Biotechnology for Biofuels

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.

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

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.

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

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

Malic acid is a C4 dicarboxylic acid traditionally produced from petroleum and widely used in the food industry. As a sustainable alternative, it can also be produced as a value-added platform chemical from biomass. Previously, the Escherichia coli strain XZ658 was engineered to produce L-malate via the carbon-fixation reductive branch of the TCA cycle. In this study, we further improved this system by relieving allosteric regulation of citrate synthase, addressing redox imbalance, and enhancing malate export. These modifications approximately doubled the L-malate titer in the final strain MO128 compared to XZ658 under simple batch fermentation conditions. The process achieved a high mass yield of 1.2 g malate g-{superscript 1} glucose, highlighting the carbon-fixation capacity of the reductive TCA pathway for fermentative malate production.

Cocoa fermentation is a spontaneous microbe-driven process in which yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) generate the flavor precursors that determine the sensory quality of chocolate. Although the microbial ecology of cocoa fermentation has been increasingly studied through culture-independent methods, the effect of targeted nutritional interventions on community structure within geographically defined production territories has received limited attention. Here, we employed dual-marker metabarcoding (16S rRNA V4 and ITS1) with Illumina NovaSeq 6000 sequencing to characterize bacterial and fungal communities during spontaneous fermentation of Trinitario cocoa beans subjected to amino acid and zinc supplementation in the Limon province of Costa Rica. Fifteen samples were collected at 0, 24, and 48 h from control, amino acid-supplemented, and zinc-supplemented fermentations, each in duplicate. The bacterial community comprised 292 amplicon sequence variants (ASVs) representing 88 genera across 15 phyla; the fungal community comprised 1,117 ASVs representing 248 genera across 9 phyla. Firmicutes and Proteobacteria dominated the bacterial fraction, with a pronounced shift from Tatumella-dominated fresh pulp toward Weissella- and Leuconostoc-rich assemblages during fermentation. Amino acid supplementation reduced Firmicutes at 48 h while favoring Acetobacter proliferation; zinc supplementation promoted Mucoromycota and Wickerhamomyces while sustaining Liquorilactobacillus abundance. Beta diversity analyses (Aitchison distance, weighted and unweighted UniFrac) confirmed significant compositional differences between treatments (PERMANOVA, p [≤] 0.01), although alpha diversity indices did not differ between individual treatment pairs. Sparse Estimation of Correlations among Microbiomes (SECOM) revealed structured co-occurrence networks, including positive associations between Gluconobacter and Acetobacter and negative associations between Tatumella and several AAB genera. Predicted functional profiles (PICRUSt2) showed no significant pathway-level differences. Taken together, these results show that nutritional supplementation can reshape microbial community composition without reducing overall diversity. This provides a viable approach for steering fermentation outcomes in cocoa-producing territories that seek quality differentiation.

Cyanobacterial natural products are a rich source of bioactive compounds, yet their heterologous production remains challenging. This study investigates the feasibility of expressing the lyngbyatoxin A (LTXA) biosynthetic gene cluster in a fungal host. The lyngbyatoxin biosynthetic genes (ltxA, ltxB, ltxC) were individually cloned and expressed in Aspergillus oryzae NSAR1 under the control of an inducible promoter. Metabolite production was assessed using LC- MS, and transcriptional analysis was performed by RT-PCR. Codon-optimized constructs and precursor feeding experiments were employed to evaluate pathway functionality. No production of LTXA or pathway intermediates was detected upon co-expression of ltxA-C despite confirmed transcription of ltxB and ltxC. RT-PCR analysis revealed truncation of the ltxA transcript, suggesting incompatibility with fungal transcriptional or splicing machinery. In contrast, expression of a codon-optimized ltxC enabled biotransformation of indolactam V to LTXA in A. oryzae, confirming functional expression of the prenyltransferase. These results highlight transcriptional limitations as a key barrier to heterologous expression of cyanobacterial NRPS pathways in fungal hosts, while demonstrating that downstream tailoring enzymes can remain functional. This work provides insights for future engineering of fungal platforms for cyanobacterial natural product biosynthesis.

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

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

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.

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.

The efficient production of food and biochemicals using microorganisms that utilize single-carbon feedstocks presents a promising approach for advancing a circular bioeconomy. Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast already widely used in industry, making it an attractive host for such applications. Recently, K. phaffii was converted into an autotrophic strain capable of assimilating CO2 into both biomass and secreted organic acids, using energy derived from dissimilation of methanol to CO2. In these strains, methanol oxidation is catalysed by an alcohol oxidase (Aox2), which transfers electrons to oxygen without conserving reducing equivalents. To address this limitation, in this study we explored redirecting methanol dissimilation through the native alcohol dehydrogenase (Adh2), coupling methanol oxidation with NADH generation to improve carbon efficiency. By deleting AOX2 and overexpressing ADH2, we generated Adh2-based autotrophic strains that exhibited growth rates comparable to the parental strain (0.007 h-{superscript 1}), while reducing specific CO2 production by 53% and increasing biomass yield (YX/MeOH) by 59%. We further applied this strategy to convert previously developed autotrophic strains producing itaconic acid and lactic acid into Adh2-dependent strains. Optimizing ADH2 expression through multicopy integration resulted in strains with approximately two-fold higher molar carbon efficiency (Y(X+P)/CO2) while achieving elevated product titers--2.2-fold for itaconic acid and 3.8-fold for lactic acid--relative to the parental strains. Our findings demonstrate that alcohol dehydrogenase-mediated methanol dissimilation can significantly improve yield and productivity of autotrophic K. phaffii strains, with broad implications for sustainable bioproduction from one-carbon substrates.

Crop loss due to infection by pests and pathogens is a major barrier to the large-scale production of algal biofuels. Test systems have seen loss of green algae crops due to infection by the fungus-like Amoeboaphelidium occidentale FD01. While current antifungal compounds are effective in inhibiting the infection, their application raises the overall cost of the crop and lowers its economic viability as a biofuel source. Here we show that co-culturing environmentally harvested bacteria alongside algae crops can drastically lower the rate of infection in two different green algae species of interest for biofuel production. These bacteria-algae consortia increase the mean time to crop failure (MTTF) by up to 350% when tested under environmentally relevant conditions. While there was an increase in diversity over time, there was no statistically significant correlation between an increase in diversity and a longer MTTF. Community composition analysis reveals similarities between the bacterial genera growing alongside both green algae species even as bacterial harvest locations differed, although there was not a single dominant genus responsible for the increase in crop protection. These results show a promising new method of anti-fungal crop protection that can be applied to algal biofuels with no increase in fuel cost. HighlightsO_LIBacteria-algal cocultures protect against fungal pests without impact to productivity C_LIO_LIBacterial community composition is variable over time even as protection persists C_LIO_LIBacterial consortia can increase mean time to failure by 350% C_LI

Current mechanical and chemical recycling strategies address less than 10% of global plastic waste, necessitating alternative valorization routes. Biological upcycling via enzymatic depolymerization combined with microbial conversion of the resulting monomers offers a promising pathway to transform mixed plastic waste into valuable alternatives. Here, we employed a single engineered Pseudomonas putida KT2440 for simultaneous co-utilization of five plastic monomers including ethylene glycol, terephthalic acid, adipic acid, 1,4-butanediol, and L-lactic acid, which can be derived from enzymatic hydrolysis of polyethylene terephthalate (PET), polybutylene adipate-co-terephthalate (PBAT), polyester-polyurethanes (PUs), and polylactic acid (PLA). Continuous fermentation over 21 days with alternating mixed-monomer feeds achieved steady state growth and complete substrate depletion, yielding adaptive mutations that informed iterative strain improvement. Further engineering enabled the biosynthesis of (R)-3-hydroxybutyrate (R-3HB), and 0.70 g L-1 R-3HB was produced directly from enzymatic hydrolysates of blended PET, PBAT, and TPU. These results establish a viable bio-based approach for upcycling realistic mixed plastics into value-added bioproducts.

Mushroom production generates large amounts of by-products, particularly stipes, which can represent up to half of the fruiting body biomass. Due to their similar composition to mushroom caps, these residues represent a promising substrate for the development of value-added foods. In this study, oyster mushroom stipes were used as a substrate for solid-state fermentation (SSF) with a Neurospora crassa strain isolated in Albacete to produce a novel meat analogue inspired by the oncom. Fermentation generated a cohesive matrix bound by hyphae that adopted the shape of the mold and exhibited a meat-like color, although with a softer texture. Nutritional analysis revealed a product with relatively low protein content but a complete amino acid profile, enriched in dietary fiber and containing unsaturated fatty acids. These results demonstrate that SSF with N. crassa provides a strategy to upcycle oyster mushroom by-products into fiber-rich meat analogues with potential applications in sustainable food systems.

Despite its industrial importance, microbial L-lysine production has largely been confined to classical producer strains, leaving the fast-growing, non-pathogenic marine microorganism V. natriegens largely untapped as an unconventional biosynthetic platform. In this work, we established an L-lysine-overproducing V. natriegens DSM759 strain through a step-wise, systematic rational engineering strategy targeting the native biosynthetic pathway. Guided by our prior systems-level analysis of the strains genetic and regulatory architecture, we identified key metabolic bottlenecks and implemented knowledge-driven interventions to relieve pathway constraints. Central to production was alleviation of feedback inhibition in the native key regulatory enzymes, aspartate kinase (AK, lysC) and dihydrodipicolinate synthase (DHDPS, dapA). Site-directed amino-acid substitutions, replicating established E. coli feedback-resistance mechanisms, were introduced into conserved regions of the V. natriegens DSM759 enzymes, producing L-lysine-insensitive variants with kinetic parameters comparable to that of corresponding wild type enzymes. Among the tested configurations, the strain co-expressing Vn.lysC2 and Vn.dapA1:E84T reached the highest L-lysine titer (9.0{+/-}0.6 mM) and yield (0.11{+/-}0.01 molLys molGlc-1), whereas overexpression of additional L-lysine pathway genes provided no further benefit. Leveraging the hosts metabolic versatility, L-lysine synthesis was also demonstrated from the chitin-derived amino-sugar N-acetylglucosamine (0.09{+/-}0.00 molLys molGlcNAc-1), highlighting the potential to valorize chitin-rich waste streams from the seafood industry. This work establishes a minimal, rational strategy for L-lysine biosynthesis in V. natriegens DSM759 and positions it as a promising platform for sustainable amino acid production.

Cyanobacteria have garnered interest as promising biological platforms for producing renewable biofuel, chemical feedstock, and bioactive molecules. For biotechnology applications, robust well-characterized genetic tools are required for genetically modifying cyanobacteria, but these tools are often developed for specific model strains. Here, we used broad host-range RSF1010-based plasmids to characterize a set of orthogonal constitutive promoters in diverse cyanobacterial strains. The promoters are random variants of the synthetic Escherichia coli PconII promoter. A library of PconII promoters driving a fluorescent reporter gene was first evaluated in Synechococcus elongatus and found to have a wide range of gene expression levels. A set of 25 promoter variants with graded strengths was selected after characterization in S. elongatus and three additional model cyanobacterial strains. To demonstrate the utility of these promoters, we isolated new genetically tractable cyanobacterial strains with high salt and alkalinity tolerance and transferred the subset of promoters into one of these newly isolated strains. Similar to the results with model strains, the subset of promoters had a wide range of expression levels in the non-model strain. These characterized promoters expand the genetic tools available for genetic engineering of model and non-model cyanobacterial strains. ImportanceThe use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications. Research AreasMicrobial genetics, plasmids and other genetic constructs, biotechnology Journal SecctionBiotechnology

Odd-chain carboxylates such as valerate and heptanoate are ecologically relevant metabolites and promising platform chemicals, yet the factors leading to their formation during secondary lactate fermentations remain poorly understood. Here, a continuous anaerobic bioreactor was operated for 297 days under mildly acidic conditions to evaluate how lactate:propionate molar ratios shape product spectrum in lactate fermentations. Valerate was the predominant odd-chain product under all conditions, reaching concentrations up to 110 mM, while heptanoate accumulated only at low levels (<10 mM). At low lactate concentrations (10-20 g/L), product selectivity strongly depended on the lactate:propionate ratio. When lactate:propionate ratios were around 1.2 mol/mol, odd-chain products were favored, whereas higher ratios (up to 4.8 mol/mol) shifted metabolism toward caproate and butyrate formation. However, this trend was not maintained at higher lactate concentrations (30-40 g/L; lactate not fully consumed), where odd-chain selectivities remained high even at lactate:propionate ratios of 4.8 mol/mol. Pathway analysis indicated that under high-lactate conditions up to 30% of lactate was redirected toward propionate and acetate formation, likely via the acrylate pathway. Microbial community analysis revealed a stable dominance of Caproiciproducens spp., that could be correlated to valerate production. Overall, this work provides mechanistic insights into the ecology of lactate fermentations and offers a framework for steering product selectivity in engineered anaerobic systems. HighlightsValerate was the dominant product, reaching up to 110 mM. Lactate:propionate ratios drive product selectivities. High lactate concentrations activated in situ propionate formation pathways. Caproiciproducens dominance was associated with sustained valerate production.

Prodigiosin is a red pigment produced by various bacteria, including Serratia marcescens. Despite its wide and promising range of biological activities, the large-scale production of prodigiosin is currently limited by its high cost and low yields. Here we propose and optimize an innovative, low-cost, peanut-based solid culture medium that enhances the yield of prodigiosin produced by Serratia marcescens. Colorimetric assays revealed that peanut significantly stimulates prodigiosin synthesis. Further HPLC-MS analysis allowed us to unambiguously identify prodigiosin and shows that our medium specifically improves the yield of prodigiosin. Overall, our innovative culture medium could help lower prodigiosin production costs and, ultimately, open new industrial applications.