the interaction, as shown from our computational predictions. Also, using our previously developed computational tool MetQuest, we identified all the metabolic exchanges happening between these organisms by analysing the pathways spanning the two organisms. By analysing the HPLC profiles and studying the isotope labelling, we show that P. stipitis consumes the ethanol produced by S. cerevisiae when grown on glucose-rich medium under aerobic conditions, as also indicated by our in silico pathway analyses. Our approach represents an important step in understanding metabolic interactions in microbial communities through an integrated computational and experimental workflow.

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.

Metabolic engineering relies on modifying gene expression to regulate protein concentrations and reaction activities. The gene expression is controlled by the promoter sequence, and sequence libraries are used to scan expression activities and to identify correlations between sequence and activity. We introduce a computational workflow called Exp2Ipynb to analyze promoter libraries maximizing information retrieval and promoter design with desired activity. We applied Exp2Ipynb to seven prokaryotic expression libraries to identify optimal experimental design principles. The workflow is open source, available as Jupyter Notebooks and covers the steps to 1) generate a statistical overview to sequence and activity, 2) train machine-learning algorithms, such as random forest, gradient boosting trees and support vector machines, for prediction and extraction of feature importance, 3) evaluate the performance of the estimator, and 4) to design new sequences with a desired activity using numerical optimization. The workflow can perform regression or classification on multiple promoter libraries, across species or reporter proteins. The most accurate predictions in the sample libraries were achieved when the promoters in the library were recognized by a single sigma factor and a unique reporter system. The prediction confidence mostly depends on sample size and sequence diversity, and we present a relationship to estimate their respective effects. The workflow can be adapted to process sequence libraries from other expression-related problems and increase insight to the growing application of high-throughput experiments, providing support for efficient strain engineering.

In recent years, it was shown that itaconic acid can be produced from glucose with Ustilago strains at up to maximum theoretical yield. The use of acetate and formate as co-feedstocks can boost the efficiency of itaconate production with Ustilaginaceae wild-type strains by reducing the glucose amount and thus the agricultural land required for the biotechnological production of this chemical. Metabolically engineered strains (U. cynodontis Δfuz7 Δcyp3 ↑Pria1 and U. cynodontis Δfuz7 Δcyp3 PetefmttA ↑Pria1) were applied in itaconate production, obtaining a titer of 56.1 g L−1 and a yield of 0.55 gitaconate per gsubstrate. Both improved titer and yield (increase of 5.2 g L−1 and 0.04 gitaconate per gsubstrate, respectively) were achieved when using sodium formate as an auxiliary substrate. By applying the design-of-experiments (DoE) methodology, cultivation parameters (glucose, sodium formate and ammonium chloride concentrations) were optimized, resulting in two empirical models predicting itaconate titer and yield for U. cynodontis Δfuz7 Δcyp3 PetefmttA ↑Pria1. Thereby, an almost doubled itaconate titer of 138 g L−1 was obtained and a yield of 0.62 gitaconate per gsubstrate was reached during confirmation experiments corresponding to 86% of the theoretical maximum. In order to close the carbon cycle by production of the co-feed via a “power-to-X” route, the biphasic Ru-catalysed hydrogenation of CO2 to formate could be integrated into the bioprocess directly using the obtained aqueous solution of formates as co-feedstock without any purification steps, demonstrating the (bio)compatibility of the two processes.

ABSTRACT It is generally recognized that proteins constitute the key cellular component in shaping microbial phenotypes. Due to limited cellular resources and space, optimal allocation of proteins is crucial for microbes to facilitate maximum proliferation rates while allowing a flexible response to environmental changes. To account for the growth condition-dependent proteome in the constraint-based metabolic modeling of Escherichia coli, we consolidated a coarse-grained protein allocation approach with the explicit consideration of enzymatic constraints on reaction fluxes. Besides representing physiologically relevant wild-type phenotypes and flux distributions, the resulting protein allocation model (PAM) advances the predictability of the metabolic responses to genetic perturbations. A main driver of mutant phenotypes was ascribed to inherited regulation patterns in protein distribution among metabolic enzymes. Moreover, the PAM correctly reflected metabolic responses to an augmented protein burden imposed by the heterologous expression of green fluorescent protein. In summary, we were able to model the effects of important and frequently applied metabolic engineering approaches on microbial metabolism. Therefore, we want to promote the integration of protein allocation constraints into classical constraint-based models to foster their predictive capabilities and application for strain analysis and engineering purposes. IMPORTANCE Predictive metabolic models are important, e.g., for generating biological knowledge and designing microbes with superior performance for target compound production. Yet today’s whole-cell models either show insufficient predictive capabilities or are computationally too expensive to be applied to metabolic engineering purposes. By linking the inherent genotype-phenotype relationship to a complete representation of the proteome, the PAM advances the accuracy of simulated phenotypes and intracellular flux distributions of E. coli. Being equally computationally lightweight as classical stoichiometric models and allowing for the application of established in silico tools, the PAM and related simulation approaches will foster the use of a model-driven metabolic research. Applications range from the investigation of mechanisms of microbial evolution to the determination of optimal strain design strategies in metabolic engineering, thus supporting basic scientists and engineers alike.

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.

Ustilago maydis is an important plant pathogen that causes corn smut disease and serves as an effective biotechnological production host. The lack of a comprehensive metabolic overview hinders a full understanding of the organism’s environmental adaptation and a full use of its metabolic potential. Here, we report the first genome-scale metabolic model (GSMM) of Ustilago maydis (iUma22) for the simulation of metabolic activities. iUma22 was reconstructed from sequencing and annotation using PathwayTools, and the biomass equation was derived from literature values and from the codon composition. The final model contains over 25% annotated genes (6909) in the sequenced genome. Substrate utilization was corrected by BIOLOG phenotype arrays, and exponential batch cultivations were used to test growth predictions. The growth data revealed a decrease in glucose uptake rate with rising glucose concentration. A pangenome of four different U. maydis strains highlighted missing metabolic pathways in iUma22. The new model allows for studies of metabolic adaptations to different environmental niches as well as for biotechnological applications.

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.

Rhamnolipids are among the glycolipids that have been investigated intensively in the last decades, mostly produced by the facultative pathogen Pseudomonas aeruginosa using plant oils as carbon source and antifoam agent. Simplification of downstream processing is envisaged using hydrophilic carbon sources, such as glucose, employing recombinant non-pathogenic Pseudomonas putida KT2440 for rhamnolipid or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA, i.e., rhamnolipid precursors) production. However, during scale-up of the cultivation from shake flask to bioreactor, excessive foam formation hinders the use of standard fermentation protocols. In this study, the foam was guided from the reactor to a foam fractionation column to separate biosurfactants from medium and bacterial cells. Applying this integrated unit operation, the space-time yield (STY) for rhamnolipid synthesis could be increased by a factor of 2.8 (STY = 0.17 gRL/L·h) compared to the production in shake flasks. The accumulation of bacteria at the gas-liquid interface of the foam resulted in removal of whole-cell biocatalyst from the reactor with the strong consequence of reduced rhamnolipid production. To diminish the accumulation of bacteria at the gas-liquid interface, we deleted genes encoding cell-surface structures, focusing on hydrophobic proteins present on P. putida KT2440. Strains lacking, e.g., the flagellum, fimbriae, exopolysaccharides, and specific surface proteins, were tested for cell surface hydrophobicity and foam adsorption. Without flagellum or the large adhesion protein F (LapF), foam enrichment of these modified P. putida KT2440 was reduced by 23 and 51%, respectively. In a bioreactor cultivation of the non-motile strain with integrated rhamnolipid production genes, biomass enrichment in the foam was reduced by 46% compared to the reference strain. The intensification of rhamnolipid production from hydrophilic carbon sources presented here is an example for integrated strain and process engineering. This approach will become routine in the development of whole-cell catalysts for the envisaged bioeconomy. The results are discussed in the context of the importance of interacting strain and process engineering early in the development of bioprocesses.

The family of Ustilaginaceae belongs to the order of Basidiomycetes. Despite their plant pathogenicity causing, e.g., corn smut disease, they are also known as natural producers of value-added chemicals such as extracellular glycolipids, organic acids, and polyols. Here, we present 17 high-quality draft genome sequences (N50 > 1 Mb) combining third-generation nanopore and second-generation Illumina sequencing. The data were analyzed with taxonomical genome-based bioinformatics methods such as Percentage of Conserved Proteins (POCP), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) analyses indicating that a reclassification of the Ustilaginaceae family might be required. Further, conserved core genes were determined to calculate a phylogenomic core genome tree of the Ustilaginaceae that also supported the results of the other phylogenomic analysis. In addition, to genomic comparisons, secondary metabolite clusters (e.g., itaconic acid, mannosylerythritol lipids, and ustilagic acid) of biotechnological interest were analyzed, whereas the sheer number of clusters did not differ much between species.

High-titer biosurfactant production in aerated fermenters using hydrophilic substrates is often hampered by excessive foaming. Ethanol has been shown to efficiently destabilize foam of rhamnolipids, a popular group of biosurfactants. To exploit this feature, we used ethanol as carbon source and defoamer, without introducing novel challenges for rhamnolipid purification. In detail, we engineered the non-pathogenic Pseudomonas putida KT2440 for heterologous rhamnolipid production from ethanol. To obtain a strain with high growth rate on ethanol as sole carbon source at elevated ethanol concentrations, adaptive laboratory evolution (ALE) was performed. Genome re-sequencing allowed to allocate the phenotypic changes to emerged mutations. Several genes were affected and differentially expressed including alcohol and aldehyde dehydrogenases, potentially contributing to the increased growth rate on ethanol of 0.51 h–1 after ALE. Further, mutations in genes were found, which possibly led to increased ethanol tolerance. The engineered rhamnolipid producer was used in a fed-batch fermentation with automated ethanol addition over 23 h, which resulted in a 3-(3-hydroxyalkanoyloxy)alkanoates and mono-rhamnolipids concentration of about 5 g L–1. The ethanol concomitantly served as carbon source and defoamer with the advantage of increased rhamnolipid and biomass production. In summary, we present a unique combination of strain and process engineering that facilitated the development of a stable fed-batch fermentation for rhamnolipid production, circumventing mechanical or chemical foam disruption.

Ustilago maydis, a smut fungus, is an appealing model in fundamental research and an upcoming cell factory for industrial biotechnology. The genome of U. maydis has been sequenced and some synthesis pathways were biochemically described; however, the operation of the cellular metabolic network is not well-characterized. Thus, we conducted a comprehensive study to optimize the sample preparation procedure for metabolomics of U. maydis using GC-MS/MS. Due to the unique characteristics of U. maydis cell culture, two quenching solutions, different washing steps, eight extraction methods, and three derivatization conditions have been examined. The optimal method was then applied for stable isotope-assisted quantification of low molecular weight hydrophilic metabolites while U. maydis utilized different carbon sources including sucrose, glucose, and fructose. This study is the first report on a methodology for absolute quantification of intracellular metabolites in U. maydis central carbon metabolism such as sugars, sugar phosphates, organic acids, amino acids, and nucleotides. For biotechnological use, this method is crucial to exploit the full production potential of this fungus and can also be used to study other fungi of the family Ustilaginaceae.

Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. To promote industrial use of rhamnolipids, recombinant rhamnolipid production from sugars needs to be intensified. Since this remains challenging, the aim of the presented research is to utilize a multidisciplinary approach to take a step toward developing a sustainable rhamnolipid production process. Here, we developed expression cassettes for stable integration of the rhamnolipid biosynthesis genes into the genome outperformed plasmid-based expression systems. Furthermore, the genetic stability of the production strain was improved by using an inducible promoter. To enhance rhamnolipid synthesis, energy- and/or carbon-consuming traits were removed: mutants negative for the synthesis of the flagellar machinery or the storage polymer PHA showed increased production by 50%. Variation of time of induction resulted in an 18% increase in titers. A scale-up from shake flasks was carried out using a 1-L bioreactor. By recycling of the foam, biomass loss could be minimized and a rhamnolipid titer of up to 1.5 g/L was achieved without using mechanical foam destroyers or antifoaming agents. Subsequent liquid–liquid extraction was optimized by using a suitable minimal medium during fermentation to reduce undesired interphase formation. A technical-scale production process was designed and evaluated by a life-cycle assessment (LCA). Different process chains and their specific environmental impact were examined. It was found that next to biomass supply, the fermentation had the biggest environmental impact. The present work underlines the need for multidisciplinary approaches to address the challenges associated with achieving sustainable production of microbial secondary metabolites. The results are discussed in the context of the challenges of microbial biosurfactant production using hydrophilic substrates on an industrial scale.

A large variety of microorganisms produces biosurfactants with the potential for a number of diverse industrial applications. To identify suitable wild-type or engineered production strains, efficient screening methods are needed, allowing for rapid and reliable quantification of biosurfactants in multiple cultures, preferably at high throughput. To this end, we have established a novel and sensitive assay for the quantification of biosurfactants based on the dye Victoria Pure Blue BO (VPBO). The assay allows the colorimetric assessment of biosurfactants directly in culture supernatants and does not require extraction or concentration procedures. Working ranges were determined for precise quantification of different rhamnolipid biosurfactants; titers in culture supernatants of recombinant Pseudomonas putida KT2440 calculated by this assay were confirmed to be the same ranges detected by independent high-performance liquid chromatography (HPLC)-charged aerosol detector (CAD) analyses. The assay was successfully applied for detection of chemically different anionic or non-ionic biosurfactants including mono- and di-rhamnolipids (glycolipids), mannosylerythritol lipids (MELs, glycolipids), 3-(3-hydroxyalkanoyloxy) alkanoic acids (fatty acid conjugates), serrawettin W1 (lipopeptide), and N-acyltyrosine (lipoamino acid). In summary, the VPBO assay offers a broad range of applications including the comparative evaluation of different cultivation conditions and high-throughput screening of biosurfactant-producing microbial strains.

Understanding the composability of genetic elements is central to synthetic biology. Even for seemingly well-known elements such as a sigma 70 promoter the genetic context-dependent variability of promoter activity remains poorly understood. The lack of understanding of sequence to function results in highly limited de novo design of novel genetic element combinations. To address this issue, we characterized in detail concatenated “stacked” synthetic promoters including varying spacer sequence lengths and compared the transcription strength to the output of the individual promoters. The proxy for promoter activity, the msfGFP synthesis from stacked promoters was consistently lower than expected from the sum of the activities of the single promoters. While the spacer sequence itself had no activity, it drastically affected promoter activities when placed up- or downstream of a promoter. Single promoter-spacer combinations revealed a bivalent effect on msfGFP synthesis. By systematic analysis of promoter and spacer combinations, a semi-empirical correlation was developed to determine the combined activity of stacked promoters.