Applied Microbiology and Biotechnology

Probiotic-based encapsulation offers unique advantages over purified enzymes, such as increased protection from thermal-, pH-, and protease-mediated degradation, for oral therapeutic delivery applications. However, one of the major disadvantages of whole-cell systems is lower reaction rate due to substrate-product transport limitations imposed by the cell membrane and/or wall. In this work, we explore the potential of different lactic acid bacteria (LAB) - Lacticaseibacillus rhamnosus GG (LGG), Lactococcus lactis (Ll), and Lactiplantibacillus plantarum (Lp) - as expression hosts for recombinant Anabaena variabilis phenylalanine ammonia-lyase (AvPAL*). AvPAL* is used as a therapeutic to treat Phenylketonuria (PKU), a rare autosomal recessive metabolic disorder. Among the three species tested, LGG showed the highest PAL activity followed by L. lactis. Next, we attempted to overcome mass transfer limitation in whole-cell biocatalysts in two ways - expression of heterologous transporters and treatment with different chemical surfactants. Engineered strains expressing heterologous transporters exhibited approximately 3-4-fold increased PAL activity, while chemical treatment did not improve reaction rates. This work highlights the challenges and advances in realizing the potential of LAB as biotherapeutics. Impact StatementOral delivery of phenylalanine ammonia-lyase (PAL) using engineered probiotics is a promising therapeutic strategy to treat Phenylketonuria (PKU). Although PAL expression has been reported in probiotic strains of Limosilactobacillus reuteri, Lactococcus lactis, and E. coli, a systematic comparison of lactic acid bacteria (LAB) is underexplored. This study explores the potential of multiple LAB as hosts for PAL expression and investigates strategies to improve whole cell enzymatic activity. The findings from this study provide a foundation for implementing LAB-based delivery of PAL and indicate an important step towards development of probiotic platform for PKU management.

Fermentation of C1 gases is an emerging technology where waste gases are bio converted into value-added products. This study navigates the gas fermentation potential of Gordonia rubripertincta to produce carotenoids. The crucial carbon monoxide dehydrogenase (CODH) enzyme, necessary for gas uptake by the microbe, was found to be present in G. rubripertincta through blastp on NCBI website. The organism was then used for gas fermentation experiments in a continuous stirred tank reactor (CSTR) in different modes of reactor operation resulting in the production of about 500 mg pigment/g WCW (wet cell weight). Two important reactor parameters, molybdenum content and pH, were optimized for enhanced carotenoid production. Overall, G. rubripertincta was observed to be an efficient candidate organism for C1 gas fermentation. KEY HIGHLIGHTSO_LIGordonia rubripertincta synthesises aerobic carbon monoxide dehydrogenase enzyme. C_LIO_LIIt is a potential gas fermenting microbe that gives carotenoids as product. C_LIO_LIThe gas uptake efficiency of the microbe is more in fed-batch discontinued mode. C_LIO_LIIn FB-D, the resultant carotenoids are 500+9 mg/g wet cell weight (WCW). C_LIO_LIMo/pH of 20 mg/7.0 resulted in highest carotenoids, i.e., 134+41 mg/g WCW. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/722808v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8b1185org.highwire.dtl.DTLVardef@2b6f90org.highwire.dtl.DTLVardef@1a9697dorg.highwire.dtl.DTLVardef@14c9dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

As the United Kingdom (UK) targets net-zero emissions by 2050, anaerobic digestion (AD) has become a cornerstone of renewable energy infrastructure. However, mathematical models, such as the Anaerobic Digestion Model No. 1 (ADM1), often struggle with high-solids agricultural feedstocks because they rely on Chemical Oxygen Demand (COD), a metric that introduces significant experimental error. To overcome this, this study applies an established mass-based ADM1 framework tailored for the co-digestion of maize silage and cow manure sourced from a UK AD site. This study uses a parallel reactor framework, using two identical laboratory-scale reactors to physically replicate the dynamic conditions of the full-scale site. A Global Sensitivity Analysis was first conducted, identifying biomass decay and carbohydrate breakdown rates as the most influential factors affecting system stability and model accuracy. The model was calibrated using data from the first reactor and then tested against an independent second reactor subjected to significant organic loading stress. Results show high predictive capabilities, with the model achieving a R2 of 0.81 for biogas production during calibration. The model maintained high predictive accuracy during the validation test of the second physical twin, achieving an R2 of 0.85, proving that the framework is robust and not overfitted to a single dataset. While predicting rapid fluctuations in pH and alkalinity remains challenging, the mass-based approach effectively forecasts gas yields and process stability. This methodology provides a reliable foundation for robust process modelling, offering a scalable tool for the UK biogas sector to optimise AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/721061v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@92c7e2org.highwire.dtl.DTLVardef@80d723org.highwire.dtl.DTLVardef@ac3d24org.highwire.dtl.DTLVardef@1e21a51_HPS_FORMAT_FIGEXP M_FIG C_FIG

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development. Key pointsO_LIS. enterica can produce 1,3-propanediol from crude glycerol alone C_LIO_LIGlycerol-to-1,3-propanediol conversion is dependent on expression of the propanediol utilization (Pdu) pathway C_LIO_LISub-saturating concentrations of exogenous vitamin B12 can boost cell growth and 1,3-propanediol yield C_LI

Biological sequences are known to be not random. Thus, the comparison of in silico restriction fragment distributions of random and biological sequences may be an indicator of this non-randomness. Our analyses show that for most of the tested combinations of restriction enzyme and genome sequence the fragments per Megabase of the biological sequence deviate at least more then 10% from the corresponding random sequence. This deviation goes into both directions, i.e. clearly increased values are as common as clearly decreased values. Although there is no species- or restriction-enzyme-specific effect, a clear impact of the GC content both of the restriction site and of the genome sequence can be seen. In contrast to the random sequences, the genome sequences show distinct peaks in their fragment length distributions, hinting to repetitive elements such as transposons.

IntroductionThe use of Saccharomyces cerevisiae to ferment alcoholic beverages is an ancient tradition, with genetic evidence indicating origins in Neolithic Asia, although the domestication process of the species is not fully understood. Kveik is a group of traditional yeasts used in farmhouse brewing in western Norway preserved through generations of rural brewing practice. While recent studies have highlighted the distinctiveness of kveik, its precise phylogenetic position, genetic diversity, and domestication history remain unclear. ResultsWe performed whole-genome sequencing on 62 samples representing 25 unique Norwegian strains selected using cultural heritage criteria, and generated telomere-to-telomere (T2T) assemblies for representative isolates. Phylogenomic and population genetic analyses reveal that kveik forms a paraphyletic and early diverging group with respect to other domesticated S. cerevisiae strains. Most strains exhibit low within-strain diversity, strong geographic clustering, and little evidence of gene-flow or admixture. Mitochondrial genomes and Ty1 retrotransposon profiles corroborate this distinct lineage history. We further show that previously reported signals of gene flow between kveik and Asian fermentation strains are likely artifacts caused by population structure and selection. Divergence time estimates suggest that the common ancestor of beer, kveik, and other liquid-phase fermenting strains originated from ancestral populations 4,000 to 8,000 years ago. ConclusionKveik yeasts represent a relic of early S. cerevisiae domestication, shaped by ancient human practices, migrations, and the spread of agriculture. Our genomic resource sheds light on yeast evolution and domestication. They likely comprise some of the oldest domesticated lineages in continuous use until today, connecting endangered intangible cultural heritage to an early genetic origin.

Yarrowia lipolytica is a yeast of industrial interest exhibiting remarkable lipolytic and proteolytic capacities, with a high potential for white biotechnology applications. This yeast can be isolated from a wide range of natural, polluted or anthropogenic environments, including various food products. The present study aims to increase the data on Y. lipolytica phenotypic diversity by evaluating the growth parameters and secreted enzymatic activities of 28 wild-type Y lipolytica (and Yarrowia sp.) strains isolated from various environments across 10 countries. These data could facilitate the selection of appropriate strains for specific research purposes, particularly when wild-type strains are prioritized over genetically engineered ones, like for food-related applications. Notably, strain SWJ-1b exhibited an outstanding combination of favourable characteristics, with optimum (or near) performances for both growth and enzymatic parameters.

Propionic acid (HPr) accumulation is a major indicator of anaerobic digestion (AD) dysfunction, yet the relative contributions of acidity, undissociated HPr, and propionate ions (Pr-) to process inhibition remain poorly understood. We investigated these effects in mesophilic batch AD microcosms fed with municipal sewage sludge, using a comparative design involving HPr, sodium propionate (NaPr), NaCl, and HCl treatments across two series of experiments. While 20 mM HPr caused a 22% reduction in the maximal methane production rate, 81 mM HPr led to complete inhibition, with the initial pH dropping to 5.1. By contrast, 81 mM NaPr reduced methane production rate by only 40%, and 81 mM NaCl caused no inhibition, demonstrating that acidity is the dominant inhibitory factor, with Pr- exerting a secondary concentration-dependent effect. 16S rRNA gene amplicon sequencing revealed strong, compound-specific shifts in microbial community composition, affecting key functional groups including syntrophs and methanogenic archaea. The proportion of methanogens dropped from 2-3% in control reactors to less than 0.2% under 81 mM HPr, consistent with the observed methane production inhibition. Under HPr81, over 100 ASVs were differentially abundant compared to controls, a pattern largely shared with HCl-treated reactors, further confirming the predominant role of acidity. The number of differentially abundant ASVs was negatively correlated with methane production rates (R{superscript 2} = 0.97), underscoring the link between community reshaping and process impairment. These results provide a unifying framework for propionate inhibition in AD and suggest that microbial community profiling could serve as an early warning tool for process imbalance detection.

Poly({varepsilon}-caprolactone) (PCL) is a well-known biodegradable polyester and is among the few polyesters susceptible to degradation in marine environments; however, marine-derived PCL-degrading enzymes remain poorly characterized. Here, we searched for PCL-degrading enzymes from the marine bacterium Alloacanivorax gelatiniphagus JCM 18425 using a genome-based approach. Five candidate genes were predicted, and one encoded protein, designated Ag0826, was identified as a PCL depolymerase. Recombinant Ag0826 was expressed, purified, and biochemically characterized. The enzyme exhibited optimal activity at 35-40{degrees}C and pH 8.0, although it showed limited thermal stability. Substrate specificity was compared with that of leaf-branch compost cutinase (LCC), a well-characterized poly(ethylene terephthalate) (PET) hydrolase, using various polyesters. Both enzymes exhibited largely overlapping substrate ranges with respect to the presence or absence of monomer conversion activity across the tested substrates. Ag0826 slightly degraded PET to terephthalic acid, indicating potential PET-hydrolyzing activity; its conversion rate, however, was substantially lower than that of LCC, suggesting that Ag0826 exhibits a substrate preference differing from LCC. Phylogenetic analysis based on amino acid sequences revealed that Ag0826 formed a separate clade from LCC and IsPETase (from Ideonella sakaiensis). At a broader level, Ag0826 was positioned near HaloPETase1 (from Halopseudomonas pachastrellae), which has been proposed as a Type III PET hydrolase; in contrast, residues corresponding to the substrate-binding subsites were similar but not identical between the two enzymes. These results suggest that Ag0826 broadly belongs to the group of known PET hydrolases, yet it exhibits a partially distinct sequence profile even within this enzyme family.

Porcine Bacteroides thetaiotaomicron LYH5 demonstrated in vitro antimicrobial activity, suggesting probiotic potential. Due to poor gastric juice tolerance, LYH5 was encapsulated via extrusion using sodium alginate (SA) and gellan gum. Box-Behnken design optimization yielded optimal parameters: SA 1.5%, gellan gum 0.4%, CaCl2 0.9%, bacteria:glue ratio 1:4, achieving an encapsulation rate of 84.22{+/-}0.17%. Its effect on weaned piglet intestinal health was evaluated using 78 piglets (7.69{+/-}0.52 kg) randomly assigned to 4 groups for 40 days: CON (control), T (basal diet + LYH5 live bacteria, 1x10{superscript 1} CFU/mL), TJ (basal diet + LYH5 microcapsules, 1x10{superscript 1} CFU/mL, J (basal diet + empty capsules). The results of this experiment showed that compared with the control group, LYH5 microcapsule can improve the intestinal barrier function without affecting the growth performance of piglets, and provide ideas and references for the development of human next-generation probiotics (NGP). IMPORTANCEThis study addresses the key bottleneck of poor gastric acid tolerance of probiotics via microencapsulation and provides a practical reference for the development of human next-generation probiotics.

Functional dairy products are increasingly recognized for their ability to provide both essential nutrition and additional health benefits. This study aimed to develop and evaluate a synbiotic yogurt enriched with Lactobacillus plantarum as a probiotic and Stevia rebaudiana extract (1% w/v) as a prebiotic source. Thirteen lactic acid bacteria (LAB) strains were isolated from fermented dairy and vegetable samples and evaluated for probiotic potential through tests for acid and bile tolerance, hydrophobicity, aggregation abilities, and pathogen co-aggregation. Isolate PG1 (Lactobacillus plantarum) demonstrated the highest prebiotic growth stimulation index (49%) in the presence of stevia extract and was selected for yogurt formulation. Yogurt samples were prepared and stored at 4{degrees}C for 10 days. Physicochemical properties (pH, titratable acidity, and protein content), microbiological viability, total phenolic and flavonoid content, antioxidant activity (DPPH assay), and sensory attributes were monitored. The synbiotic yogurt (St-Y) showed enhanced functional properties, with a total phenolic content of 16.67 {micro}g GAE/g, a flavonoid content of 6.28 {micro}g QE/g, and 57.84% antioxidant activity. Additionally, it showed improved protein content and superior sensory scores compared to control samples. These findings suggest that S. rebaudiana fortified probiotic yogurt can serve as a nutritious, antioxidant-rich, and sensory-acceptable functional dairy product.

Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes the interaction with negatively charged components of the yeast cell surface, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated the interaction of chitosan with the cell surface and its permeabilizing capacity in three yeast species displaying distinct susceptibility profiles, Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii. We evaluated the correlation between differential susceptibility and chitosan association at the cell surface, as well as cell permeabilization, by integrating growth analyses with surface-binding assays, including FITC-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell surface and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization as a method to measure the activity of selected enzymes in situ, demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast.

Microbial fuel cell offers a promising approach to improve wastewater quality and generate bioenergy from dark fermented effluents. In this study, the use of dark-fermented palm oil mill effluent as an electron donor for bioelectricity generation was investigated using a double-chambered microbial fuel cell (MFC). The MFCs were operated at room temperature (29 {+/-} 2{degrees}C), anode electrolytes adjusted to pH 7, and a chemical catholyte as the oxidizing agent. The maximum power {+/-} 8.07 mW/m2 and 155.16 {+/-} 12.88 mA/m2, respectively, were generated from the MFCs inoculated with sludge, which was 5.9 times higher than control without inoculum. Microbial community analysis revealed the enrichment of fermentative and electrogenic representative taxa from the phyla Bacillota, Bacteroidota and Pseudomonadota on the anode electrodes. Optimizations of the running conditions were carried out, suggesting the optimum parameters of 0.5 k{Omega} external resistance, anolyte initial pH 9, and 75% DFPOME substrate concentration. Operation under the optimized conditions increased current production, wastewater treatment, and Coulombic efficiency compared to the non-optimized conditions. Multiple configurations were also evaluated, showing higher cumulative voltage, power, and current densities with the stacked MFC connections, compared to single MFC units. Parallel circuit connection produced higher power and current density than serial connection. This study demonstrated the feasibility of MFC as a promising downstream treatment for biohydrogen production processes, towards higher treatment efficiency and resource recovery.

Organic amendments provide crops with nutrients, but can also add pollutants. Yet the fate of micronutrients such as zinc (Zn) and pollutants such as cadmium (Cd) in soil-crop systems is difficult to predict because of the complexity of amendments added to soils. We performed pot and incubation experiments to determine whether the soil availability, uptake and transfer to grain of Zn and Cd in wheat (Triticum aestivum) are linked to the composition of amendments. Three amendments with highly diverse chemical properties, including varied organic matter (OM) degradability, were applied to a non-contaminated, arable soil. Stable isotopes of 70Zn and 106Cd were used to trace metals taken up from inputs versus soil in wheat biomass. We found the amendment most enriched in rapidly degradable OM (poultry manure) led to the highest wheat uptake of input-derived Zn i.e., 87{+/-}14 mg Zn (kg soil)-1. This was 2.5 times higher than input-derived Zn uptake from the most degraded amendment (compost). We did not observe an increase in soil available Zn with amendment application. Thus, biotic processes resulting from soil-plant-microbial interactions led to the increase in wheat uptake of input-derived Zn with amendment enrichment in rapidly degradable OM. Amendments led to minimal uptake of input-derived Cd in wheat and did not increase soil available Cd. Furthermore, we found no significant increase in grain Zn and Cd concentrations with amendments compared to the control. Our results highlight how amendment OM composition affects soil availability and wheat uptake of Zn and Cd with organic amendment application.

BackgroundSuccinic acid (SA) is a four-carbon dicarboxylic acid of considerable industrial relevance, with applications spanning the food, chemical, and pharmaceutical sectors. The remarkable acid tolerance of the yeast Yarrowia lipolytica makes it a promising microbial cell factory for SA production. Numerous metabolic engineering strategies have focused on disrupting genes encoding the succinate dehydrogenase (SDH) complex to enhance SA accumulation. However, such a modification is associated with impaired growth and the accumulation of by-products, notably acetic acid (AA). ResultsTo improve growth capacity, SA productivity, and reduce AA formation in Y. lipolytica SDH5-deficient strains (Sdh5{Delta}), carbon flux from glycolysis was partially redirected toward the pentose phosphate pathway by overexpression of the native genes encoding glucose-6-phosphate dehydrogenase (ZWF1) and 6-phosphogluconate dehydrogenase (GND1), thereby enhancing NADPH generation. The resulting strain was further engineered to increase NADH availability for the mitochondrial electron transport chain by overexpressing genes encoding either a mutated NADPH-dependent malate dehydrogenase (TfMdh) from Thermus flavus or the soluble transhydrogenase (EcSthA) from Escherichia coli, enabling indirect conversion of NADPH to NADH. This strategy resulted in 2-fold and 2.2-fold increase in SA productivity and titre, respectively, compared to the Sdh5{Delta}-ALE strain during bioreactor cultivation on glucose-based media. Moreover, AA accumulation was reduced 1.2-fold, while growth rates were significantly improved. ConclusionsThe proposed engineering strategies, especially heterologous expression of EcSthA, partly alleviated energy limitations in Y. lipolytica Sdh5{Delta} strain, resulting in improved SA productivity and growth performance.

Lignin-derived aromatics are abundant in depolymerized lignin but remain remain untilized as carbon sources for commercial production of bulk chemicals. Among these aromatics, p-coumaric acid can be funnelled through the {beta}-ketoadipate pathway toward cis,cis-muconic acid (ccMA), a precursor of bio-based adipic and terephthalic acids. However, efficient ccMA production by Acinetobacter baylyi ADP1 is constrained by toxicity of catechol (the immediate precursor of ccMA), inefficient channelling of protocatechuate (PCA) metabolism towards ccMA production, and absence of PCA decarboxylase for converting PCA to catechol. Therefore, in this study, we engineered a modular co-culture system, combining engineered strains of A. baylyi and E. coli, for ccMA production from synthetic p-coumaric acid. Deletion of catB and catC genes and overexpression of catA in A. baylyi GJS_catA strain enabled near-stoichiometric conversion of catechol to ccMA ([~]90% carbon yield) with titres up to 56.4 mM ([~] 8 g/L) under controlled fed-batch feeding. The strain was further engineered (A. baylyi GJS2_catA) to convert p-coumaric acid to PCA. Due to the inactivity of heterologous PCA decarboxylase (aroY gene) in A. baylyi, this gene was incorporated in E. coli where it exhibited activity through PCA to catechol conversion. Upon its production by E.coli_aroY in the co-culture, catechol is instantaneously converted to ccMA by A. baylyi GJS2_catA strain. In a two-step process, 22 mM p-coumaric acid was initially converted to 20.6 mM PCA (A. baylyi GJS2_catA), which was further converted to catechol (E.coli_aroY) and finally to 18.55 mM ccMA (2.63 g L-{superscript 1}) by A. baylyi GJS2_catA. This process was validated by the valorization of lignin-derived p-coumaric acid to ccMA. While the modular strategy developed in this study substantially improves ccMA titres, it also highlights the bottlenecks in A. baylyi metabolic pathway engineering for lignin valorization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/709578v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a83daborg.highwire.dtl.DTLVardef@168c6b6org.highwire.dtl.DTLVardef@1ce0abdorg.highwire.dtl.DTLVardef@23200b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Yeasts ability to invade surfaces has important implications for infections and food contamination. Invasive growth in yeast is influenced by genetic and environmental factors. In this exploratory study, we investigated the effects of sodium sulfide, gene deletions, and environmental conditions on the invasive behaviour of the wine yeast strain AWRI 796. Sodium sulfide enhanced invasion in the (parent) AWRI 796 strain under nitrogen-limiting conditions, although its effect was obscured by experimental variability and pre-culture conditions. Genetic factors had a major effect on the overall invasive phenotype, with deletion of key genes suppressing invasion. Most gene-deletion mutants did not significantly affect how the colony responded to sulfide. In addition to sulfide and genotype, environmental conditions also influenced invasive behaviour. The pre-2xSLAD pre-culture condition was best for detecting sulfide-induced growth, and later plate washing time and decreased nutrient levels enhanced invasiveness. Our experimental design and findings provide a framework for understanding the determinants of yeast invasiveness, which may inform future studies on filamentous yeast behaviour.

In this study, we employ a two-stage dynamic metabolic control strategy to enhance the NADPH dependent biosynthesis of ethylene glycol from xylose in engineered E. coli. We evaluated the use of metabolic valves to dynamically reduce the enzymes involved in competitive pathways which compete for substrates with ethylene glycol biosynthesis, as well as regulatory pathways aimed at increasing NADPH fluxes. The performance of our initial strains with limits in pathway expression levels was improved by the addition of competitive valves, but not by increases in NADPH flux. In contrast, improving pathway expression levels, led to strains improved significantly by our regulatory valves which improved NADPH flux, but not by the competitive valves. This is consistent with a central hypothesis that faster pathways in and of themselves can compete with other metabolic fluxes by being faster and are better aided by regulatory changes capable of change rates elsewhere in metabolism. In this case in NADPH flux. Lastly, upon scale up to fed-batch bioreactors, our optimized strain, featuring dynamic control of two regulatory valves produced 140 g/L of EG in 70 hours at 92% of the theoretical yield.

Reducing the cost and environmental impact of cell culture media is an important goal for cultivated meat, the process of generating meat in vitro using proliferating animal cells. While prior approaches have demonstrated the use of microbial lysates to replace expensive animal-based fetal bovine serum (FBS) in media, these formulations still rely on large quantities of growth factors such as fibroblast-like growth factor 2 (FGF2). Here, we demonstrate the use of FGF2-expressing Vibrio natriegens to create whole-cell lysates that replace both FBS and FGF2 in cell culture media for cultivated meat applications. This medium, named "VN40FGF", supports rapid proliferation of immortalized bovine muscle satellite cells (iBSCs) in the absence of supplemented FGF2. Cells grown in VN40FGF maintain phenotype and differentiation capacity. We also demonstrate that engineered V. natriegens can grow in spent cell culture media, further improving sustainability and economics, and reducing potential eutrophication concerns associated with waste disposal. Our approach combines multiple strategies for reducing the total number of media inputs, demonstrating opportunities for more economical and sustainable cell culture, especially for cultivated meats.

Zymomonas mobilis is an ethanologenic Alphaproteobacterium with many interesting characteristics for fundamental research and applied microbial engineering. Although genetic engineering has been established for Z. mobilis since the 1980s, a rich set of inducible transcriptional regulators is still unavailable. In this work, seven different chemically inducible promoters have been systematically tested for their functionality in Z. mobilis. In particular, for the first time, NahR-PsalTTC, VanRAM-PvanCC, CinRAM-Pcin and LuxR-PluxB have been characterized in Z. mobilis, alongside the commonly used regulator-promoter pairs TetR-Ptet and LacI-PlacT7A1_O3O4, and the less commonly used XylS-Pm. All promoters investigated in this work are compatible with the Golden Gate modular cloning framework Zymo-Parts. Characterization was carried out with a shuttle vector backbone based on pZMO7, which has so far been rarely used for applications in Z. mobilis but seems to be completely stable without selection and generates high and uniform levels of expression. From the experimental results presented, it can be concluded that VanRAM-PvanCC and CinRAM-Pcin are particularly promising for broad use in the Z. mobilis community. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/712268v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16579e6org.highwire.dtl.DTLVardef@1262533org.highwire.dtl.DTLVardef@15456a2org.highwire.dtl.DTLVardef@3af98_HPS_FORMAT_FIGEXP M_FIG C_FIG