Pediocin, produced by certain lactic acid bacteria, has high antibacterial activity against Listeria monocytogenes and is therefore expected to be used as a peptide food preservative. In this study, a rapid and efficient method for recovering pediocin with antibacterial activity from a fusion protein with green fluorescent protein (GFP) produced by Escherichia coli was developed. After cell lysis, the concentrated fusion protein contained in the insoluble fraction was solubilized and desalted, and pediocin was cleaved from GFP by a protease factor Xa treatment. As pediocin exhibited strong adsorption to various membranes and a column matrix, and was presumed to adsorb to GFP, it was difficult to efficiently collect the pediocin with column purification and filtrations including ultrafiltration. Heating the mixture in boiling water was found to be an effective method to selectively remove GFP followed by centrifugation. Because pediocin is highly thermostable, soluble pediocin with high antilisterial activity was retained in the recovered supernatant. This study demonstrates that recombinant pediocin can be recovered rapidly and efficiently by the heat denaturation of cleaved fusion partner and centrifugation.
Extracellular environments regulate cell proliferation, differentiation, and function through dynamic interactions between cells and the extracellular matrix (ECM), as well as through soluble biochemical cues such as growth factors, all of which are governed by coupled biochemical reactions and mass transport processes. To engineer such environments in a controllable manner, we developed azide-cyclooctyne clickable alginate microcapsules that allow bioactive peptides to be introduced by in situ conjugation via strain-promoted azide-alkyne cycloaddition (SPAAC). Osteoblastic cells were encapsulated in cyclooctyne-modified alginate microcapsules, and branched bone morphogenetic protein 2 (BMP-2) mimetic peptides bearing azide groups were added to the culture medium. The peptides diffused into the microcapsules and selectively reacted with cyclooctyne groups without nonspecific reactions, resulting in spatially confined accumulation controlled by diffusion and click reaction kinetics. This localized enrichment of osteogenic signals promoted early osteogenic differentiation of the encapsulated cells, as indicated by activation of the Col1a1-GFP reporter. From a bioreaction engineering perspective, the present system demonstrates an in situ conjugation strategy for spatiotemporal regulation of bioactive molecules within microstructured materials, enabling modulation of early osteogenic responses without direct chemical modification of cells. This bioinspired in situ conjugation approach based on the click chemistry provides a versatile platform for biomedical, bioindustrial, and biochemical applications.
Postoperative adhesions occur frequently after abdominal surgery and gynecological surgeries, causing serious complications. For applications in laparoscopic surgery, we developed a thermally cross-linked gelatin film-cut (GC) by dividing a thermally cross-linked gelatin film (GF) into small fragments. In this study, we examined the basic biological properties of the GC and conducted anti-adhesion tests using a rat cecum abrasion model to evaluate the effects of film fragmentation, focusing on the influence of fragment size, dosage and film thickness. Solubility test results showed that GC degraded more slowly than its film counterparts. Furthermore, as the thickness and the size of the GC increased, its degradation rate decreased. In addition, the results of the collagenase degradation test demonstrated that the GC degraded more slowly than the unfragmented film and that as the thickness and size of the GC increased, its degradation rate decreased. The results of the rat anti-adhesion test demonstrated that compared with GF, GC was significantly less effective in preventing adhesion. However, compared with the untreated group, several GCs showed significantly lower adhesion scores. Some of the GCs demonstrated anti-adhesion effect similar to or better than those of hyaluronic acid and carboxymethyl cellulose (HA/CMC). In summary, increased fragment size, reduced film thickness, and an elevated dosage of GC improved anti-adhesion efficacy. Thus, GC exhibits excellent anti-adhesion effects when its geometry, thickness, and dosage are optimized. Consequently, anti-adhesive GC improves the clinical applicability of anti-adhesion materials, particularly in laparoscopic surgery.
Migrasomes are extracellular vesicles secreted by migrating cells that mediate intercellular communication and contribute to inflammation and cancer progression. We have previously demonstrated that IL-6 expression is induced in cells that have taken up migrasomes derived from IL-6 amplifier activated cells, suggesting that migrasomes are involved in the propagation of inflammation. However, the molecular changes occurring within migrasomes upon IL-6 amplifier remain unknown. In this study, migrasome-enriched samples were extracted from IL-6 amplification-induced human breast cancer MDA-MB-231 cells by ultracentrifugation, and comparative proteomics analysis was performed. Shotgun proteomics detected a total of 1984 proteins, of which 1339 were detected under all conditions. In the cytokine-stimulated migrasomes, 330 proteins up-regulated and 149 proteins down-regulated compared to starvation condition. Changes associated with IL-6 amplification included the IL-6 pathway kinase JAK1, proteins involved in vesicular transport, and SNARE family members syntaxin-3 and syntaxin-4. After immunostaining, the fluorescence of JAK1 in migrasomes under stimulation condition was approximately 1.5-fold higher than those under starvation condition, consistent with the proteomic result. Furthermore, functional enrichment analysis revealed enhanced expression of pathways related to oxidative phosphorylation, endoplasmic reticulum protein processing, and cell organization. These findings indicate that IL-6 amplifier activation is associated with coordinated changes in migrasome proteins, including those involved in inflammatory signaling and trafficking. This study provides a proteomic understanding of migrasome-mediated inflammatory communication in the tumor microenvironment.
Disease-suppressive soils, in which soilborne pathogens are naturally suppressed, offer a promising model for sustainable crop protection, particularly in organic farming systems where chemical disease control options are limited. Although disease suppression in these soils is considered to rely on biological control, the underlying mechanisms remain poorly understood. In this study, we investigated soil from a long-term organically managed field in Shiga Prefecture, Japan, where soilborne disease incidence has remained consistently low, to identify bacterial community features potentially associated with this field. The 16S rRNA gene amplicon sequencing indicated that this soil harbored a bacterial community distinct from those of nearby agricultural soils. Following the application of organic compounds, the genus Lysobacter, a taxon with known antagonistic activity against plant pathogens, was markedly enriched in response to proteinaceous organic inputs. This enrichment was consistent across sampling times and specific to certain proteinaceous organic inputs, whereas minimal effects were observed on chitin, N-acetyl-d-glucosamine, or cysteine. Broader soil surveys indicated that Lysobacter enrichment was not strictly associated with whether soils had been managed under organic or conventional farming practices. Stepwise multiple regression analysis identified 10 co-occurring bacterial genera that were strongly associated with Lysobacter abundance. These findings highlight condition-dependent Lysobacter enrichment as a characteristic microbial response to proteinaceous organic amendments in this low-disease-incidence field and provide microbial insights that may inform microbiome-based strategies for sustainable soil management.
Green Awak banana (Musa paradisiaca cv. Awak) is a tropical fruit widely cultivated in Indonesia and valued for its high resistant starch content, which contributes to improved glycemic control, gut health, and potential prebiotic effects. The growing prevalence of gluten intolerance and celiac disease has increased demand for naturally gluten-free alternatives, positioning banana flour as a promising substitute for wheat-based products. This study evaluated the influence of different drying techniques, including sun-drying, oven-drying, and freeze-drying, on the metabolomic profile, functional properties, and available carbohydrate of green Awak banana. Metabolite profiling was conducted using gas chromatography mass spectrometry (GC-MS), while multivariate analysis and in vitro available carbohydrate assays were performed to assess biochemical variation among drying treatments. GC-MS metabolite profiling annotated 79 metabolites, and multivariate analysis showed clear separation among drying treatments. Pathway enrichment highlighted starch and sucrose metabolism as a primary pathway affected by drying. Sun-dried samples exhibited the highest available carbohydrate despite lower free glucose intensity, indicating preservation of enzyme-accessible polymeric carbohydrates. In contrast, oven-dried samples showed higher low-molecular-weight sugars but reduced available carbohydrate, likely due to thermal modification. These results demonstrate that drying methods alter carbohydrate functionality rather than total carbohydrate content, supporting the potential of sun-dried green Awak banana as a functional, slowly digestible carbohydrate ingredient.
Diazotrophic l-glutamate (L-Glu) fermentation, independent of the Haber-Bosch process, is a sustainable strategy. Following our successful production of L-Glu from aerial nitrogen by an engineered Klebsiella pasteurii NG13 overproducing citrate synthase and citrate transporters (+CC strain), we herein examined L-Glu transporters in this bacterium. Extracellular L-Glu accumulation was abolished in a K. pasteurii strain in which a gene homologous to Escherichia coli ybjL was deleted, suggesting that the YbjL homologous protein (herein designated as GluE) was the main exporter of the +CC strain for diazotrophic L-Glu production. However, the overexpression of gluE did not increase diazotrophic L-Glu production, suggesting that the export of L-Glu was not a rate-limiting step in diazotrophic L-Glu production in the +CC strain. To sustain and increase diazotrophic L-Glu production, we constructed a triple knockout K. pasteurii +CC strain lacking the putative gltS, gltP, and gltIJKL orthologs, all the counterparts of which encode L-Glu importers in E. coli. In glucose-fed cultures, while L-Glu produced extracellularly by the +CC strain decreased during a long-term cultivation, the triple knockout strain sustained L-Glu production over the culture period and finally reached 2.67 g L-1. These results suggest the potential of the practical application of our system to the production of nitrogen-containing compounds from aerial nitrogen.
Sago starch is an important carbohydrate resource with a potential relevance for future food security. Flavor characteristics of sago starch play a critical role in product acceptability. To date, the influence of the milling grade and contribution of varietal differences of raw materials on its flavor characteristics, which are produced using similar processing methods, has not been sufficiently explored. In this study, we investigated the effects of varietal differences and milling grades on the sensory and metabolites profile of sago starch samples produced from two different sago varieties that have morphological differences, Tuni variety (Metroxylon rumphii) (spiny) and Molat variety (Metroxylon sagu Rottb.) (spinless), and three different milling process: local grade (milled using rotary shifter 100 mesh size), consumer grade (milled using rotary shifter 120 mesh size) and export grade (milled using vibrating screen 120 mesh size). The analyses were performed through integrated metabolomics and sensory quantitative descriptive analysis. Under similar production methods, varietal differences exerted a stronger influence than the milling grade on both the sensory and metabolite profiles of sago starch samples. Non-volatile metabolites were largely unaffected by the milling grade, whereas volatile compounds exhibited greater sensitivity to milling differences. Partial least squares regression analysis further revealed that sugars and specific volatile metabolites contributed to desirable and off-flavor characteristics between the two varieties. Overall, these findings provide the preliminary insight that varietal differences are the primary determinants of the flavor characteristics of sago starch, rather than milling grades, when the sago starch samples are produced using similar processing methods.
Tyrosine production in Corynebacterium glutamicum has mainly been improved because of the use of feedback-resistant enzymes and redirection of carbon flux. However, the genetic basis of the classical feedback-deregulated phenotype and contribution of post-transcriptional regulation remain unclear. In this study, we identified the mutation responsible for the feedback-deregulated phenotype of the aroG-csm region in the phenylalanine-producing C. glutamicum strain KY10694, examined the effect of RNase J deficiency on tyrosine production, and developed a stepwise strategy to further improve tyrosine production. Sequence analysis revealed a single nucleotide substitution in aroG that resulted in an A178V amino acid substitution, whereas no mutation was detected in csm. Enzyme assays showed that the KY10694-derived aroG-csm gene products converted the feedback response of chorismate mutase activity to phenylalanine and tyrosine. In a 2 L jar fermentor cultivation, the strain harboring the KY10694-derived aroG-csm genes produced 5.45 mM tyrosine, and deletion of rnj, which encodes RNase J, increased tyrosine production to 10.0 mM. Transcriptome analysis of the RNase J-deficient strain revealed broad changes in the expression of genes involved in the central carbon metabolism and aromatic amino acid biosynthesis. Further redirection of carbon flux by deletion of pheA and subsequent deletion of ppc, which encode prephenate dehydratase and phosphoenolpyruvate carboxylase, respectively, increased tyrosine production to 21.7 and 24.8 mM, respectively. These results demonstrate that integrating pathway deregulation through AroG A178V, regulatory rewiring through RNase J deficiency, and rational carbon flux redirection is an effective strategy for improving tyrosine production in C. glutamicum.
The anoxygenic phototrophic bacterium Cereibacter sphaeroides produces molecular hydrogen (H2) by nitrogenase using light energy and electrons derived from organic compounds, the process of which is known as photo-fermentative H2 production. An objective of photo-fermentative H2 production is inhibition by ammonia since expression of nitrogenase is repressed under nitrogen-sufficient conditions. Therefore, we have previously constructed the ΔhupcbbP∗ strain that can produce H2 from acetate in the presence of 10 mM ammonium chloride. In this study, we observed that the ΔhupcbbP∗ strain can also produce H2 during photoheterotrophic growth with glucose and 10 mM ammonium chloride. For further genetic manipulation, we constructed a plasmid-free strain CsHydtrc2, which produced similar amounts of H2 with those of the ΔhupcbbP∗ strain during photoheterotrophic growth with glucose in the presence of ammonia. Furthermore, supplementation of sodium bicarbonate to culture medium stabilized culture pH and markedly enhanced H2 production of the CsHydtrc2 strain. Gene deletion analyses revealed that the Entner-Doudoroff pathway, but not the Embden-Meyerhof pathway, is essential for H2 production by the CsHydtrc2 strain with glucose. Plasmid expression of genes involved in glucose metabolism suggested that the glucose incorporation is the rate-limiting step in the glucose metabolism in the CsHydtrc2 strain under photoheterotrophic growth conditions.
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) is a biodegradable copolymer whose mechanical properties can be tuned by the 3-hydroxyhexanoate (3HHx) fraction. However, current industrial production largely relies on established hosts and plant oil-based feedstocks. Here, we developed Rhodobacter capsulatus SB1003 as a new PHBH-producing platform by focusing on the two key determinants of copolymer formation: polyhydroxyalkanoate (PHA) synthase substrate specificity and intracellular monomer supply. A PHA synthase with broad-substrate specificity was integrated into the native phaC locus to generate a heterologous phaC strain. All PHA production experiments were performed under anaerobic photoheterotrophic conditions in 8-mL screw-cap tubes containing 7.6 mL of medium and illuminated with continuous white light. During butyrate cultivation under these conditions, the engineered strain accumulated polymer up to 41.5% of cell dry weight and incorporated detectable 3HHx, whereas the wild type showed no 3HHx incorporation. To increase 3HHx-CoA availability from butyrate, we introduced C4-to-C6 precursor-supply modules involving β-ketothiolase (BktB)/β-ketoacyl-CoA reductase (PhaB) and crotonyl-CoA carboxylase/reductase (Ccr)/ethylmalonyl-CoA decarboxylase (Emd), but these modifications led to only marginal improvements in the 3HHx fraction. In contrast, supplying C6 or longer fatty acids under the same conditions markedly increased 3HHx incorporation; cultivation on hexanoate yielded PHBH containing 32.4 mol% 3HHx. Collectively, this study demonstrates PHBH biosynthesis in R. capsulatus and indicates that limited 3HHx-CoA supply rather than polymerization capacity is the primary bottleneck, providing a foundation for further pathway and host optimization toward flexible PHBH production from diverse substrates.
The constant dearth of transplantable tissues and organs in India required the development of substitute biomaterials for tissue engineering. Plant-based decellularized scaffolds have become attractive options because of their abundance, ethical acceptability, architectural diversity, and lower risks of zoonotic transmission. Curcuma longa leaves were investigated in this study as a possible source of cellulose-based scaffolding for use in biomedical applications. After cuticle removal, an immersion decellularization technique utilizing sodium dodecyl sulphate (SDS) and triton-X-100 was developed to successfully remove cellular and nuclear material while maintaining leaf parenchyma architecture. Histology, DAPI staining, scanning electron microscopy, and a notable decrease in leftover DNA content all demonstrated efficient decellularization. When contrasted with native leaves, the resultant decellularized C. longa leaf scaffolds showed significant increase in porosity, water vapor transmission rate and swelling percent, and significantly lower contact angle with an optimum surface roughness promoting cell adhesion. Mechanical test manifest higher tensile strength with decreased stiffness. Fourier transform infrared spectra of leaf scaffold reveals persistence of different components except cuticle but the intensity of different peaks was decreased. The leaf scaffolds showed superior hemocompatibility and excellent compatibility with Madin-Darby canine kidney cells (MDCK) which is demonstrated by cell attachment and proliferation. MTT assay of seeded scaffold showed significantly higher metabolically active cell. In vivo subcutaneous implantation of decellularized scaffolds showed host tissue incorporation, accumulation of collagen, and neovascularization. C. longa leaf scaffolds can be utilized as cost effective and sustainable biomaterials for soft tissue engineering and regenerative medicine.
Carbohydrate-binding modules (CBMs) promote enzyme-substrate association, enhancing catalytic activity toward soluble and insoluble substrates. In this study, we characterized the three tandem domains of laminarinase LA-Lam from Pseudoalteromonas sp. LA as the first members of the CBM115 family. Affinity gel electrophoresis (AGE) showed that the three domains bound Laminaria digitata laminarin. Depletion isotherm assays revealed that binding affinity for curdlan followed the order CBM115_C > CBM115_M > CBM115_N, whereas CBM115_C exhibited the highest affinity for yeast β-glucan. Kinetic analyses demonstrated that the CBMs enhanced laminarin hydrolysis, but the number of CBMs had little additional effect on catalytic efficiency. The predicted structural models of the three CBMs indicated similarities to CBMs in families 4, 6, and 102. The differing binding affinities for curdlan and yeast β-glucan among the three CBMs may reflect variations in the region surrounding the substrate-binding cleft.
Genome-modifying enzymes, such as recombinases and CRISPR-associated nucleases, enable targeted gene insertion when delivered transiently to minimize off-target effects. Precise genome engineering requires controlled enzyme activity, as well as efficient donor DNA transfer. Integrase-defective lentiviral vectors (IDLVs) provide a promising platform for transient episomal DNA transfer; however, their integration efficiency depends on complementary genome-targeting strategies. Here, we engineered Cre-loaded IDLVs (Cre-IDLVs) that co-package lentiviral vector genomes together with bioactive Cre recombinase. Cre was inserted into the Gag region of an integrase-defective gag-pol construct, allowing for efficient encapsidation and protease-mediated release during virion maturation without compromising the viral titer. The resulting particles carried donor cassettes flanked by heterospecific loxP sites. When applied to CHO founder cells harboring compatible genomic loxP landing pads, Cre-IDLVs efficiently mediated recombination-mediated cassette exchange, producing the highest number of G418-resistant colonies among the plasmid ratios tested. Genomic PCR and sequencing confirmed precise locus-specific insertion without detectable random integration in the analyzed clones. These findings establish Cre-IDLVs as a streamlined dual-delivery platform that couples transient recombinase activity with episomal donor DNA transfer. This hybrid lentiviral strategy provides a programmable approach for controlled and site-specific genome modification in mammalian cells.
Substrate specificity of amino acid racemases is a key determinant of their biological function, yet the structural principles governing this specificity, particularly with respect to substrate side-chain structure, remain poorly understood. In this study, we investigated whether substrate specificity in the pyridoxal 5'-phosphate (PLP)-independent 2,4-diaminobutyrate racemase PddB can be progressively redirected through minimal substitutions, guided by structural and phylogenetic variation within a conserved enzyme scaffold. Comparative analyses identified residues in the distal region of the substrate-binding pocket as candidate determinants for substrate accommodation. Substitutions at three positions (92, 164, and 206) progressively shifted substrate preference from C4 to C5, and ultimately to C6 diamino acids. These stepwise transitions involved the acquisition of activity toward previously unrecognized substrates, accompanied by loss of the original specificity in the triple mutant. Catalytic efficiencies of active variants toward their preferred substrates were maintained within the same order of magnitude, indicating that catalytic competence was preserved despite substantial changes in substrate preference. Structural modeling indicates that these shifts arise from remodeling of the distal binding pocket, thereby altering its capacity to accommodate substrates of different chain lengths without perturbing the catalytic machinery. These findings demonstrate that substrate specificity in this enzyme family can be redirected in a stepwise manner through minimal substitutions, and that such changes define a plausible evolutionary trajectory toward alternative substrate specificities within a conserved scaffold. This study provides insights into the structural basis of substrate specificity in diamino acid racemases and highlights the evolutionary accessibility of alternative substrate specificities within this scaffold.
Lignin is a complex aromatic polymer and a major component of lignocellulosic biomass, whose sustainable utilization is critical for the development of a low-carbon society. The white-rot fungus Phanerochaete sordida YK-624 exhibits high lignin-degrading activity. However, the pathways underlying the metabolism of lignin-derived aromatic compounds by this fungus remain unclear. Here, we combined genomic, transcriptomic, and metabolomic analyses to elucidate the catabolism of lignin-derived aromatic compounds in P. sordida YK-624. Multi-omics data suggest that this strain primarily metabolizes lignin-derived aromatic compounds using 1,2,4-trihydroxybenzene (THB) as a central intermediate, subsequently converting it first into 3-hydroxy-cis,cis-muconic acid (HMA) and then into 3-hydroxyhex-2-enedioic acid. The genes Psthbd and Pshmar 1 or 2, encoding putative THB dioxygenase and HMA reductase in P. sordida YK-624, were heterologously expressed in Escherichia coli. Functional assays confirmed that recombinant PsTHBD catalyzes the dioxygenation of THB and catechol, whereas PsHMAR2 exhibits HMA reductase activity. In contrast, PsHMAR1 showed no detectable activity, suggesting differences in cofactor binding or catalytic function. Our results demonstrate the involvement of PsTHBD and PsHMAR2 in THB degradation and identify for the first time an HMA reductase gene in white-rot fungi. This study advances our understanding of the catabolism of lignin-derived aromatic compounds and highlights the potential of P. sordida YK-624 as a platform for lignin valorization in biotechnological applications.
Methylorubrum extorquens AM1 has been extensively studied as a platform for biomanufacturing using a methanol feedstock. Given that lanthanide (Ln) ions are recognized as important metals related to methylotrophy of methylotrophic bacteria, this study applied adaptive laboratory evolution to M. extorquens under Ln-supplemented conditions to obtain strains with robust growth at high concentrations of methanol. The cultivation results indicated a possible Ln-dependent mechanism for methanol tolerance in this methylotroph. The parent strain was unable to grow at 5% (v/v) methanol under Ln-free conditions, but exhibited slight yet definite growth with La3+. Repeated passaging under high methanol concentrations resulted in isolation of evolved strains that acquired additional Ln-independent methanol tolerance, thereby could grow at high concentrations of methanol up to 7% with La3+. The identification of the mutations by whole-genome resequencing and reconstitution of the identified mutations in the parent strain suggested an association between impaired function of proton-transport protein and high methanol tolerance of M. extorquens. At 5% methanol, the evolved strain exhibited a cell yield comparable to that of the parent strain at 0.5% methanol, while its PHA content was approximately 2.5-fold higher, resulting in enhanced production of PHA from methanol. No increase in PHA production was observed between the parent and the evolved strain at 0.5% methanol, indicating that the acquired ability to grow at high methanol concentrations contributed to the enhanced PHA production. This study demonstrates the importance of Ln for methanol tolerance of M. extorquens and the advantage of high-methanol conditions for bioproduction using methylotrophs.
Over the past decade, CRISPR-based technologies have revolutionized our capacity to manipulate genomes, thereby reshaping the landscape of functional genomics research. Among the CRISPR toolkit, CRISPR/Cas9-mediated homology-directed repair (HDR) enables precise genome editing with predefined mutations, rendering it an indispensable tool for gene functional analysis, disease model construction, and the development of gene therapy strategies. Nevertheless, despite the robust efficiency of CRISPR/Cas9 in mediating gene knockouts, HDR-dependent gene knock-in (KI) remains a major bottleneck due to its inherently low efficiency. Herein, we report that the co-expression of PALB2 with the CRISPR/Cas9 nuclease could trigger an enhanced HDR effect. Specifically, the fusion of Cas9 with PALB2 elevated KI efficiency by approximately 1.7-fold in human HEK293T cells. Furthermore, this Cas9-PALB2 fusion strategy exhibited cross-cell-type efficacy, demonstrating its broad applicability beyond a single cell line. Notably, the combined application of the Cas9-PALB2 fusion system and Nocodazole further boosted KI efficiency to a remarkable 25.5%. Collectively, these findings establish the Cas9-PALB2 fusion as a highly potent and versatile strategy to augment HDR-mediated KI efficiency, highlighting its substantial potential for widespread utilization in applications that demand high-fidelity genome editing.
Sophorolipid, a biosurfactant which can be fermentatively produced by the yeast Starmerella bombicola using sugars and lipids as carbon sources, exhibits several advantageous properties over synthetic detergents, such as biodegradability and low toxicity. While some life cycle assessment (LCA) studies have been reported to evaluate the environmental impact of sophorolipid production, carbon footprint estimations remain limited. In this study, we combined small-scale experimental data with large-scale simulation data to estimate the carbon footprint of the fermentative production of sophorolipid by S. bombicola. Our LCA analysis estimated that sophorolipid production in a 1-L bench-top bioreactor emits 685 kg CO2 eq per kg of product. A scale-up simulation of the carbon footprint resulted in a marked decrease in CO2 emission from sophorolipid production in larger scales. Through sensitivity analysis, we identified key hotspots in the production process, particularly related to production scale.
