Bioresource Technology

{beta}-1,3-Glucan phosphorylases capable of utilizing glucose as a priming substrate are key biocatalysts for the synthesis of functional {beta}-1,3-glucan. In this study, we identified THA_1941 from Thermosipho africanus (Ta{beta}GP) as a GH161 {beta}-1,3-glucan phosphorylase exhibiting robust synthetic activity towards glucose, as confirmed by 13C nuclear magnetic resonance, liquid chromatography-mass spectrometry, and sequence and structural analyses. Ta{beta}GP displayed exceptional thermostability, retaining 93% of its activity at 60 {degrees}C for 180 h, and showed broad pH tolerance ranging from pH 5.0 to 10.0, surpassing the performance of previously reported homologs. In addition, Ta{beta}GP exhibited broad substrate flexibility, accepting both - and {beta}-linked disaccharides, and demonstrated strong resistance to metal ions and lignocellulose-derived inhibitors. In the presence of 150 mM glucose 1-phosphate as the donor substrate, Ta{beta}GP synthesized {beta}-1,3-glucan with a tunable average degree of polymerization (10-32), depending on the concentration of glucose used as the primer. The combination of thermostability, inhibitor resistance, and substrate versatility makes Ta{beta}GP a promising biocatalyst for the economically viable and environmentally sustainable synthesis of {beta}-1,3-glucan from non-food biomass sources.

Raffinose family oligosaccharides (RFOs) are abundant in side streams from food and feed production from legumes, and the transition to plant-based diets increases the volume of such side streams. RFOs in the diet tend to have negative impacts on the consumers gut (e.g., nausea, bloating, diarrhoea), and in many ways, RFOs are comparable to lactose as a side stream from the dairy industry and symptoms associated with lactose intolerance. On the contrary, galactooligosaccharides (GOS) are recognized as prebiotics, and in this study we used a {beta}-galactosidase from Niallia circulans to produce potential prebiotics from RFOs (acceptors) and lactose (donor), which we hypothesized to have a lower fermentability than unmodified RFOs. The transglycosylation reactions resulted in RFO-based -{beta}-GOS, with NMR characterization showing ({beta}1-4) galactosylations on the non-reducing galactose end of RFOs as the major product. In reactions with RFOs, the characteristics were comparable to reactions with lactose alone and the new -{beta}-GOS products made up the largest fraction (by weight). A screening of 11 relevant gut and food microbe strains revealed that the gut commensal Bacteroides ovatus metabolised these modified oligosaccharides for growth whereas other strains grew only after adaption and others did not use them at all. This implies that mixed-linkage -{beta}-GOS are less fermentable by some microbes compared to raffinose, while other (beneficial) bacteria can still ferment them. The enzymatic synthesis established here is an interesting approach to upgrade abundant food side streams towards new prebiotics in a world where functional foods and food waste reduction receive increasing attention. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/731070v1_ufig1.gif" ALT="Figure 1000"> View larger version (22K): org.highwire.dtl.DTLVardef@18e0e62org.highwire.dtl.DTLVardef@1525b4borg.highwire.dtl.DTLVardef@1e7be88org.highwire.dtl.DTLVardef@18df278_HPS_FORMAT_FIGEXP M_FIG C_FIG

To understand how extracellular polymer substances (EPS) as additives promotes methanogenesis, batch tests of methane production potential in anaerobic reactors with the addition of EPS or not were conducted. Research showed that EPS increased remarkably methane production during anaerobic digestion (36.5% increase compared with the control). EPS enriched functional microorganisms such as Firmicutes, Actinobacteria, Synergistetes, and Chloroflexi. Among them, 8.86% OTUs from the important hydrolysis and acidification phyla, which may be an important reason for the enhanced methanogenic capacity of anaerobic granular sludge. Additionally, EPS also improved the abundance of cytochrome c (c-Cyts), accelerating the direct interspecies electron transfer (DIET) between syntrophic bacteria and methanogens, thus enhancing the methane production. Interestingly, the average particle size, volatile suspended solids/total suspended solids (VSS/TSS) and EPS content of anaerobic granular sludge (AnGS) in the EPS reactor were approximately equal to that of the control reactor during the anaerobic digestion, illustrating that EPS could not affect the physicochemical properties of AnGS. Therefore, these results suggested that EPS mainly played a role in the form of conductive materials in the anaerobic digestion process. Compared with conductive materials, EPS as biomass conductive materials was not only environmentally friendly and economical but also no secondary pollution.\n\nImportanceCompared with the reported conductive materials, EPS has the potential of biodegradation, electron transfer and no significant secondary pollution. Besides, there are few studies on the utilization of EPS resources, especially the effect of EPS as an additive on anaerobic digestion performance. To clarify whether EPS as conductive materials or carbon source promotes methanogenesis. Therefore, in this study, we investigated the influence of EPS as an additive on the methanogenic capacity, physical-chemical properties, microbial community structure and metabolic function of anaerobic granular sludge (AnGS), and preliminarily expatiate the influence mechanism of EPS as an additive on methanogenesis. At the meantime, the research is expected to provide new solutions for the improvement of anaerobic digestion performance and disposal of waste mud.

Plastic pollution from fossil-based materials is a major global environmental challenge. Microbial-derived bioplastics, such as polyhydroxybutyrate (PHB), offer a promising biodegradable alternative. However, the high substrate and operational costs of PHB production remain a major barrier to large-scale deployment. Optimizing PHB synthesis requires navigating a multidimensional design space of metabolic, nutritional, and operational variables that is impractical to explore experimentally. Here, we developed an integrated computational framework that combines a genome-scale metabolic model (GEM) of Rhodopseudomonas palustris, machine-learning surrogate modeling, Pareto multi-objective optimization, and thermodynamics-based flux analysis (TFA) to identify cost-efficient and biologically feasible PHB production strategies. Experimental and literature-derived medium compositions were translated into mechanistic constraints, enabling the GEM to generate metabolically coherent synthetic datasets that augmented sparse experimental observations. CatBoost surrogate models trained on this hybrid dataset accurately predicted PHB synthesis across thousands of hypothetical conditions, and Pareto optimization revealed operating regimes that balance PHB productivity with nutrient cost. TFA validated the thermodynamic feasibility of these strategies and refined pathway usage, reinforcing thiolase-initiated routing into PHB biosynthesis and suppressing infeasible {beta}-oxidation-like redox loops. Overall, this hybrid GEM-ML-TFA framework identifies metabolic bottlenecks, engineering targets, and cost-optimal nutrient regimes for phototrophic PHB production, providing a scalable blueprint for rational process and strain design.

The increasing accumulation of polyethylene terephthalate (PET) waste has prompted the development of sustainable biotechnological strategies for its degradation and valorisation. This study presents an integrated approach combining enzymatic PET depolymerization by Yarrowia lipolytica, engineered to express and secrete the cutinase HiC and the lipase CalB, with the microbial valorization of PET-derived monomers, terephthalic acid (TPA) and ethylene glycol (EG), by Rhodococcus jostii RHA1. Y. lipolytica was further engineered for xylose metabolism, enabling enzyme production from low-cost lignocellulose-derived substrates. Enzymatic assays with HiC and CalB crudes effectively hydrolysate PET to TPA and EG, demonstrating functional enzymatic activity without purification steps. In addition, R. jostii RHA1 was able to use as substrate the released monomers and accumulated intracellular lipids. Overall, this work demonstrates the feasibility of coupling the production of PET degrading enzymes and microbial lipid, using an abundant monosaccharide, with the assimilation of the PET degradation products to also produce microbial lipids. This modular system provides a promising framework for the sustainable upcycling of plastic waste into value-added bioproducts within a circular economy.

The primary challenge in utilizing palm kernel meal (PKM, an agricultural by-product) as non- ruminant livestock feed is its high fibre content, predominantly in the form of mannan. Microbial fermentation offers an economically favourable alternative to enzyme supplementation for breaking down fibre in lignocellulosic biomass. In a recent study, we have isolated and characterized an undomesticated strain (Bacillus subtilis F6) that is able to secrete mannanase. In this work, the mannanase production was substantially improved by optimizing multiple regulatory elements controlling the mannanase expression. Mannanase GmuG, sourced from B. subtilis F6 and verified for its hydrolytic activity on PKM fibre, was expressed using a replicative plasmid (pBE-S). The recombinant strain of B. subtilis F6 exhibited 1.9-fold increase in the mannanase activity during solid-state fermentation. Optimization of signal peptide and ribosome binding site further enhanced mannanase activity by 3.1-fold. Subsequently, promoter screening based on highly transcribed genes in B. subtilis F6 resulted in a significant 5.4-fold improvement in mannanase activity under the nprE promoter. The nprE promoter was further refined by eliminating specific transcription factor binding sites, enhancing the mannanase activity further by 1.8-fold. Notably, a substantial 35-40% reduction in PKM fibre content was observed after 30 h of fermentation using the recombinant strains. Lastly, the highest mannanase-producing strain was examined for scaled-up fermentation. The impacts of fermentation on fibre and protein contents, as well as the surface morphology of PKM, were analysed. The outcomes of this study offer an efficient method for robust mannanase expression in B. subtilis and its potential application in the biotransformation of PKM and other mannan-rich bioresources for improved feed utilization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/602432v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@10fbb9corg.highwire.dtl.DTLVardef@1e619fborg.highwire.dtl.DTLVardef@1b3bc0corg.highwire.dtl.DTLVardef@fec816_HPS_FORMAT_FIGEXP M_FIG C_FIG

The accumulation of plastic waste necessitates innovative strategies that convert polymer carbon into value-added products. Here, we present a sequential yeast-bacterial workflow for the integrated valorisation of glycerol and amorphous polyethylene terephthalate (amPET) into triacyl glyceride (TAGs) and polyhydroxyalkanoates (PHAs). First, an engineered obese strain of Yarrowia lipolytica was cultivated on glycerol, for the simultaneous production of intracellular lipids, up to 42.9% of its cell dry weight, and secretion of a PET-depolymerizing enzymatic cocktail, composed of the cutinase from Mycothermus thermophilus (HiC) and the lipase B from Moesziomyces antarcticus (CALB). The resulting enzymatic crude hydrolysed amPET, and the released degradation products--terephthalic acid (TPA) and ethylene glycol (EG)-- served as feedstocks for a bacterial consortium composed of Comamonas testosteroni RW31 and Pseudomonas putida JM37, which naturally assimilate TPA and EG, respectively. This consortium successfully upcycled the released monomers into intracellular polyhydroxybutyrate (PHB) and medium-chain-length PHAs. Furthermore, fluorescent strains of both bacteria enabled the development of a semi-quantitative method for monitoring the consortium population dynamics. Overall, this study provides a robust proof-of-concept for a circular bioeconomy approach, successfully coupling glycerol-based enzyme and lipid production with the downstream biological conversion of PET-derived monomers into valuable bioplastics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/729029v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1575eedorg.highwire.dtl.DTLVardef@1195aeborg.highwire.dtl.DTLVardef@1a01bb0org.highwire.dtl.DTLVardef@79f4f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cellobiose is an important disaccharide used in food, health, and biorefinery applications, but its efficient enzymatic production from crystalline cellulose remains challenging. In this study, crystalline cellulose was dissolved in ionic liquids and regenerated by dilution, and subsequently hydrolyzed at 80{degrees}C using a highly thermostable cellobiohydrolase, HmCel6A-3SNP. The enzyme retained activity in the presence of low concentrations of ionic liquids. Among the pretreatment conditions tested, cellulose treated with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) showed the highest enzymatic digestibility. After washing to remove residual ionic liquid, the reaction produced reducing sugars at levels 1.5-fold higher than those obtained in the presence of 10% [Bmim]Cl, with cellobiose accounting for approximately 96% of the products. Under the optimized conditions, the hydrolysis yield reached [~]36% after 48 hr. Structural analyses using birefringence imaging, electron microscopy, and Fourier transform infrared spectroscopy indicated that higher-order structural changes in regenerated cellulose strongly influence enzymatic accessibility. These results demonstrate the potential of combining ionic-liquid pretreatment with thermostable enzymes for selective cellobiose production from cellulose.

Extremophilic red microalgae are promising platforms for sustainable biotechnology, combining robust growth under selective thermoacidophilic conditions with production of thermostable phycobiliproteins and carbon-rich biomass. However, reactor-dependent effects on growth, product formation and biomass composition remain insufficiently resolved. Here, we systematically evaluated the extremophilic red microalga Cyanidioschyzon merolae across cultivation scales and reactor formats and benchmarked its performance against the well-established Galdieria javensis and Limnospira platensis. In small-scale multi-cultivator photobioreactors and microfluidic growth chambers, C. merolae showed superior growth, reaching a maximum growth rate of 0.034 {+/-} 0.001 h-1 and 8.3 {+/-} 0.3 g l-1 cell dry weight. Microfluidic cultivation enabled growth analysis at single-cell resolution and matched growth rates obtained in photobioreactors. To identify scalable production strategies, C. merolae was further cultivated in a flat-panel photobioreactor and a custom-designed internally illuminated photobioreactor. The custom-designed photobioreactor delivered the highest biomass concentration and productivity, yielding 11.5 {+/-} 0.6 g l-1 cell dry weight and 1.07 {+/-} 0.06 g l-1 d-1, and comparable yields with regard to R-phycocyanin and R-allophycocyanin. Biomass analysis revealed substantial carbon and nitrogen contents, starch accumulation up to > 20 % of cell dry weight, and fatty acids dominated by palmitic, linoleic and oleic acids. Despite its reduced cell wall fraction, C. merolae contained structurally diverse, cultivation-dependent polysaccharides. These results establish C. merolae as a versatile chassis for thermostable pigment production and renewable feedstock generation, highlighting photobioreactor design as a key determinant of productivity and biomass quality.

PHB (poly-hydroxy-butyrate) represents a promising bioplastic variety with good biodegradation properties. Furthermore, PHB can be produced completely carbon-neutral when synthesized in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This model strain has a long history of various attempts to further boost its low amounts of produced intracellular PHB of ~15 % per cell-dry-weight (CDW). We have created a new strain that lacks the regulatory protein PirC (gene product of sll0944), which causes a rapid conversion of the intracellular glycogen pools to PHB under nutrient limiting conditions. To further improve the intracellular PHB content, two genes from the PHB metabolism, phaA and phaB from the known production strain Cupriavidus necator, were introduced under the regime of the strong promotor PpsbA2. The created strain, termed PPT1 ({Delta}sll0944-REphaAB), produced high amounts of PHB under continuous light as well under day-night rhythm. When grown in nitrogen and phosphor depleted medium, the cells produced up to 63 % / CDW. Upon the addition of acetate, the content was further increased to 81 % / CDW. The produced polymer consists of pure PHB, which is highly isotactic. The achieved amounts were the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.

Macroalgae (or seaweeds) are the dominant primary producers in marine vegetated coastal habitats and largely contribute to global ocean carbon fluxes. They also represent attractive renewable production platforms for biofuels, food, feed, and bioactives, notably due to their diverse and peculiar polysaccharides and carbohydrates. Among seaweeds, brown algae produce alginates and sulfated fucans as constituents of their cell wall, and the photoassimilates laminarin and mannitol for carbon storage. Availability of brown algal genomes, including those of the kelp Saccharina japonica and the filamentous Ectocarpus sp., has paved the way for biochemical characterization of recombinant enzymes involved in their polysaccharide and carbohydrates synthesis, notably mannitol. Biosynthesis of mannitol in brown algae starts from fructose-6-phospate, which is converted into mannitol-1-phosphate (M1P), and this intermediate is then hydrolysed by a haloacid dehalogenase type M1P phosphatase (M1Pase) to produce mannitol. We report here the biochemical characterization of a second M1Pase in Ectocarpus sp after heterologous expression in Escherichia coli. (EsM1Pase1). Our results show that both Ectocarpus M1Pases were redox sensitive, with EsM1Pase1 being active only in presence of reducing agent. Such catalytic properties have not been observed for any of the M1Pase characterized so far. EsM1Pases were specific to mannitol, in contrast to S. japonica M1Pases that can use other phosphorylated sugars as substrates. Finally, brown algal M1Pases grouped into two well-supported clades, with potential different subcellular localization and physiological role(s) under diverse environmental conditions and/or stages of life cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Polyethylene terephthalate (PET) waste remains a major environmental challenge due to its recalcitrance and low economic value. Here, we present an integrated biochemical approach that couples glycolysis with a synthetic microbial consortium to upcycle post-consumer PET (pcPET) into polyhydroxyalkanoates (PHA). Glycolysis efficiently depolymerized pcPET into bis(2-hydroxyethyl) terephthalate (BHET) in 2 h, circumventing the limitations of in vivo PET degradation. We engineered a two-species microbial consortium composed of Comamonas testosteroni RW31, able to metabolise terephthalic acid, and Pseudomonas putida JM37, able to consume ethylene glycol, each modified for the extracellular secretion of PET- and MHET-hydrolases, employing different plasmid architectures. This division of labour enabled rapid BHET hydrolysis and the subsequent upcycling of the released monomers into PHAs. The combination of the different strains allowed to select C. testosteroni pSEVA354-MHETase and P. putida pSEVA234-PETase as the best consortium, based on growth and PHAs content. Overall, this work proposes a strategy for PET waste depolymerisation and valorisation, highlighting the potential of mixed chemical and biological approaches and the use of non-conventional microbial chassis within engineered consortia.

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

The colony-forming, green microalga Botryococcus braunii secretes petroleum-like hydrocarbons, which enables the non-destructive continuous in situ extraction, milking, of these extracellular products during culture growth without cell lysis. This work compares the suitability of 15 different solvents, including alkanes, halogenated solvents, and green solvents, for in situ extraction of B. braunii race B (Showa strain) hydrocarbons after acclimation to moderate salinity stress. After 24 h of extraction, bio-based terpene green solvents such as {gamma}-terpinene showed the highest hydrocarbon recovery, around 10-fold greater than with conventional alkane solvents. Brominated alkanes and liquid perfluorocarbons (FCs) formed a lower phase to algal cultures rather than an upper phase as with other solvents, but only bromodecane effectively captured extracellular hydrocarbons similar to conventional alkane solvents. However, bromodecane and all green solvents were too toxic for two-phase continuous culture contact hydrocarbon milking, leading to 33-100% chlorophyll content loss. To overcome the biologically adverse effects of these solvents with suitable hydrocarbon recovery, future research should focus on their application in short-term extraction period milking systems to minimize algal-solvent contact and enable continuous extraction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/563540v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1072e6borg.highwire.dtl.DTLVardef@787c8forg.highwire.dtl.DTLVardef@39af4dorg.highwire.dtl.DTLVardef@123ed79_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 {+/-} 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 {+/-} 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/500214v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1662a84org.highwire.dtl.DTLVardef@717f66org.highwire.dtl.DTLVardef@1633b36org.highwire.dtl.DTLVardef@1c46c7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neurotoxin {beta}-N-methylamino-L-alanine (BMAA) has been deemed a pathogenic factor for human neurodegenerative diseases. It is an important issue to disclose the biosynthesis mechanism of BMAA in marine diatoms. In the present study, the iron (Fe) limitation (1/3 x Fe) was found to suppress the growth of diatoms but stimulate the production of BMAA-containing proteins, maximum 7.7 fold in Thalassiosira minima. Transcriptome analysis showed that energy metabolism, protein biosynthesis and carbon fixation functions were mainly affected by the Fe limitation in the diatom. Analysis of subcellular distribution of BMAA showed that BMAA-containing proteins were mainly detected in the endoplasmic reticulum and the Golgi apparatus. Combination results of the responses of the diatom to Fe deficiency and co-culture with cyanobacteria in our previous study, we speculate that cysteine embedded in peptide chains and methylamine produced by the diatom itself are possibly catalyzed by the cysteine synthase (cysK) to form the BMAA structure in situ. Spiked methylamine in culture media significantly stimulated the production of BMAA, and BMAA amounts were correlated with the expression of cysK gene in different diatoms. The reduced ubiquitination-mediated proteolysis and vesicle trafficking precision through the COPII system would aggravate the accumulation of BMAA-containing proteins in the diatom. Significance StatementWith the detection of neurotoxin BMAA in diverse marine diatoms, the pathogenic risk of BMAA has been further concerned to human neurodegenerative diseases such as Alzheimers disease. Interestingly, BMAA-containing proteins are the dominant forms of this neurotoxin in diatoms. It is a keystone issue to disclose the biosynthesis mechanism of BMAA in marine diatoms. We found Fe-limitation could stimulate the production of BMAA-containing proteins in diatoms and explored its biosynthesis using transcriptomics in this study. Results suggested that cysteine embedded in peptides and methylamine in cytoplasm were catalyzed by the cysteine synthase (cysK) to form BMAA. This study hints that the biosynthesis of BMAA would be improved by the worldwide prevalence of iron deficiency in the coastal waters.

Rhodosporidium toruloides has emerged as a prominent candidate for producing single-cell oil from cost-effective feedstocks. In this study, the capability of R. toruloides to produce punicic acid (PuA), a representative plant unusual fatty acid, was investigated. The introduction of acyl lipid desaturase and conjugase (PgFADX) allowed R. toruloides to accumulate 3.7% of total fatty acids as PuA. Delta-12 acyl lipid desaturase (PgFAD2) and diacylglycerol acyltransferase 2 were shown to benefit PuA production. The strain with PgFADX and PgFAD2 coexpression accumulated 12% of its lipids as PuA from glucose, which translated into a PuA titer of 451.6 mg/L in shake flask condition. Utilizing wood hydrolysate as the feedstock, this strain produced 6.4% PuA with a titer of 310 mg/L. Taken together, the results demonstrated that R. toruloides could serve as an ideal platform for the production of plant-derived high-value conjugated fatty acid using agricultural and forestry waste as feedstock.

Itaconic acid is a versatile bio-based platform chemical produced from sugar-based feedstocks, linking its production to arable land use. As global food demand rises, alternative carbon sources that decouple industrial biotechnology from agriculture are required. The C2 compound acetate can be derived from lignocellulosic biomass and industrial side streams. Emerging routes enable the direct synthesis of acetate from C1 carbon sources such as CO2, CO, and methane. Here, we show that the smut fungus Ustilago maydis can efficiently produce itaconic acid using acetate as the sole carbon source. To overcome weak-acid toxicity and pH-related stress, a combined pH-stat and DO-triggered feeding strategy was applied in a 1 L-scale fed-batch bioreactor, enabling an itaconate titer of 97 g L-1 and an overall yield of 0.41 g g-1. Key performance indicators were comparable to those of a glucose-based reference process. Despite substantially lower biomass formation on acetate, biomass-specific production rates were markedly higher than on glucose, indicating highly efficient channeling of carbon toward product formation. Overall, our results establish acetate as a competitive and sustainable feedstock for fungal itaconic acid production and position acetate-based processes as a viable route toward land-free biotechnology.

There is a strong need to develop technologies that reduce anthropogenic pollution and the dependence on nonrenewable Earth resources. One way of doing so is by harnessing biological systems for replacing the production of fossil-fuel based goods with low-environmental-impact alternatives. Recently, progress was made in engineering the model organism E. coli to grow using CO2 and formate as its only carbon and energy sources using the reductive glycine pathway (rGlyP). Here, we use this engineered strain of E. coli as a host system for the production of polyhydroxybutyrate (PHB), a biologically derived and biodegradable plastic. We confirmed the production of PHB in this strain using Nile red fluorescent microscopy, transmission electron microscopy, and GC measurements. Since formate can be efficiently generated from CO2 by electrochemical reduction using renewable energy sources, this study serves as a proof of concept for the emerging field of electro-bioproduction.

Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that carry pectinolytic enzymes. Pectate lyases that cleave -1,4-galacturonosidic linkage of pectin are widely used in industries, such as paper making and fruit softening. However, reports on pectate lyases with high thermostability are few. Two pectate lyases (CbPL3 and CbPL9) from a thermophilic bacterium Caldicellulosiruptor bescii were investigated. Although these two enzymes belonged to different families of polysaccharide lyase, both were Ca2+-dependent. Similar biochemical properties were shown under optimized conditions 80 {degrees}C-85 {degrees}C and pH 8-9. However, the degradation products on pectin and polygalacturonic acids (pGA) were different, revealing the distinct mode of action. A concanavalin A-like lectin/glucanase (CALG) domain, located in the N-terminus of two CbPLs, shares 100% amino acid identity. CALG-truncated mutant of CbPL9 showed lower activities than the wild-type, whereas the CbPL3 with CALG knock-out portion was reported with enhanced activities, thereby revealing the different roles of CALG in two CbPLs. I-TASSER predicted that the CALG in two CbPLs is structurally close to the family 66 carbohydrate binding module (CBM66). Furthermore, substrate-binding assay indicated that the catalytic domains in two CbPLs had strong affinities on pectate-related substrates, but CALG showed weak interaction with a number of lignocellulosic carbohydrates, except sodium carboxymethyl cellulose and sodium alginate. Finally, scanning electron microscope analysis and total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for degradation of plant biomass. ImportanceThermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium Caldicellulosiruptor bescii. The two enzymes had high optimum temperature, low optimum pH, and good thermostability at evaluated temperature. A family-66 carbohydrate binding module (CBM66) was identified in two CbPLs with sharing 100% amino acid identity. Deletion of CBM66 obviously decreased the activity of CbPL9, but increase the activity and thermostability of CbPL3, suggesting the different roles of CBM66 in two enzymes. Moreover, the degradation products by two CbPLs were different. These results revealed these enzymes could represent a potential pectate lyase for applications in paper and textile industries.