Fungal iterative type I polyketide synthases (iPKSs) generate structurally diverse natural products. A subset of these assembly line enzymes, exemplified by LovB from Aspergillus terreus, contains a C-terminal condensation domain bearing a noncanonical HRxxxDG motif. While these LovB-like iPKSs are widely distributed in fungi, the majority remain uncharacterized, leaving both their associated polyketide products and the function of their unusual condensation domains largely unexplored. Here, we report the characterization of a LovB-like iPKS from Aspergillus clavatus. Heterologous reconstitution of this PKS system in Aspergillus nidulans led to the discovery of clavaic acid, a previously undescribed polyketide featuring a trimethylated trans-decalin core and an all-E-configured carboxytriene side chain. Functional analysis of the condensation domain in vivo demonstrated that it is essential for clavaic acid biosynthesis. Surprisingly, whereas the conserved arginine residue within the HRxxxDG motif was dispensable for product formation, the conserved aspartate residue was strictly required. These findings expand our understanding of LovB-like iPKSs and establish this enzyme family as a promising source of cryptic fungal polyketides. One-sentence summary Genome-guided discovery of a trans-decalin-containing polyketide encoded by a LovB-like polyketide synthase in Aspergillus clavatus.
The trend toward concentrated animal feeding operations (CAFOs) has served to concentrate not only livestock animals but the waste they produce to comparatively smaller areas. The point-source nature of this waste is an opportunity for the recovery and valorization of the nitrogen therein. Such a process would be viable on small to intermediate scales and require minimal inputs at the CAFO. In this study, we demonstrate the potential of the biopolymer cyanophycin to serve as a medium for manure-nitrogen recovery. In the first step, genetically modified strains of Escherichia coli produce intracellular cyanophycin from mock manure hydrolysates. Next, cyanophycin is recovered from microbial biomass via acid solubilization and base precipitation using electrochemically generated acids and bases. Finally, to improve both the yield and recoverable fraction of cyanophycin produced, we leverage the tunability of our genetically engineered system to probe the impacts of cyanophycin synthetase solubility, N-domain activity, and cyanophycin molecular weight on cyanophycin recoverability. Collectively, this work serves as a proof of concept for nitrogen recovery from agricultural waste, aligning with global sustainability initiatives. One sentence summary Mock manure-derived substrates can be converted to cyanophycin and recovered, supporting the feasibility of nitrogen recovery at potentially decentralized sites of waste production.
Uncontrolled, fast-growing nontuberculous mycobacteria (NTM) in metalworking fluids (MWFs) can cause dermal lesions, hypersensitivity pneumonitis, and other health complications in exposed metalworkers. Although biocides are routinely used to control microbial growth in MWF, they are often ineffective because they are unsuitable for the physical or chemical conditions of the system, added infrequently, or ineffective against the microorganisms present. Given the increasing concern for NTM in industrial settings, there is a growing need for MWF-compatible (i.e., nonoxidizing) biocides that are effective against NTM. Here, we evaluated the efficacy of four nonoxidizing biocides against fast-growing NTM species in water-based MWF. Results from a 72-hr laboratory challenge indicated that biocides containing 2-(thiocyanomethylthio)benzothiazole (TCMTB), commonly used as a fungicide, were most effective at controlling Mycobacterium chelonae, M. fortuitum, and field and laboratory strains of M. immunogenum. In subsequent field trials at nine MWF plants where workers had reported symptoms of dermal and lung illnesses, TCMTB consistently reduced viable NTM counts, as measured through both culture-based assays and microscopy. However, dosages needed to be increased two- to four-fold relative to the laboratory experiments to control NTM populations in systems with established biofilms. Due to the observed success of TCMTB against NTM in field context, the manufacturing plants involved in this study continued regular application of TCMTB as a preventative treatment. No additional outbreaks were observed throughout our follow-up period, which, in several cases, spanned approximately 10 years. The results suggest that TCMTB may provide an efficacious method for treating and preventing NTM contamination in MWF systems. Future studies should explore the mechanism of action of the sulfur-rich chemical structure of TCMTB to potentially guide the development and synthesis of related compounds with anti-NTM properties. One sentence summary 2--(thiocyanomethylthio)benzothiazole kills fast-growing nontuberculous mycobacteria, including Mycobacterium immunogenum, and prevents worker infections based on laboratory, field trial, and long-term (6-10 years) industrial studies.
Resveratrol is a high-value polyphenolic compound widely utilized in nutraceutical, cosmetic, and pharmaceutical applications. However, most microbial production systems rely on glucose as the primary carbon source, which limits flexibility for integrating alternative and renewable feedstocks. In this study, we developed an engineered Escherichia coli platform to investigate resveratrol biosynthesis under xylose-supporting conditions, using food-grade D-xylose as the carbon source. A heterologous pathway consisting of Populus tomentosa 4-coumarate: CoA ligase (Pt4CL) and Arachis hypogaea stilbene synthase (AhSTS) was introduced to convert externally supplied p-coumaric acid (PCA) into resveratrol. To improve precursor availability, intracellular malonyl-CoA supply was enhanced by introducing matB and matC from Streptomyces coelicolor A3(2) and overexpressing the acetyl-CoA carboxylase complex (ACC) from E. coli. Xylose assimilation was further strengthened by expressing xylE, xylA, and xylB, while carbon catabolite repression was alleviated using CRISPR interference (CRISPRi) targeting the glucose transporter gene ptsG. Under shake-flask conditions, the engineered strain produced up to 23.9 mg/L resveratrol from food-grade D-xylose, accompanied by near-complete xylose consumption and 93%-94% precursor conversion. This corresponded to an overall fermentation yield of approximately 12.5 mg resveratrol per g xylose consumed. Similar titers (27 mg/L) were obtained in a 5-L bioreactor, indicating stable pathway performance under controlled fermentation conditions. Overall, these results show that E. coli can be engineered to support efficient precursor-to-product conversion under xylose-supported conditions, providing a useful proof-of-concept framework for integrating alternative carbon sources into microbial production platforms for aromatic compounds.One sentence summary An engineered Escherichia coli system integrates xylose utilization, malonyl-CoA pathway optimization, and CRISPRi regulation to support resveratrol biosynthesis under xylose-supported conditions.
Knowledge of protein acid sensitivity remains sparse and is largely derived from low-throughput, enzyme-specific assays. We used a scalable framework to map acid stability across the Escherichia coli proteome to assess the acid stability of 1,675 unique proteins, estimating pH50 values for over 90% of them. The parameter pH50 was defined as the pH value at which only 50% of the initial protein remains in solution following acid treatment. Proteome-wide pH50 values ranged from 2.28 to 6.33 (median 5.11). Approximately 9% of detected proteins remained stable across all tested pH conditions. Our results align with published data and the assay of citrate synthase (GltA) performed here. Protein acid stability differed significantly by subcellular localization: periplasmic proteins were relatively more abundant in the acid-stable group, cytoplasmic proteins were abundant at pH50 values 4.5-5.5, and inner membrane proteins at higher pH50 between 5.5 and 6.0. Outer membrane proteins were too few to draw strong conclusions regarding enrichment within specific pH50 groups. Notably, the periplasmic binding protein of the molybdate ABC transporter (ModA), was enriched after incubation at low pH. Estimated pH50 values showed no correlation with protein isoelectric point and molecular weight. Together, this work provides the first proteome-wide map of protein acid stability and establishes a general framework for studying different chemical stressors.One sentence summary This paper presents a quantitative map of acid stability of E. coli proteins and established a procedure for the study of protein acid sensitivity that is adaptable to other stressors and other organisms.
Many strategies to create a circular bioeconomy have been proposed. To be successful, CO2 must be reduced with renewable energy into chemical building blocks, from which the chemical industry can be supported. Circular strategies include leveraging photosynthesis to produce sugar and lipid intermediates or renewable electricity to produce hydrogen or other electron carriers to support CO2 reduction. Acetogens can anaerobically reduce CO2 with H2 to produce mixtures of small organic molecules in gas fermentations. We previously demonstrated that acetate, a common product of gas fermentation, can be converted to the model oleochemical dodecanol in engineered Escherichia coli. Here, we explored the conversion of ethanol and mixtures of ethanol and acetate to the same model oleochemicals. Co-feeding ethanol can supply both carbon and additional reducing power relative to acetate alone. In this work, we engineered E. coli to catabolize ethanol and expressed two distinct ethanol metabolism pathways in different operons and combined them with improved engineered acetate activation. We evaluated the performance of these operons in dodecanol-producing strains when fed ethanol or acetate and found ethanol to be a better carbon source when judged by product titers. The engineered strains fed ethanol produced about two-fold more dodecanol than the strains fed acetate. This increase was in part, due to change in product distribution. Cells fed ethanol produced predominantly dodecanol, whereas cells fed acetate generated a mixture of dodecanol and dodecanoic acid. Dodecanol titers were further improved by employing feeding strategies in controlled bioreactors.
Successful development of a predictive digital twin or digital shadow enabling improved batch planning and process optimization of industrial fungal fermentation relies on the fidelity of oxygen transfer rate modeling. Such models depend, among other factors, on viscosity. Traditional process models use mechanistic approaches to describe the apparent viscosity but face challenges due to its inherent complex behavior, requiring many assumptions and often relying on tedious offline rheological measurements. This article presents a novel model development framework, which significantly reduces associated experimental costs and eliminates the need for offline rheological measurement. First, a method for developing a model for the oxygen mass transfer coefficient (kLa) at pilot scale is presented, reducing experimental effort from nine to two fermentations while achieving an R² of 0.92 with online data compared to an R² of 0.67 with offline data. Alongside the mechanistic model, which links biological yields to the fungal growth rate, three machine learning algorithms were evaluated as a data-driven soft sensor for predicting online viscosity across different strains and scales. The outcome is a hybrid model that requires less manual lab work for its development while predicting the dynamics of six pilot-scale fermentations under a variety of operating conditions with a modest improvement in accuracy. Results emphasize the importance of integrating online sensor technologies into mathematical model development and highlight their role in advancing data-driven methods. By lowering the costs and efforts of model development, this study contributes to the long-term vision of automated model development for industrial applications. One-sentence summary This study presents a novel hybrid modeling approach that minimizes experimental efforts by replacing offline viscosity measurements with online dynamic viscosity data to predict oxygen transfer for industrial fungal fermentation processes at a 550 L pilot scale, enabling more time, and cost-efficient bioprocess model development.
To realize the full potential of biomanufacturing, the breadth of industrial microbes used to consume diverse feedstock and generate bioproducts needs to expand. As such, portable tools are required that can be used by multiple hosts for straightforward genomic manipulation and precise gene expression. Here, we demonstrate the co-utilization of two synthetic biology tools to achieve these goals: cis-repressors (CRs) and serine recombinase-assisted genome engineering (SAGE). CRs are small, noncoding RNAs that are placed upstream of the target gene to modulate bacterial translation rates at varying, discrete levels. SAGE uses site-specific serine recombinases to catalyze highly efficient, unidirectional insertion of DNA into the chromosome of diverse organisms. We used SAGE to integrate a suite of CRs into the industrially relevant hosts Pseudomonas putida, Corynebacterium glutamicum, and Cupriavidus necator. Using a fluorescent reporter as a readout of CR functionality, we found that CR performance across these backgrounds was similar-providing a range of translational repression up to 100-fold. Overall, these results demonstrate the high portability of CRs across bacterial genetic backgrounds, which ideally can be used in future microbial engineering efforts pertinent to biomanufacturing. One-Sentence Summary: Fine tuning translation across phylogenetically diverse microorganisms using riboregulators and serine recombinase-assisted genome engineering.
Ornithine is a nonproteinogenic amino acid with various health benefits, making it a promising target for functional food development. In this study, we developed the yeast Saccharomyces cerevisiae strains with elevated intracellular ornithine and identified the genetic mutation responsible for this trait. An ornithine-rich mutant was successfully isolated by chemical mutagenesis and selection for resistance to canavanine, a toxic arginine analog. Whole-genome sequencing revealed a heterozygous point mutation in the ARG6 gene encoding N-acetylglutamate kinase, a key enzyme in ornithine biosynthesis. This mutation caused a Gly351Asp substitution located in a conserved linker region between functional domains. Introduction of this substitution, along with introducing alternative amino acids at the same site, consistently increased ornithine levels in various strain backgrounds. Structural modeling suggested that this substitution could affect the enzyme conformation or inter-domain interactions. These results establish a practical nongenetically modified breeding strategy for enhancing ornithine production in yeast, which will facilitate the development of ornithine-enriched fermented beverages such as craft beer.One-sentence summary The ARG6 mutant increases intracellular ornithine and enables value-added craft beer brewing.
Medium-chain alcohol dehydrogenases/reductases (MDRs) are crucial to the biosynthesis of plant monoterpene indole alkaloids (MIAs), yet studies have focused solely on their catalytic activities with strictosidine aglycone derivatives and NADPH as the redox cofactor. Here, we combined computational and experimental approaches to characterize the substrate and cofactor promiscuity of five MDRs recently identified in the medicinal plant kratom, including MsMDR4, MsMDR11, MsTHAS (tetrahydroalstonine synthase), MsDCS1 (dihydrocorynantheine synthase), and MsHYS (heteroyohimbine synthase). In vitro and yeast-based biochemical analyses showed that MsMDR4, MsMDR11, and MsTHAS catalyze the aldehyde reduction for cinnamaldehyde and 8-oxogeraniol, while MsMDR11 and MsTHAS also catalyze the alcohol oxidation for cinnamyl alcohol and 8-hydroxygeraniol. In particular, MsMDR11 exhibits 8-hydroxygeraniol oxidoreductase activity, producing 8-oxogeranial, an upstream intermediate in the strictosidine pathway. Additionally, all five enzymes can accept NADH/NAD + as redox cofactors, though with decreased productivity. Swapping the reducing cofactor from NADPH to NADH further alters the distribution of MIAs produced by MsDCS1 and MsHYS. Together, kratom MDRs exhibit great plasticity and potential for future enzyme engineering. One-sentence summary Computational and biochemical analyses revealed that previously identified kratom medium-chain dehydrogenase/reductases exhibit broad substrate and cofactor promiscuity.
Increasing interest in the bioeconomy has spurred the development of integrated methods to convert organic waste streams, particularly starch-rich substrates, into bioethanol. However, starch-based ethanol fermentations are vulnerable to bacterial contamination, particularly by lactic acid bacteria (LAB). Severe contamination can cause significant economic losses due to stuck fermentations and ethanol plant shutdowns. Although bacterial contamination can be managed with antibiotics, this approach is not cost-effective at an industrial scale and may increase the risk of selecting for antibiotic-resistant strains. Natural antimicrobial peptides (AMPs) can inhibit LAB contaminants in yeast fermentations, but commercial applications are limited by their low abundance and high production costs. Engineering Saccharomyces cerevisiae to produce recombinant AMPs might provide a cost-effective strategy to control LAB, thereby boosting ethanol yields during fermentation. Despite a comprehensive toolkit for gene expression in S. cerevisiae, only a few successful cases of bacteriocin expression have been reported. Since starch-to-ethanol fermentation is a key application for recombinant AMPs, this review explores strategies to optimize the expression of bacteriocin-encoding genes in S. cerevisiae. The ideal scenario would be a single yeast strain capable of producing amylases for starch hydrolysis, fermenting glucose to ethanol, and expressing bacteriocins to inhibit LAB contaminants. One-sentence summary: Yeast strains can produce heterologous antimicrobial peptides that help prevent contaminating bacteria from interfering with the starch-to-ethanol fermentation process.
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by various classes of bacteria, exhibiting broad-spectrum activity that makes them promising candidates for applications in food preservation and medicine. Their inherent stability under extreme pH, temperature, and salinity conditions further supports their functional versatility. However, the widespread industrial application of bacteriocins remains constrained by the low titres typically achieved during fermentation. Despite extensive efforts to optimize production using batch and fed-batch fermentation strategies, the resulting titres remain inadequate for economically viable large-scale manufacturing. This review aims to provide a comprehensive overview of novel process strategies developed over the past two decades to enhance bacteriocin yields during fermentation. One of the primary challenges is the inhibition of microbial growth due to the accumulation of lactic acid or the bacteriocin itself during production. To address this, in situ product removal techniques-such as co-cultivation with lactic acid-consuming microorganisms, in situ adsorption, filtration, and foam fractionation-have been explored, yielding notable improvements in bacteriocin titres. Additionally, stress-induced production strategies involving biological (e.g., co-culture with competing microbes), chemical (e.g., salinity and pH stress), and physical (e.g., agitation, temperature, and aeration stress) stimuli have also demonstrated success in enhancing bacteriocin synthesis. This review underscores the importance of these innovative fermentation approaches and highlights the need for further research focused on scaling up such processes. Advancing these strategies is critical to realizing the full potential of bacteriocins in food safety, antimicrobial therapy, and broader biomedical applications. One-sentence summary This review provides a comprehensive view of the novel fermentation strategies developed in the last two decades to overcome low bacteriocin titres during fermentation such as in situ lactate removal, in situ bacteriocin removal, and stress-led induction.
Post-harvest algae biomass is prone to degradation, resulting in mass loss and compositional changes, and preservation is vital to economic viability of algal products. Effective storage solutions are needed to mitigate for seasonal productivity variations (long-term storage) and to keep post-harvest biomass stable until processing. Ensiling has emerged as a long-term storage solution capable of preserving biomass up to 6 months with little loss without the energy demands of drying. Organic acids produced during ensiling lower biomass pH and prevent growth of degradative bacteria such as Clostridia. However, losses are front-loaded with a majority occurring within the first week before organic acids can accumulate. Currently, there is no information on the stability of algae biomass within the first 24 hr post-harvest or methods available to ensure stability during this period. Freshly harvested Tetradesmus obliquus UTEX 393 biomass was stored in three conditions: ambient atmosphere, anaerobic atmosphere without treatment, and anaerobic atmosphere with citric acid amendment. Citric acid treatment limited mass loss to 1% after 28 days, while untreated biomass experienced 4% mass loss after just 4 hr and 18% mass loss after 4 weeks. The carbohydrate fraction was most affected, with minimal changes to the elemental composition of biomass across treatments. Bacilli bacteria, including lactic acid bacteria, increased in abundance under all storage conditions. Untreated biomass showed a rise in Clostridia, but none were found in citric acid-treated biomass. After 28 days, organic acid composition differed significantly among treatments, with succinic acid being accumulated to 30% of dry cellular weight in citric acid treated UTEX 393 biomass. Citric acid treatment effectively mitigates biomass loss and, surprisingly, promotes substantial production of succinic acid. The unexpected autofermentation of UTEX 393 biomass to a versatile intermediate chemical such as succinic acid at high titers with minimal energy input could contribute to the economic viability of algae cultivation for fuels and chemicals. One-Sentence Summary Tetradesmus obliquus biomass is susceptible to degradation immediately after harvest; citric acid treatment preserves biomass while stimulating succinic acid accumulation.
In the present study, the economic evaluation of fermentation produced butyric acid (HBu) from corn was carried out (capacity 40 × 103 metric tons HBu∙yr-1) and two recovery processes (distillation and adsorption) were compared. Distillation is a longstanding process that has been effectively used to recover volatile chemicals. Recently, we developed an adsorption process that can be used to recover HBu cost competitively. The direct fixed capital (DFC) of the two processes (distillation & adsorption) were (in US dollars) $73.96 × 106 and $43.91 × 106, respectively. It is reported that the production of HBu from corn requires supplementing with amylase enzymes to the corn mash. For the two processes (distillation & adsorption) the annual operating costs were $37.67 × 106 and $31.83 × 106, respectively. The utilities annual costs for the two processes were $47.38 × 106 and $5.91 × 106, respectively. Corn price is one of the most important factors that influence HBu selling price, but other factors that could lower production costs include plant capacity. Factors such as tax on profit, interest rates on borrowed capital and plant life have marginal or low impact on HBu selling price. The significance of this paper is that HBu produced from corn and recovered by adsorption could be sold for $1.22 to 0.85∙kg-1. The selling price of fermentation produced HBu could further be reduced considerably if agricultural biomass such as wheat straw is used for production. One sentence summary Butyric acid production by fermentation and process economics.
Biomanufacturing can play a pivotal role in the transition away from fossil fuel dependence for the production of chemicals and fuels. There is growing interest in inexpensive alternative bioproduction feedstocks from renewable sources that avoid competing with food production for land use. Ethylene glycol (EG), a C2 compound that can be recovered from plastic waste or derived from carbon dioxide, is gaining attention as a carbon source for microbial processes. Here, we systematically evaluate natural and synthetic metabolic pathways for EG assimilation using theoretical modeling approaches. We analyzed five pathways for their maximum theoretical yields, thermodynamic favourability, enzyme costs, and orthogonality to cell growth and identify favourable traits for each of these pathways for a given product. Our results reveal distinct trade-offs between pathway types. Synthetic pathways achieved higher theoretical yields for biomass and most bioproducts, with synthetic glycolaldehyde assimilation (SAGA) pathways showing the best overall yields and the synthetic acetyl-CoA assimilation (SACA) pathway demonstrating the highest thermodynamic favourability and lowest enzyme costs. Among natural pathways, the glycerate pathway exhibited favourable thermodynamics and moderate enzyme costs comparable to synthetic alternatives, while being particularly advantageous for glycolate production despite carbon losses. The β-hydroxyaspartate cycle (BHAC) showed the poorest thermodynamic performance and highest enzyme burden. However, natural pathways exhibited equal or higher orthogonality to growth-associated reactions, making them potentially suitable for dynamically controlled production systems. These findings provide guidance for selecting optimal EG assimilation strategies based on target products and process requirements, supporting the development of sustainable bioprocesses utilizing this promising unconventional feedstock. One-sentence summary This article reviews and compares metabolic pathways for the utilization of the compound EG in the context of bioproduction.
The dynamics and impact of microbial contaminants in industrial sugarcane bioethanol production in Brazil were investigated through a two-year metagenomic study across two biorefineries. Shotgun metagenomic sequencing revealed that temporal shifts in the contaminant microbiome dynamics within production seasons were more pronounced than inter-annual or inter-mill variations. While Saccharomyces spp. dominated, bacterial communities, primarily within the Firmicutes phylum and dominated by the genera Lactobacillus , Limosilactobacillus , and Bacillus , exhibited dynamic changes. Correlation analyses with industrial process parameters revealed a complex interplay: lower Lactobacillus levels in one mill were associated with increased ethanol yield, whereas higher levels in another mill correlated with reduced yeast viability and increased flocculation. The presence of Limosilactobacillus was linked to decreased yeast viability, whereas Bacillus showed potential for inhibiting both Lactobacillus and Limosilactobacillus . These findings highlight the nuanced and species-specific impacts of bacterial contaminants on bioethanol production, underscoring the need for strain-level functional studies and targeted interventions to optimize fermentation efficiency and stability in industrial settings.
Designer cellulosomes (DCs) are engineered multienzyme complexes inspired by natural cellulosomes, designed to improve lignocellulose breakdown. Their modular architecture enables the spatial colocalization of diverse catalytic activities, potentially enhancing depolymerization efficiency compared to free enzymes. Although conceptually promising, little is known about how they perform on complex lignocellulosic substrates. In this study, we developed a tetravalent DC using a modular VersaTile assembly approach, incorporating endoglucanase, cellobiohydrolase, β-glucosidase, and endoxylanase activities. The process involved (i) delineating catalytic modules from Cellvibrio japonicus enzymes, (ii) generating docking enzyme variants via combinatorial cloning, and (iii) selecting optimal candidates based on expression, activity, and cohesin-dockerin binding before assembling them onto a scaffoldin with four cohesins and a cellulose-binding module. The resulting DC was tested on two industrially relevant substrates: agro-industrial wheat fibers and genome-edited low-lignin poplar biomass under controlled laboratory conditions. It achieved cellulose-to-glucose conversion yields of 24.98% (150 pmol DC/ml) and 0.82% (200 pmol DC/ml), respectively, under the test conditions. By comparing the saccharification efficiencies of the enzymes in their free and complexed forms, we found that colocalization on a common scaffoldin significantly enhanced synergistic activity. This effect was most pronounced under low enzyme concentrations and when acting on complex lignocellulosic substrates, increasing glucose release compared to free enzymes. These observations highlight that the benefits of colocalization are substrate-dependent and occur under conditions that mimic the natural environment of biomass degradation, conditions that differ from typical industrial settings. This work advances our understanding of DC behavior on real-world substrates, providing essential insights for evaluating their economic viability in industrial applications. One-sentence summary Natural-like conditions helped customized DC release more sugars from biomass than standard industrial setups.
Microbial synthesis of carotenoids has garnered significant attention as an eco-friendly alternative to conventional synthetic methods and its facile extraction for impressive yield. This study delves into the efficacy of carotenoid production from a red yeast strain, as well as the biodiversity of yeast species from Thai flowers. The research involved the collection of flower samples within Thailand, along with 12 yeast species from 10 genera of Ascomycetes and 4 genera of Basidiomycetes which were isolated by identifying the D1/D2 domain of the large subunit rRNA gene. Unexpectedly, Rhodotorula paludigena SWU-FKT03 emerged as the top performing yeast strain, boasting an impressive carotenoid production rate of 183.30 ± 5.00 mg/L among the 36 red yeast strains isolated. Subsequently, a further investigation was performed, focusing on optimized culture conditions for carotenoid production from this yeast strain. The results were promising, as carotenoid production surged to 288.27 mg/L when 20 g/L of glucose and 10 g/L of monosodium glutamate served as the carbon and nitrogen sources, respectively. These findings underscore the potential of the R. paludigena SWU-FKT03 as a high-yield carotenoid producer when cultivated in shaking flasks, exhibiting a three-fold increase in carotenoid content when under optimized conditions. These results hint at the potential of this approach for future large-scale carotenoid production. One-sentence summary A novel red yeast, Rhodotorula paludigena SWU-FKT03, isolated from Thai floral ecosystems, demonstrated high-yield carotenoid production of 288.27 mg/L after fermentation optimization, establishing a significant potential for industrial applications.
Successful design of an industrial biological product such as a vaccine requires an efficient, robust and reproducible process. In this study, we investigated conditions for process optimization of a promising recombinant factor H binding protein (fHbp)-Porin A (PorA) chimeric protein-based vaccine candidate against Meningococcal B serogroup in Escherichia coli B834 strain using the inducible T7-lac promoter system. Random screening of components of complex culture media for growth and expression using IPTG as an inducer, showed inconsistency when analyzed using Plackett-Burman design (PBD). Further analysis identified galactose present either in tryptone (as lactose) or soy-phytone, has caused strong autoinduction and is surmised as an interfering factor for IPTG induction. Synergistic combination of soy-phytone and yeast extract under conditions of controlled growth and auto-induction gave a good correlation between the cell growth and expression of the fHbp-PorA chimeric protein when evaluated using PBD and central composite design. Using response optimization conditions, an optimized media was attained. A shake flask study was performed to validate the optimized media, and later, a fed-batch fermentation at 10 L scale was established to prove the scalability, consistency, and product quality using the optimized media. One-sentence summary This study reports optimization of cell growth and expression conditions for a recombinant chimeric meningococcal protein in E. coli that assures its industrial scale production and suitability for preclinical and clinical studies as a vaccine component against Meningococcal B serogroup.
Here, we present the first complete, fully phased diploid genome of type strain Yarrowia lipolytica YB-423 (=ATCC 18942™), constructed using a combination of Oxford Nanopore long-read and Illumina short-read sequencing. Yarrowia lipolytica is an industrially relevant yeast species known for its metabolic versatility, particularly its ability to degrade hydrophobic compounds and express useful products such as fatty acids. Despite its growing use in biotechnology, a high-quality genome assembly of the species' diploid type-strain has been lacking. The assembly and annotations presented here span six chromosomes of paired "haplotigs" and a mitochondrial genome, capturing large-scale structural variations and prominent levels of genome-wide heterozygosity. Variant analysis revealed 13,908 heterozygous alleles, of which 3,201 alleles were distributed among 1,237 protein-coding genes. Gene set enrichment analysis showed that these variants are enriched among genes involved in transmembrane transport, suggesting a role in environmental adaptability. Comparative analysis of matched haplotigs for the same chromosome uncovered multiple inversions and transpositions, as well as allele-specific insertions of retrotransposons, providing new insights into the structural complexity and evolutionary dynamics of the genome. The fully phased, finished diploid genome of Y. lipolytica YB-423 represents a crucial step toward unlocking the full genetic potential of Y. lipolytica. Our work will provide a valuable foundation for future comparative and functional genomics and strain engineering studies, particularly for industrial microbiology and biotechnology applications. One-sentence summary This study presents the first fully phased diploid genome of Yarrowia lipolytica type strain YB-423, revealing extensive structural variation and heterozygosity that enhance understanding of its genetic adaptability and industrial potential.
