Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells.

Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 µg/ml) and (14.80 ± 0.13 µg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.

Harnessing Raman spectroscopy for cell therapy bioprocessing.

Cell therapy manufacturing requires precise monitoring of critical parameters to ensure product quality, consistency and to facilitate the implementation of cost-effective processes. While conventional analytical methods offer limited real-time insights, integration of process analytical technology tools such as Raman spectroscopy in bioprocessing has the potential to drive efficiency and reliability during the manufacture of cell-based therapies while meeting stringent regulatory requirements. The non-destructive nature of Raman spectroscopy, combined with its ability to be integrated on-line with scalable platforms, allows for continuous data acquisition, enabling real-time correlations between process parameters and critical quality attributes. Herein, we review the role of Raman spectroscopy in cell therapy bioprocessing and discuss how simultaneous measurement of distinct parameters and attributes, such as cell density, viability, metabolites and cell identity biomarkers can streamline on-line monitoring and facilitate adaptive process control. This, in turn, enhances productivity and mitigates process-related risks. We focus on recent advances integrating Raman spectroscopy across various manufacturing stages, from optimizing culture media feeds to monitoring bioprocess dynamics, covering downstream applications such as detection of co-isolated contaminating cells, cryopreservation, and quality control of the drug product. Finally, we discuss the potential of Raman spectroscopy to revolutionize current practices and accelerate the development of advanced therapy medicinal products.

Management of experimental workflows in robotic cultivation platforms.

In the last decades, robotic cultivation facilities combined with automated execution of workflows have drastically increased the speed of research in biotechnology. In this work, we present the design and deployment of a digital infrastructure for robotic cultivation platforms. We implement a workflow management system, using Directed Acyclic Graphs, based on the open-source platform Apache Airflow to increase traceability and the automated execution of experiments. We demonstrate the integration and automation of experimental workflows in a laboratory environment with a heterogeneous device landscape including liquid handling stations, parallel cultivation systems, and mobile robots. The feasibility of our approach is assessed in parallel E. coli fed-batch cultivations with glucose oscillations in which different elastin-like proteins are produced. We show that the use of workflow management systems in robotic cultivation platforms increases automation, robustness and traceability of experimental data.

Valorization of Strawberry Juice Production Wastewater: Possibilities for Polyphenols Recovery and Plant Biostimulant Production.

Fruit juice production is one of the most important branches of the food and beverage industry, considering both the market size and demand. It is also one of the largest generators of industrial wastewater, considering the large consumption of fresh water during fruit processing. Hence, the appropriate treatment strategies are of the utmost importance to minimize the environmental footprint of food industry effluents. This study aimed to investigate the valorization routes for strawberry juice production wastewater (SJPW), both in terms of nutrient recovery and a circular approach to its utilization as a medium for plant biostimulant production. The results show a low antioxidant capacity and low content of polyphenols in SJPW; however, promising results were obtained for the in vitro seed germination and tomato growth promotion when investigating a biostimulant based on Bacillus sp. BioSol021, which was cultivated using SJPW in a lab-scale bioreactor, with root and shoot length improvements of approximately 30% and 25%, respectively, compared to the control samples. The plant growth promotion (PGP) traits indicated the ability of IAA production, in a concentration of 8.55 ± 0.05 mg/L, and the enzymatic activity was evaluated as through the enzymatic activity index (EAI), achieving the following: 2.26 ± 0.04 for cellulolytic activity, 2.49 ± 0.08 for hemicellulolytic activity, 2.91 ± 0.16 for pectinolytic activity, and 1.05 ± 0.00 for proteolytic activity. This study opens a new chapter of possibilities for the development of techno-economically viable circular bioprocess solutions aimed at obtaining value-added microbial products for sustainable agriculture based on the valorization of food industry effluents thus contributing to more sustainable food production at both the agricultural and industrial levels.

Exploiting phenotypic heterogeneity to improve production of glutathione by yeast.

BACKGROUND: Gene expression noise (variation in gene expression among individual cells of a genetically uniform cell population) can result in heterogenous metabolite production by industrial microorganisms, with cultures containing both low- and high-producing cells. The presence of low-producing individuals may be a factor limiting the potential for high yields. This study tested the hypothesis that low-producing variants in yeast cell populations can be continuously counter-selected, to increase net production of glutathione (GSH) as an exemplar product. RESULTS: A counter-selection system was engineered in Saccharomyces cerevisiae based on the known feedback inhibition of gamma-glutamylcysteine synthetase (GSH1) gene expression, which is rate limiting for GSH synthesis: the GSH1 ORF and the counter-selectable marker GAP1 were expressed under control of the TEF1 and GSH-regulated GSH1 promoters, respectively. An 18% increase in the mean cellular GSH level was achieved in cultures of the engineered strain supplemented with D-histidine to counter-select cells with high GAP1 expression (i.e. low GSH-producing cells). The phenotype was non-heritable and did not arise from a generic response to D-histidine, unlike that with certain other test-constructs prepared with alternative markers. CONCLUSIONS: The results corroborate that the system developed here improves GSH production by targeting low-producing cells. This supports the potential for exploiting end-product/promoter interactions to enrich high-producing cells in phenotypically heterogeneous populations, in order to improve metabolite production by yeast.

Optimization, characterization and biosafety of carotenoids produced from whey using Micrococcus luteus.

This study aimed to optimize the production of carotenoid pigments from Micrococcus luteus (ATCC 9341) through the statistical screening of media components and the characterization of antimicrobial, antioxidant, cytogenetic and cytotoxic activities. A BOX-Behnken design was used to assess the effects of whey concentration, inoculum size, pH, temperature, and agitation speed on carotenoid yield. The optimum combination increased production to 2.19 g/L, with a productivity of 0.045 g L-1 h-1 and a productivity yield of 0.644 g/g, as confirmed by an observed carotene production of 2.19 g/L. The final response surface model fitting the data had an R2 of 0.9461. High-performance liquid chromatography (HPLC) analysis identified 12 carotenoid pigment compounds produced by M. luteus. The extracts displayed moderate antimicrobial efficacy against Gram-positive bacteria such as Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538), and E. faecalis (ATCC 19433), with inhibition zone diameters (IZD) of 29.0, 14.0, and 37.0 mm, respectively, at 1000 µg/mL. However, its effectiveness against Gram-negative bacteria is limited. In comparison, tetracycline exhibited greater antimicrobial potency. The IC50 value of carotenoids was used to indicate the antioxidant activity. IC50 value from the DPPH assay was 152.80 mg/100mL. An IC50 cytotoxicity value greater than 300 µg/mL was found against normal mouse liver cells, with over 68% cell viability even at 300 µg/mL, indicating low toxicity. Histological structure studies revealed normal myocardial muscle tissue, lung tissue, and kidney tissue sections, whereas liver tissue sections revealed ballooning degeneration of hepatocytes and disorganization of hepatic cords. Cytogenetic parameters revealed that the carotene treatment group had a mitotic index (70%) lower than that of the control but higher than that of the positive control, mitomycin, and did not substantially increase numerical (1.2%) or structural aberrations compared with those of the control, suggesting a lack of genotoxic effects under the experimental conditions. In conclusion, optimized culture conditions enhanced carotenoid yields from M. luteus, and the extracts displayed promising bioactivity as moderate antibiotics against certain gram-positive bacteria and as antioxidants. The high IC50 values demonstrate biosafety. Overall, this bioprocess for enhanced carotenoid production coupled with bioactivity profiling and low cytotoxicity support the application of M. luteus carotenoids.

Inline Raman spectroscopy as process analytical technology for SARS-CoV-2 VLP production.

The present work focused on inline Raman spectroscopy monitoring of SARS-CoV-2 VLP production using two culture media by fitting chemometric models for biochemical parameters (viable cell density, cell viability, glucose, lactate, glutamine, glutamate, ammonium, and viral titer). For that purpose, linear, partial least square (PLS), and nonlinear approaches, artificial neural network (ANN), were used as correlation techniques to build the models for each variable. ANN approach resulted in better fitting for most parameters, except for viable cell density and glucose, whose PLS presented more suitable models. Both were statistically similar for ammonium. The mean absolute error of the best models, within the quantified value range for viable cell density (375,000-1,287,500 cell/mL), cell viability (29.76-100.00%), glucose (8.700-10.500 g/), lactate (0.019-0.400 g/L), glutamine (0.925-1.520 g/L), glutamate (0.552-1.610 g/L), viral titer (no virus quantified-7.505 log10 PFU/mL) and ammonium (0.0074-0.0478 g/L) were, respectively, 41,533 ± 45,273 cell/mL (PLS), 1.63 ± 1.54% (ANN), 0.058 ± 0.065 g/L (PLS), 0.007 ± 0.007 g/L (ANN), 0.007 ± 0.006 g/L (ANN), 0.006 ± 0.006 g/L (ANN), 0.211 ± 0.221 log10 PFU/mL (ANN), and 0.0026 ± 0.0026 g/L (PLS) or 0.0027 ± 0.0034 g/L (ANN). The correlation accuracy, errors, and best models obtained are in accord with studies, both online and offline approaches while using the same insect cell/baculovirus expression system or different cell host. Besides, the biochemical tracking throughout bioreactor runs using the models showed suitable profiles, even using two different culture media.

Exploring the potential of biosurfactants produced by fungi found in soil contaminated with petrochemical wastes.

Biosurfactants are a diverse group of compounds derived from microorganisms, possessing various structures and applications. The current study was seeking to isolate and identify a new biosurfactant-producing fungus from soil contaminated with petrochemical waste. The bioprocess conditions were optimized to maximize biosurfactant production for Aspergillus carneus OQ152507 using a glucose peptone culture medium with a pH of 7.0 and a temperature of 35 °C. The carbon source was glucose (3%), and ammonium sulfate (0.25%) was utilized as the nitrogen source. For Aspergillus niger OQ195934, the optimized conditions involved a starch nitrate culture medium with a pH of 7.0 and a temperature of 30 °C. The carbon source used was sucrose (3.5%), and ammonium sulfate (0.25%) served as the nitrogen source. The phenol-H2SO4 and phosphate tests showed that the biosurfactants that were extracted did contain glycolipid and/or phospholipid molecules. They showed considerable antimicrobial activity against certain microbes. The obtained biosurfactants increased the solubility of tested polyaromatic hydrocarbons, including fluoranthene, pyrene, anthracene, and fluorine, and successfully removed the lubricating oil from contaminated soil and aqueous media surface tension reduction. Based on the obtained results, A. carneus and A. niger biosurfactants could be potential candidates for environmental oil remediation processes.

Monitoring small-scale bioreactor studies for media development using polarized total synchronous fluorescence spectroscopy (pTSFS) and synchronous light scattering (SyLS).

Biopharmaceutical process development often involves the use of small-scale bioreactors (SSBR) for optimizing media formulations and process conditions during scale up to commercial scale production. Two key process parameters (CPP) used in SSBR studies are protein titre and viable cell density (VCD). Here, we explore the efficacy of parallel polarized total synchronous fluorescence spectroscopy (TSFS||) and Synchronous Light Scattering (SyLS||) to qualitatively monitor these CPPs and quantitatively predict titre and VCD for a large-scale cell culture media optimization SSBR study. The study involved 71 different media formulations (50+ components each), and the bioprocess was run for 13 days or more. Samples were extracted at set times (Day 0, 3, 9, and 13) and clarified by centrifugation. TSFS|| spectra showed significant emission changes along with increased light scatter over the course of the bioprocess. SyLS|| measurements strongly correlated with particle size data obtained from Dynamic Light Scattering but did not correlate well with VCD probably because of the centrifugation-based sample preparation. Statistical and principal component analysis (PCA) of the pTSFS data showed that spectral variation was greater between media formulations than due to the evolving bioprocess. This prevented the development of accurate global prediction models for media performance (e.g., predicting product titre at day 9 from media spectra measured at day 0). However, classification methods were successfully used to select media subsets with better quantitative prediction accuracy based on spectral similarities. A practical binary (high/low performance) classification model based on Support Vector Machines was generated for media formulation screening. Combining emission and scatter measurements with multivariate data analysis provides a more holistic, multi-attribute bioprocess monitoring method that minimizes the need to use different offline analytical methods. This methodology can be used to monitor process trajectories and deviations, and ultimately be used to predict bioprocess CPPs when implemented on production scale processes where there is much less compositional variation in the media. We believe this SSBR-pTSFS/SyLS approach will provide a valuable resource to develop the design/parameter space for in-process monitoring at production scale from early-stage process/media development studies.

Elevated endoplasmic reticulum pH is associated with high growth and bisAb aggregation in CHO cells.

Chinese hamster ovary (CHO) bioprocesses, the dominant platform for therapeutic protein production, are increasingly used to produce complex multispecific proteins. Product quantity and quality are affected by intracellular conditions, but these are challenging to measure and often overlooked during process optimization studies. pH is known to impact quality attributes like protein aggregation across upstream and downstream processes, yet the effects of intracellular pH on cell culture performance are largely unknown. Recently, advances in protein biosensors have enabled investigations of intracellular environments with high spatiotemporal resolution. In this study, we integrated a fluorescent pH-sensitive biosensor into a bispecifc (bisAb)-producing cell line to investigate changes in endoplasmic reticulum pH (pHER). We then investigated how changes in lactate metabolism impacted pHER, cellular redox, and product quality in fed-batch and perfusion bioreactors. Our data show pHER rapidly increased during exponential growth to a maximum of pH 7.7, followed by a sharp drop in the stationary phase in all perfusion and fed-batch conditions. pHER decline in the stationary phase was driven by an apparent loss of cellular pH regulation that occurred despite differences in redox profiles. Finally, we found protein aggregate levels correlated most closely with pHER which provides new insights into product aggregate formation in CHO processes. An improved understanding of the intracellular changes impacting bioprocesses can ultimately help guide media optimizations, improve bioprocess control strategies, or provide new targets for cell engineering.

Chemometrics and analytical blank on the at-line monitoring of Zika-VLP production using near-infrared spectroscopy.

The Zika disease caused by the Zika virus was declared a Public Health Emergency by the World Health Union (WHO), with microcephaly as the most critical consequence. Aiming to reduce the spread of the virus, biopharmaceutical organizations invest in vaccine research and production, based on multiple platforms. A crescent vaccine production approach is based on virus-like particles (VLP), for not having genetic material in its composition, hypoallergenic and non-mutant character. For bioprocess, it is essential to have means of real-time monitoring, which can be assessed using process analysis techniques such as Near-infrared (NIR) spectroscopy, that can be combined with chemometric methods, like Partial-Least Squares (PLS) and Artificial Neural Networks (ANN) for prediction of biochemical variables. This work proposes a biochemical Zika VLP upstream production at-line monitoring model using NIR spectroscopy comparing sampling conditions (with or without cells), analytical blank (air, ultrapure water), and spectra pre-processing approaches. Seven experiments in a benchtop bioreactor using recombinant baculovirus/Sf9 insect cell platform in serum-free medium were performed to obtain biochemical and spectral data for chemometrics modeling (PLS and ANN), composed by a random data split (80 % calibration, 20 % validation) for cross-validation of the PLS models and 70 % training, 15 % testing, 15 % validation for ANN. The best models generated in the present work presented an average absolute error of 1.59 × 105 cell/mL for density of viable cells, 2.37 % for cell viability, 0.25 g/L for glucose, 0.007 g/L for lactate, 0.138 g/L for glutamine, 0.18 g/L for glutamate, 0,003 g/L for ammonium, and 0.014 g/L for potassium.

Bioconversion of L-Tyrosine into p-Coumaric Acid by Tyrosine Ammonia-Lyase Heterologue of Rhodobacter sphaeroides Produced in Pseudomonas putida KT2440.

p-Coumaric acid (p-CA) is a valuable compound with applications in food additives, cosmetics, and pharmaceuticals. However, traditional production methods are often inefficient and unsustainable. This study focuses on enhancing p-CA production efficiency through the heterologous expression of tyrosine ammonia-lyase (TAL) from Rhodobacter sphaeroides in Pseudomonas putida KT2440. TAL catalyzes the conversion of L-tyrosine into p-CA and ammonia. We engineered P. putida KT2440 to express TAL in a fed-batch fermentation system. Our results demonstrate the following: (i) successful integration of the TAL gene into P. putida KT2440 and (ii) efficient bioconversion of L-tyrosine into p-CA (1381 mg/L) by implementing a pH shift from 7.0 to 8.5 during fed-batch fermentation. This approach highlights the viability of P. putida KT2440 as a host for TAL expression and the successful coupling of fermentation with the pH-shift-mediated bioconversion of L-tyrosine. Our findings underscore the potential of genetically modified P. putida for sustainable p-CA production and encourage further research to optimize bioconversion steps and fermentation conditions.

Modulating antibody N-glycosylation through feed additives using a multi-tiered approach.

Glycosylation of recombinant proteins is a post-translational modification that affects multiple physicochemical and biological properties of proteins. As such, it is a critical quality attribute that must be carefully controlled during protein production in the pharmaceutical industry. Glycosylation can be modulated by various conditions, including the composition of production media and feeds. In this study, the N-glycosylation-modulating effects of numerous compounds, including metal enzyme cofactors, enzyme inhibitors, and metabolic intermediates, were evaluated. Chinese hamster ovary cells producing three different IgG antibodies were cultivated in a fed-batch mode. First, a one-factor-at-a-time experiment was performed in 24-well deep well plates to identify the strongest modulators and appropriate concentration ranges. Then, a full response surface experiment was designed to gauge the effects and interactions of the 14 most effective hit compounds in an Ambr® 15 bioreactor system. A wide range of glycoform content was achieved, with an up to eight-fold increase in individual glycoforms compared to controls. The resulting model can be used to determine modulator combinations that will yield desired glycoforms in the final product.

Optimization of exopolysaccharide production from the novel Enterococcus species, using statistical design of experiment.

The Exopolysaccharide (EPS) producing novel strains of Enterococcus previously isolated from the vaginal source of pregnant women were selected based on ropy structure formation. The two selected strains, E.villorum SB-2 and E.rivorum S22-3, were found to be producing 2.87 g/l and 3.14 g/l EPS, respectively, in the minimal media (M17 media) after 24-hour fermentation under anaerobic condition. Both the strains have probiotic properties and have the potential to be used for industrial applications. The production media and fermentation conditions were optimized to enhance the EPS production using the one-factor method, Placket-Burman factorial designing and Central composite design (CCD) of Response surface methodology (RSM). The most relevant factors affecting the EPS yield were sucrose, yeast extract and pH for E.villorum SB2 and sucrose, yeast extract and magnesium sulfate for the E.rivorum S22-3 as determined by Placket-Burman design, whose concentrations were further optimized using CCD. The optimized fermentation conditions gave the total EPS of 9.76 g/l (4 times the initial production) from E.villorum SB-2 and 7.74 g/l (2.5 times the initial production) from E.rivorum S22-3, respectively, after 36-hour incubation at 37 °C. These optimization studies might be helpful during scale-up process for the industrial scale production of these exopolysaccharide.

Bioprocesses for lactic acid production from organic wastes toward industrialization-a critical review.

Lactic acid (LA) is a crucial chemical which has been widely used for industrial application. Microbial fermentation is the dominant pathway for LA production and has been regarded as the promising technology. In recent years, many studies on LA production from various organic wastes have been published, which provided alternative ways to reduce the LA production cost, and further recycle organic wastes. However, few researchers focused on industrial application of this technology due to the knowledge gap and some uncertainties. In this review, the recent advances, basic knowledge and limitations of LA fermentation from organic wastes are discussed, the challenges and suitable envisaged solutions for enhancing LA yield and productivity are provided to realize industrial application of this technology, and also some perspectives are given to further valorize the LA fermentation processes from organic wastes. This review can be a useful guidance for industrial LA production from organic wastes on a sustainable view.

The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes.

BACKGROUND: The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled. RESULTS: Using different growth rates in the range of 0.075 and 0.4 h-1, highest surfactin titres of 36 g/L were reached at 0.25 h-1 with production yields YP/S of 0.21 g/g and YP/X of 0.7 g/g, while growth rates lower than 0.2 h-1 resulted in insufficient and slowed biomass formation as well as surfactin production (YP/S of 0.11 g/g and YP/X of 0.47 g/g for 0.075 h-1). In contrast, feeding rates higher than 0.25 h-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1. CONCLUSIONS: Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates.

Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process.

Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearized plasmid DNA. Then, to develop EGFP- and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single-cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVVs that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies.

Investigating biological mechanisms of colour changes in sustainable food systems: The role of Starmerella bacillaris in white wine colouration using a combination of genomic and biostatistics strategies.

This study explores the biological mechanisms behind colour changes in white wine fermentation using different strains of Starmerella bacillaris. We combined food engineering, genomics, machine learning, and physicochemical analyses to examine interactions between S. bacillaris and Saccharomyces cerevisiae. Significant differences in total polyphenol content were observed, with S. bacillaris fermentation yielding 6 % higher polyphenol content compared to S. cerevisiae EC1118. Genomic analysis identified 12 genes in S. bacillaris with high variant counts that could impact phenotypic properties related to wine color. Notably, SNP analysis revealed numerous missense and synonymous variants, as well as stop-gained and start-lost variants between PAS13 and FRI751, suggesting changes in metabolic pathways affecting pigment production. Besides that, high upstream gene variants in SSK1 and HIP1R indicated potential regulatory changes influencing gene expression. Fermentation trials revealed FRI751 consistently showed high antioxidant activity and polyphenol content (Total Polyphenol: 299.33 ± 3.51 mg GAE/L, DPPH: 1.09 ± 0.01 mmol TE/L, FRAP: 0.95 ± 0.02 mmol TE/L). PAS13 exhibited a balanced profile, while EC1118 had lower values, indicating moderate antioxidant activity. The Weibull model effectively captured nitrogen consumption dynamics, with EC1118 serving as a reliable benchmark. The scale parameter delta for EC1118 was 23.04 ± 2.63, indicating moderate variability in event times. These findings highlight S. bacillaris as a valuable component in sustainable winemaking, offering an alternative to chemical additives for maintaining wine quality and enhancing colours profiles. This study provides insights into the biotechnological and fermented food systems applications of yeast strains in improving food sustainability and supply chain, opening new avenues in food engineering and microbiology.

Cultivation optimization promotes ginsenoside and universal triterpenoid production by engineered yeast.

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.