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Ginseng, a cornerstone of traditional herbal medicine in Asia, garnered significant attention for its therapeutic potential. Central to its pharmacological effects are ginsenosides, the primary active metabolites, many of which fall within the dammarane-type and share protopanaxadiol as a common precursor. Challenges in extracting protopanaxadiol and ginsenosides from ginseng arise due to their low concentrations in the roots. Emerging solutions involve leveraging microbial cell factories employing genetically engineered yeasts. Here, we optimized the fermentation conditions via the Design of Experiment, realizing 1.2 g/L protopanaxadiol in simple shake flask cultivations. Extrapolating the optimized setup to complex ginsenosides, like compound K, achieved 7.3-fold (0.22 g/L) titer improvements. Our adaptable fermentation conditions enable the production of high-value products, such as sustainable triterpenoids synthesis. Through synthetic biology, microbial engineering, and formulation studies, we pave the way for a scalable and sustainable production of bioactive compounds from ginseng., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Biofilm formation on implant-abutment surfaces can cause inflammatory reactions. Ethical concerns often limit intraoral testing, necessitating preliminary in vitro or animal studies. Here, we propose an in vitro model using human saliva and hypothesize that this model has the potential to closely mimic the dynamics of biofilm formation on implant-abutment material surfaces in vivo., Methods: A saliva stock was mixed with modified Brain-Heart-Infusion medium to form biofilms on Titanium-Aluminum-Vanadium (Ti6Al4V) and Yttria-partially Stabilized Zirconia (Y-TZP) discs in 24-well plates. Biofilm analyses included crystal violet staining, intact cell quantification with BactoBox, 16S rRNA gene analysis, and short-chain fatty acids measurement. As a control, discs were worn in maxillary splints by four subjects for four days to induce in vivo biofilm formation., Results: After four days, biofilms fully covered Ti6Al4V and Y-TZP discs both in vivo and in vitro, with similar cell viability. There was a 60.31% overlap of genera between in vitro and in vivo biofilms in the early stages, and 41% in the late stages. Ten key oral bacteria, including Streptococcus, Haemophilus, Neisseria, Veillonella, and Porphyromonas, were still detectable in vitro, representing the common stages of oral biofilm formation., Conclusion: This in vitro model effectively simulates oral conditions and provides valuable insights into biofilm dynamics., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting nonmodel bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in nonmodel bacteria. As a test strain, we use Cupriavidus necator H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO 2 and hydrogen. However, its low electroporation efficiency hampers the high-throughput genetic engineering required to develop C. necator into an industrially relevant host organism. Thus, conjugation has often been the method of choice for introducing exogenous DNA, especially when introducing large plasmids or suicide plasmids. We first propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of nonmodel bacteria.

Background: Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii., Results: High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time., Conclusions: These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii., (© 2024. The Author(s).)

Polyphosphate (polyP) is an intriguing molecule that is found in almost any organism, covering a multitude of cellular functions. In industry, polyP is used due to its unique physiochemical properties, including pH buffering, water binding, and bacteriostatic activities. Despite the importance of polyP, its analytics is still challenging, with the gold standard being 31 P NMR. Here, we present a simple staining method using the fluorescent dye JC-D7 for the semi-quantitative polyP evaluation in yeast extracts. Notably, fluorescence response was affected by polyP concentration and polymer chain length in the 0.5-500 µg/mL polyP concentration range. Hence, for polyP samples of unknown chain compositions, JC-D7 cannot be used for absolute quantification. Fluorescence of JC-D7 was unaffected by inorganic phosphate up to 50 mM. Trace elements (FeSO 4  > CuSO 4  > CoCl 2  > ZnSO 4 ) and toxic mineral salts (PbNO 3 and HgCl 2 ) diminished polyP-induced JC-D7 fluorescence, affecting its applicability to samples containing polyP-metal complexes. The fluorescence was only marginally affected by other parameters, such as pH and temperature. After validation, this simple assay was used to elucidate the degree of polyP production by yeast strains carrying gene deletions in (poly)phosphate homeostasis. The results suggest that staining with JC-D7 provides a robust and sensitive method for detecting polyP in yeast extracts and likely in extracts of other microbes. The simplicity of the assay enables high-throughput screening of microbes to fully elucidate and potentially enhance biotechnological polyP production, ultimately contributing to a sustainable phosphorus utilization., (© 2024 The Author(s). Yeast published by John Wiley & Sons Ltd.)

Genome-scale metabolic models (GEMs) can facilitate metabolism-focused multi-omics integrative analysis. Since Yeast8, the yeast-GEM of Saccharomyces cerevisiae, published in 2019, has been continuously updated by the community. This has increased the quality and scope of the model, culminating now in Yeast9. To evaluate its predictive performance, we generated 163 condition-specific GEMs constrained by single-cell transcriptomics from osmotic pressure or reference conditions. Comparative flux analysis showed that yeast adapting to high osmotic pressure benefits from upregulating fluxes through central carbon metabolism. Furthermore, combining Yeast9 with proteomics revealed metabolic rewiring underlying its preference for nitrogen sources. Lastly, we created strain-specific GEMs (ssGEMs) constrained by transcriptomics for 1229 mutant strains. Well able to predict the strains' growth rates, fluxomics from those large-scale ssGEMs outperformed transcriptomics in predicting functional categories for all studied genes in machine learning models. Based on those findings we anticipate that Yeast9 will continue to empower systems biology studies of yeast metabolism., (© 2024. The Author(s).)

Adaptive laboratory evolution (ALE) is a widely used microbial strain development and optimization method. ALE experiments, to select for faster-growing strains, are commonly performed as serial batch cultivations in shake flasks, serum bottles, or microtiter plates or as continuous cultivations in bioreactors on a laboratory scale. To combine the advantages of higher throughput in parallel shaken cultures with continuous fermentations for conducting ALE experiments, a new Continuous parallel shaken pH-auxostat (CPA) was developed. The CPA consists of six autonomous parallel shaken cylindrical reactors, equipped with real-time pH control of the culture medium. The noninvasive pH measurement and control are realized by biocompatible pH sensor spots and a programmable pump module, to adjust the dilution rate of fresh medium for each reactor separately. Two different strains of the methylotrophic yeast Ogataea polymorpha were used as microbial model systems for parallel chemostat and pH-auxostat cultivations. During cultivation, the medium is acidified by the microbial activity of the yeast. For pH-auxostat cultivations, the growth-dependent acidification triggers the addition of fresh feed medium into the reactors, leading to a pH increase and thereby to the control of the pH to a predetermined set value. By controlling the pH to a predetermined set value, the dilution rate of the continuous cultivation is adjusted to values close to the washout point, in the range of the maximum specific growth rate of the yeast. The pH control was optimized by conducting a step-response experiment and obtaining tuned PI controller parameters by the Chien-Hrones-Reswick (CHR) PID tuning method. Two pH-auxostat cultivations were performed with two different O. polymorpha strains at high dilution rates for up to 18 days. As a result, up to 4.8-fold faster-growing strains were selected. The increased specific maximum growth rates of the selected strains were confirmed in subsequent batch cultivations., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Solving the plastic crisis requires high recycling quotas and technologies that allow open loop recycling. Here a biological plastic valorization approach consisting of tandem enzymatic hydrolysis and monomer conversion of post-consumer polyethylene terephthalate into value-added products is presented. Hydrolysates obtained from enzymatic degradation of pre-treated post-consumer polyethylene terephthalate bottles in a stirred-tank reactor served as the carbon source for a batch fermentation with an engineered Pseudomonas putida strain to produce 90mg/L of the biopolymer cyanophycin. Through fed-batch operation, the fermentation could be intensified to 1.4 g/L cyanophycin. Additionally, the upcycling of polyethylene terephthalate monomers to the biosurfactants (hydroxyalkanoyloxy)alkanoates and rhamnolipids is presented. These biodegradable products hold significant potential for applications in areas such as detergents, building blocks for novel polymers, and tissue engineering. In summary, the presented bio-valorization process underscores that addressing challenges like the plastic crisis requires an interdisciplinary approach., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aims: To qualitatively evaluate the experiences and emotional responses of elderly individuals with type 2 diabetes regarding the use of an interactive virtual assistant device., Methods and Results: This qualitative study included elderly individuals who were diagnosed with type 2 diabetes and who had been using the Smart Speaker EchoDot 3rd Gen (Amazon Echo ® ) device for three months. A structured face-to-face interview with open-ended questions was conducted to evaluate their experiences and emotional responses associated with the device. Data analysis was performed using inductive thematic content analysis with deductive coding followed by narrative synthesis to present the overall perceptions of the participants. Thirty individuals with a mean diabetes duration of 17.1 ± 9.45 years and a mean age of 71.9 ± 5.1 years were interviewed to ensure saturation of responses. Three major themes were identified through response analysis: (1) Emotional response to user experience; (2) Humanization feelings in human-device interactions; (3) Diabetes-related self-care. Overall, participants experienced a wide range of feelings regarding the use of the interactive virtual assistant device, predominantly with positive connotations, highlighting aspects of humanization of technology and its use, and experiencing assistance in self-care related to diabetes., Conclusion: Our results highlight the overwhelmingly positive emotional responses and strong sense of humanization expressed by elderly individuals with diabetes toward an interactive virtual assistant device. This underscores its potential to improve mental health and diabetes care, although further studies are warranted to fully explore its impact., (© 2024. Springer-Verlag Italia S.r.l., part of Springer Nature.)

Rhamnolipids (RLs) are amphiphilic compounds of bacterial origin that offer a broad range of potential applications as biosurfactants in industry and agriculture. They are reported to be active against different plant pests and pathogens and thus are considered promising candidates for nature-derived plant protection agents. However, as these glycolipids are structurally diverse, little is known about their exact mode of action and, in particular, the relation between molecular structure and biological activity against plant pests and pathogens. Engineering the synthesis pathway in recombinant Pseudomonas putida strains in combination with advanced HPLC techniques allowed us to separately analyze the activities of mixtures of pure mono-RLs (mRLs) and of pure di-RL (dRLs), as well as the activity of single congeners. In a model system with the plant Arabidopsis thaliana and the plant-parasitic nematode (PPN) Heterodera schachtii we demonstrate that RLs can significantly reduce infection, whereas their impact on the host plant varied depending on their molecular structure. While mRLs reduced plant growth even at a low concentration, dRLs showed a neutral to beneficial impact on plant development. Treating plants with dRLs triggered an increased reactive oxygen species (ROS) production, indicating the activation of stress-response signaling and possibly plant defense. Pretreatment of plants with mRLs or dRLs prior to application of flagellin (flg22), a known ROS inducer, further increased the ROS response to flg22. While dRLs stimulated an elevated flg22-induced ROS peak, a pretreatment with mRLs resulted in a prolonged synthesis of ROS indicating a generally elevated stress level. Neither mRLs nor dRLs induced the expression of plant defense marker genes of salicylic acid, jasmonic acid, and ethylene pathways. Detailed studies on dRLs revealed that even high concentrations up to 755 ppm of these molecules have no lethal impact on H. schachtii infective juveniles. Infection assays with individual dRL congeners showed that the C10-C8 acyl chained dRL was the only congener without effect, while dRLs with C10-C12 and C10-C12:1 acyl chains were most efficient in reducing nematode infection even at concentrations below 2 ppm. As determined by phenotyping and ROS measurements, A. thaliana reacted more sensitive to long-chained dRLs in a concentration-dependent manner. Our experiments show a clear structure-activity relation for the effect of RLs on plants. In conclusion, functional assessment and analysis of the mode of action of RLs in plants and other organisms require careful consideration of their molecular structure and composition., Competing Interests: Declaration of competing interest This work is part of the patent application “Method of Pathogen Control” with SB, ASSS, FMWG, CM, TT, and LMB being inventors [WO2022238567A1]. The other authors have nothing to declare., (Copyright © 2024. Published by Elsevier Inc.)

Squalene is a natural triterpenoid and a biosynthetic precursor of steroids and hopanoids in microorganisms, plants, humans, and other animals. Squalene has exceptional properties, such as its antioxidant activity, a high penetrability of the skin, and the ability to trigger the immune system, promoting its application in the cosmetic, sustenance, and pharmaceutical industries. Because sharks are the primary source of squalene, there is a need to identify low-cost, environment friendly, and sustainable alternatives for producing squalene commercially. This shift has prompted scientists to apply biotechnological advances to research microorganisms for synthesizing squalene. This review summarizes recent metabolic and bioprocess engineering strategies in various microorganisms for the biotechnological production of this valuable molecule., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Engineering in Life Sciences published by Wiley‐VCH GmbH.)

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., (© 2024. The Author(s).)

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., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for Pseudomonas putida KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data. Homologous recombination constructs were designed to insert heterologous genes into intergenic sites in these regions, allowing condition-independent gene expression. Ten potential landing pads were characterized using four different msfGFP expression cassettes. An insulated probe sensor was used to study locus-dependent effects on recombinant gene expression, excluding genomic read-through of flanking promoters under changing cultivation conditions. While the reproducibility of expression in the landing pads was very high, the msfGFP signals varied strongly between the different landing pads, confirming a strong influence of the genomic context. To showcase that the identified landing pads are also suitable candidates for heterologous gene expression in other Pseudomonads, four equivalent landing pads were identified and characterized in Pseudomonas taiwanensis VLB120. This study shows that genomic "hot" and "cold" spots exist, causing strong promoter-independent variations in gene expression. This highlights that the genomic context is an additional parameter to consider when designing integrable genomic cassettes for tailored heterologous expression. The set of characterized genomic landing pads presented here further increases the genetic toolbox for deep metabolic engineering in Pseudomonads.

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., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Itaconic acid is a platform chemical with a range of applications in polymer synthesis and is also discussed for biofuel production. While produced in industry from glucose or sucrose, co-feeding of glucose and acetate was recently discussed to increase itaconic acid production by the smut fungus Ustilago maydis. In this study, we investigate the optimal co-feeding conditions by interlocking experimental and computational methods. Flux balance analysis indicates that acetate improves the itaconic acid yield up to a share of 40% acetate on a carbon molar basis. A design of experiment results in the maximum yield of 0.14 itaconic acid per carbon source from 100  g L - 1 $\,\text{g L}{}^{-1}$ glucose and 12  g L - 1 $\,\text{g L}{}^{-1}$ acetate. The yield is improved by around 22% when compared to feeding of glucose as sole carbon source. To further improve the yield, gene deletion targets are discussed that were identified using the metabolic optimization tool OptKnock. The study contributes ideas to reduce land use for biotechnology by incorporating acetate as co-substrate, a C2-carbon source that is potentially derived from carbon dioxide., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Polyphosphates, chains of polymerized phosphate subunits, are used as food additives for various applications such as conservation, water retention, and pH buffering. Currently, the value chain of phosphates is linear, based on mining fossil phosphate rock, which is anticipated to be depleted in a few hundred years. With no replacement available, a transition to a circular phosphate economy, to which biological systems can contribute, is required. Baker's yeast can hyperaccumulate phosphate from various phosphate-rich waste streams and form polyphosphates, which can be used directly or as polyphosphate-rich yeast extract with enhanced properties in the food industry. By maturing the technology to an industrial level and allowing upcycled waste streams for food applications, substantial contributions to a sustainable phosphate economy can be achieved., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest. LMB filed two patent applications: “Zusammensetzung, enthaltend getrocknetes Polyphosphat und Verfahren zur Gewinnung von Polyphosphat aus polyphosphat-haltigen Hefezellen dazu” (DE 10 2019 131 561.1) and “Polyphosphatreiche Hefeextrakte und Herstellverfahren dazu” (DE 10 2018 130 081.6, PCT/EP2019/082709)., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed., Results: This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall., Conclusions: This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Background: With one in five individuals aged 65 or older living with type 2 diabetes worldwide, it is crucial to acknowledge and address the challenges faced by this population. In this context, our study aims to evaluate the efficacy of a behavioral intervention model delivered through a smart speaker on mental health and diabetes self-care in the elderly with diabetes., Methods: This is a single-center, pragmatic, parallel two-arm open randomized clinical trial involving elderly patients with type 2 diabetes. We plan to enroll a total of 112 individuals who will be randomized 1:1 to receive the Smart Speaker EchoDot 3rd Gen device (Amazon Echo®) for home use (intervention arm) or to maintain usual care (control arm). The primary outcome is mental distress, assessed using the 20-item Self Reporting Questionnaire (SRQ-20) after a 12-week intervention period. Secondary outcomes include quality of life, adherence to diabetes self-care behaviors, perception of stress, glycemic control, blood pressure, and lipid profile. Analysis of covariance (ANCOVA) will be used to evaluate the effects of the intervention on the outcomes., Discussion: This study assesses the effectiveness of an interactive virtual assistance system for enhancing mental health and glycemic control among elderly individuals with type 2 diabetes. The findings may introduce smart speakers as a valuable tool for promoting diabetes-related self-care in this population., Trial Registration: ClinicalTrials.gov NCT05329376. Registered on 15 April 2022. Enrollment began on 20 June 2023 and the last update of protocol was on 13 December 2023., (© 2024. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Haala, Dielentheis-Frenken, Brandt, Karmainski, Blank and Tiso.)

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-MS 2 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., (© 2024 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Chemically defined mineral media are widely used in bioprocesses, as these show less batch to batch variation compared with complex media. Nonetheless, the recommended media formulations often lead to the formation of precipitants at elevated pH values. These precipitates are insoluble and reduce the availability of macronutrients to the cells, which can result in limiting growth rates and lower productivity. They can also damage equipment by clogging pipes, hoses, and spargers in stirred tank fermenters. In this study, the observed precipitate was analyzed via X-ray fluorescence spectroscopy and identified as the magnesium ammonium phosphate salt struvite (MgNH 4 PO 4  × 6H 2 O). The solubility of struvite crystals is known to be extremely low, causing the macronutrients magnesium, phosphate, and ammonium to be bound in the struvite crystals. Here, it was shown that struvite precipitates can be redissolved under common fermentation conditions. Furthermore, it was found that the struvite particle size distribution has a significant effect on the dissolution kinetics, which directly affects macronutrient availability. At a certain particle size, struvite crystals rapidly dissolved and provided unlimiting growth conditions. Therefore, struvite formation should be considered during media and bioprocess development, to ensure that the dissolution kinetics of struvite are faster than the growth kinetics., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

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., (© 2024. The Author(s).)

Background: One carbon (C1) molecules such as methanol have the potential to become sustainable feedstocks for biotechnological processes, as they can be derived from CO 2 and green hydrogen, without the need for arable land. Therefore, we investigated the suitability of the methylotrophic yeast Ogataea polymorpha as a potential production organism for platform chemicals derived from methanol. We selected acetone, malate, and isoprene as industrially relevant products to demonstrate the production of compounds with 3, 4, or 5 carbon atoms, respectively., Results: We successfully engineered O. polymorpha for the production of all three molecules and demonstrated their production using methanol as carbon source. We showed that the metabolism of O. polymorpha is well suited to produce malate as a product and demonstrated that the introduction of an efficient malate transporter is essential for malate production from methanol. Through optimization of the cultivation conditions in shake flasks, which included pH regulation and constant substrate feeding, we were able to achieve a maximum titer of 13 g/L malate with a production rate of 3.3 g/L/d using methanol as carbon source. We further demonstrated the production of acetone and isoprene as additional heterologous products in O. polymorpha, with maximum titers of 13.6 mg/L and 4.4 mg/L, respectively., Conclusion: These findings highlight how O. polymorpha has the potential to be applied as a versatile cell factory and contribute to the limited knowledge on how methylotrophic yeasts can be used for the production of low molecular weight biochemicals from methanol. Thus, this study can serve as a point of reference for future metabolic engineering in O. polymorpha and process optimization efforts to boost the production of platform chemicals from renewable C1 carbon sources., (© 2023. The Author(s).)

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., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Pseudomonas sp. LFM693 is a 2-methylisocitrate lyase (prpB) disrupted mutant. This enzyme catalyzes a step in the 2-methylcitrate cycle, the only known and described pathway for propionate oxidation in this organism. The affected mutants can efficiently produce PHA containing even and odd-chain length hydroxyalkanoates (HA even/odd ) in the presence of propionate and glucose. In this study, a constant fed-batch configuration was utilized to control the composition of PHA and decrease the toxicity of propionate. The incorporation of HA odd into the copolymer was linear, ranging from 7 to approximately 30 %, and correlated directly with the propionate/glucose molar ratio in the feeding solution. This allowed for the molecular composition of the mclPHA to be fine-tuned with minimum process monitoring and control. The average PHA content was 52 % cell dry weight with a molar composition that favored 3-hydroxyalkanoates containing C8, C9, and C10. The conversion factor of propionate to HA odd varied between 0.36 and 0.53 mol·mol -1 (Y HAodd/prop .), which are significantly lower than the theoretical maximum efficiency (1.0 mol·mol -1 ). These results along with the lack of 2-methylisocitrate as a byproduct provides further support for the evidence that the mutant prpB - is still capable of oxidizing propionate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A novel biosurfactant was discovered to be synthesized by the marine bacterium Alcanivorax borkumensis in 1992. This bacterium is abundant in marine environments affected by oil spills, where it helps to degrade alkanes and, under such conditions, produces a glycine-glucolipid biosurfactant. The biosurfactant enhances the bacterium's attachment to oil droplets and facilitates the uptake of hydrocarbons. Due to its useful properties expected, there is interest in the biotechnological production of this biosurfactant. To support this effort analytically, a method combining reversed-phase high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) was developed, allowing the separation and identification of glycine-glucolipid congeners. Accurate mass, retention time, and characteristic fragmentation pattern were utilized for species assignment. In addition, charged-aerosol detection (CAD) was employed to enable absolute quantification without authentic standards. The methodology was used to investigate the glycine-glucolipid production by A. borkumensis SK2 using different carbon sources. Mass spectrometry allowed us to identify congeners with varying chain lengths (C 6 -C 12 ) and degrees of unsaturation (0-1 double bonds) in the incorporated 3-hydroxy-alkanoic acids, some previously unknown. Quantification using CAD revealed that the titer was approximately twice as high when grown with hexadecane as with pyruvate (49 mg/L versus 22 mg/L). The main congener for both carbon sources was glc-40:0-gly, accounting for 64% with pyruvate and 85% with hexadecane as sole carbon source. With the here presented analytical suit, complex and varying glycolipids can be identified, characterized, and quantified, as here exemplarily shown for the interesting glycine-glucolipid of A. borkumensis., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Karmainski, Dielentheis-Frenken, Lipa, Phan, Blank and Tiso.)

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., Competing Interests: Author J-FS was employed by the company Bayer AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fendt, Ghallab, Myllys, Hofmann, Hassan, Hobloss, González, Brackhagen, Marchan, Edlund, Seddek, Abdelmageed, Blank, Schlender, Holland, Hengstler and Kuepfer.)

Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on the protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum , Escherichia coli , and Pseudomonas putida . Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enabled the production of an unnatural polyketide in each of these hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. In this work, we not only highlight the significance of codon optimization but also establish the groundwork for the high-throughput assembly and characterization of PKS pathways in alternative hosts.

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., (© 2023. The Author(s).)

Pseudomonas sp. LFM046 produces polyhydroxyalkanoates of medium-chain length. When carbohydrates are used, only monomers of even-length chains (3HA even ) are generated. Propionate, when used as a co-substrate, enabled the synthesis of odd-chain length monomers, albeit with poor yields (Y 3HAodd/prop. ) of approx. 10 % . A mini-Tn5 mutant (LFM693) was generated by interrupting the prpB gene, which encodes the 2-methylisocitrate lyase responsible for one step in propionate oxidation via 2-methylcitrate cycle. At low propionate concentrations of 0.1-0.3 g.L - 1 , LFM693 showed higher Y 3HAodd/prop . In contrast, the propionyl incorporation into the polymer was less efficient at high propionate concentrations. The excretion of 2-methylisocitrate partially explained this reduced efficiency. However, mass balances and 13 C isotopomer analysis revealed significant propionate oxidation in LFM693 of 21 % and 46 % for propionate concentrations of 0.5 and 1.0 g.L - 1 , respectively. Experiments using C1-labeled ( 13 C) propionate revealed an alternative propionate oxidation pathway (α-oxidation, β-oxidation, or 3-methylmalate) whose genes are yet to be revealed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Background & Aims: Growing evidence supports a role of gut-derived metabolites in nonalcoholic fatty liver disease (NAFLD), but the relation of endotoxin levels with gut permeability and NAFLD stage remains unclear. This systematic review with meta-analysis aims to provide further insights., Methods: PubMed, Embase, and Cochrane Library were searched for studies published until January 2022 assessing blood endotoxins in patients with NAFLD. Meta-analyses and univariate/multivariate meta-regression, as well as correlation analyses, were performed for endotoxin values and potential relationships to disease stage, age, sex, parameters of systemic inflammation, and metabolic syndrome, as well as liver function and histology., Results: Forty-three studies were included, of which 34 were used for meta-analyses. Blood endotoxin levels were higher in patients with simple steatosis vs liver-healthy controls (standardized mean difference, 0.86; 95% confidence interval, 0.62-1.11) as well as in patients with nonalcoholic steatohepatitis vs patients with nonalcoholic fatty liver/non-nonalcoholic steatohepatitis (standardized mean difference, 0.81; 95% confidence interval, 0.27-1.35; P = .0078). Consistently, higher endotoxin levels were observed in patients with more advanced histopathological gradings of liver steatosis and fibrosis. An increase of blood endotoxin levels was partially attributed to a body mass index rise in patients with NAFLD compared with controls. Nevertheless, significant increases of blood endotoxin levels in NAFLD retained after compensation for differences in body mass index, metabolic condition, or liver enzymes. Increases in blood endotoxin levels were associated with increases in C-reactive protein concentrations, and in most cases, paralleled a rise in markers for intestinal permeability., Conclusion: Our results support blood endotoxin levels as relevant diagnostic biomarker for NAFLD, both for disease detection as well as staging during disease progression, and might serve as surrogate marker of enhanced intestinal permeability in NAFLD. Registration number in Prospero: CRD42022311166., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have no conflicts of interest to declare., (© 2023 The Author(s).)

Fibrodysplasia ossificans progressive (FOP) is an ultra-rare genetic disorder that is caused by a mutation in the ACVR1 gene and provokes severe heterotopic ossification. Since flares of the disease are associated with inflammation, it is assumed that JAK inhibitors can control active FOP due to blocking multiple signaling pathways., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The overall challenge of establishing a sustainable anthropogenic phosphate cycle did not change in the last decade, rather the need to act becomes ever more urgent. Here, I briefly highlight some developments in (poly)phosphate research within the last 10 years and dare a crystal ball look for topics that might help us towards a sustainable P society., (© 2023 The Author. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

The potential of nonmodel organisms for industrial biotechnology is increasingly becoming evident since advances in systems and synthetic biology have made it possible to explore their unique traits. However, the lack of adequately characterized genetic elements that drive gene expression impedes benchmarking nonmodel with model organisms. Promoters are one of the genetic elements that contribute significantly to gene expression, but information about their performance in different organisms is limited. This work addresses this bottleneck by characterizing libraries of synthetic σ 70 -dependent promoters controlling the expression of msf GFP, a monomeric, superfolder green fluorescent protein, in both Escherichia coli TOP10 and Pseudomonas taiwanensis VLB120, a less explored microbe with industrially attractive attributes. We adopted a standardized method for comparing gene promoter strength across species and laboratories. Our approach uses fluorescein calibration and adjusts for cell growth variation, enabling accurate cross-species comparisons. The quantitative description of promoter strength is a valuable expansion of P. taiwanensis VLB120's genetic toolbox, while the comparison with the performance in E. coli facilitates the evaluation of P. taiwanensis VLB120's potential as a chassis for biotechnology applications.

The structure-property relationship of rhamnolipids, RLs, well-known microbial bioamphiphiles (biosurfactants), is explored in detail by coupling cryogenic transmission electron microscopy (cryo-TEM) and both ex situ and in situ small-angle X-ray scattering (SAXS). The self-assembly of three RLs with reasoned variation of their molecular structure (RhaC10, RhaC10C10, and RhaRhaC10C10) and a rhamnose-free C10C10 fatty acid is studied in water as a function of pH. It is found that RhaC10 and RhaRhaC10C10 form micelles in a broad pH range and RhaC10C10 undergoes a micelle-to-vesicle transition from basic to acid pH occurring at pH 6.5. Modeling coupled to fitting SAXS data allows a good estimation of the hydrophobic core radius (or length), the hydrophilic shell thickness, the aggregation number, and the surface area per RL. The essentially micellar morphology found for RhaC10 and RhaRhaC10C10 and the micelle-to-vesicle transition found for RhaC10C10 are reasonably well explained by employing the packing parameter (PP) model, provided a good estimation of the surface area per RL. On the contrary, the PP model fails to explain the lamellar phase found for the protonated RhaRhaC10C10 at acidic pH. The lamellar phase can only be explained by values of the surface area per RL being counterintuitively small for a di-rhamnose group and folding of the C10C10 chain. These structural features are only possible for a change in the conformation of the di-rhamnose group between the alkaline and acidic pH.

Enzyme-based plastic degradation and valorization of the plastic-derived monomers has emerged as a potent option to address the plastic waste dilemma. Obstacles in implementing the enzymatic degradation of plastics in industry are here summarized, and strategies to overcome these obstacles are discussed to exploit the full potential of enzymatic plastic degradation toward a sustainable plastic economy., Competing Interests: Declaration of interests There are no interests to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Lactate has gained increasing attention as a platform chemical, particularly for the production of the bioplastic poly-lactic acid (PLA). While current microbial lactate production processes primarily rely on the use of sugars as carbon sources, it is possible to envision a future where lactate can be produced from sustainable, non-food substrates. Methanol could be such a potential substrate, as it can be produced by (electro)chemical hydrogenation from CO 2 . Methods: In this study, the use of the methylotrophic yeast Ogataea polymorpha as a host organism for lactate production from methanol was explored. To enable lactate production in Ogataea polymorpha , four different lactate dehydrogenases were expressed under the control of the methanol-inducible MOX promoter. The L-lactate dehydrogenase of Lactobacillus helveticus performed well in the yeast, and the lactate production of this engineered strain could additionally be improved by conducting methanol fed-batch experiments in shake flasks. Further, the impact of different nitrogen sources and the resulting pH levels on production was examined more closely. In order to increase methanol assimilation of the lactate-producing strain, an adaptive laboratory evolution experiment was performed. Results and Discussion: The growth rate of the lactate-producing strain on methanol was increased by 55%, while at the same time lactate production was preserved. The highest lactate titer of 3.8 g/L in this study was obtained by cultivating this evolved strain in a methanol fed-batch experiment in shake flasks with urea as nitrogen source. This study provides a proof of principle that Ogataea polymorpha is a suitable host organism for the production of lactate using methanol as carbon source. In addition, it offers guidance for the engineering of methylotrophic organisms that produce platform chemicals from CO 2 -derived substrates. With reduced land use, this technology will promote the development of a sustainable industrial biotechnology in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wefelmeier, Schmitz, Haut, Otten, Jülich and Blank.)

Paracoccus spp. are Gram-negative, coccoid bacteria, fascinating for their ability to grow in highly diverse environments while producing commercially relevant products. This study describes the draft genome sequence of the halotolerant, alkaliphilic, and thermotolerant carotenoid-producing type strain Paracoccus bogoriensis BOG6 T ., Competing Interests: The authors declare no conflict of interest.