Biotechnology for Biofuels最新文献

Background

Glycoside Hydrolase family 2 (GH2) is one of the largest and most functionally diverse carbohydrate-active enzyme families. This functional diversity is an obstacle to accurate functional prediction by family assignment and has led to the accumulation of erroneous annotations in non-curated databases.

Results

We explored the sequence space of the GH2 family using Sequence-Similarity Networks coupled with closeness centrality to identify 23 subfamilies. The analysis suggests that the GH2 family evolved via multiple duplications followed by neofunctionalization events, with two main activities, β-glucuronidase and β-galacturonidase, re-emerging from likely flexible/reversible ancestors, while an early diverging branch gave birth to several subfamilies with unique activities. To increase the predictive power of subfamily assignments, we biochemically characterized seven members of four of the five subfamilies without previously reported activity.

Conclusions

The GH2 subfamilies showing high functional homogeneity will enable more precise functional predictions, while our work highlights subfamilies that require further biochemical and structural investigations.

To stay within the planetary boundaries circularizing economy by utilizing residues is key. Bioprocesses can use abundant, but complex biogenic residues, giving access to various value-added products. To advance circularization, the feasibility of exploiting diverse biogenic residues as feedstocks for different, yet specific, bioprocesses needs to be assessed. Exemplifying the national level in Germany, we categorized biogenic residues compiled in the DE Biomass Monitor regarding their composition and feedstock potential in a resource matrix, detailing their constituents and the quality of available data. Three biotechnological processes, making use of lignin, non-fibrous carbohydrates, and oil, respectively, served as model processes to assess the biogenic production potential. By developing material flows based on state-of-the-art conversion routes, we found that residue-based production via all three example processes could meet national demands of specific polymer bricks, medium chain carboxylates, and platform chemicals, respectively, when mobilizing only 20–30% of possible raw materials. The accruing side streams underline the importance of cluster approaches early in bioprocess development. Specific challenges for fully exploiting the potential of biogenic residues were identified, including legal and acceptance issues, the need for considered biomass decomposition in interweaved production lines, and residue availability and management. This study provides an example-based framework for integrating biogenic residues with biotechnological production, using the resource matrix and an initial material-to-product estimation to advance a circular bioeconomy.

Background

Overcoming lignocellulose recalcitrance to enzymatic or chemical processing is a prerequisite for biorefinery applications. Expansins and loosenins are non-lytic proteins that could assist reducing this recalcitrance by disrupting the intermolecular contacts between plant cell wall components. Here, immunolocalization with fluorescence and transmission electron microscopy (TEM) were used to study the ability of a Bacillus subtilis expansin-like protein (BsEXLX1), a Phanerochaete carnosa loosenin protein (PcaLOOL12) and a fusion protein of PcaLOOL12 with the carbohydrate-binding module 63 (CBM63) of BsEXLX1 (i.e., PcaLOOL12-CBM63) to bind secondary cell walls (SCW) of aspen fibres, including fresh aspen wood, milled wood fibres (MWF) and MWF subjected to subcritical water extraction.

Results

The immunofluorescence labelling of fresh wood samples showed a weak signal for PcaLOOL12 and a strong signal for BsEXLX1 and PcaLOOL12-CBM63, suggesting the importance of CBM63 for protein adsorption to SCW components. TEM analysis after immunogold labelling revealed the presence of BsEXLX1 and PcaLOOL12-CBM63 in all secondary cell wall layers. Pretreatment of wood samples with the proteins reduced the binding of glucomannan- and glucuronoxylan-specific monoclonal antibodies. Similarly, protein adsorption to MWF was higher before subcritical water extraction. Together, these results suggest the adsorption of BsEXLX1 and PcaLOOL12-CBM63 to SCWs was mediated at least in part by their interaction with hemicelluloses.

Conclusions

Our study demonstrates that microbial expansin-related proteins can bind to the secondary walls of aspen wood through potential interaction of CBM63 with hemicelluloses.

This study introduces a novel hybrid photobioreactor system that integrates an open raceway pond (ORWP) with a Nested-bottled photobioreactor (NB-PBR) in a closed-loop configuration to enhance microalgal biomass production and CO₂ fixation. The system facilitates continuous culture circulation, improving mass transfer and mixing efficiency while ensuring optimal light exposure and CO₂ dissolution. This design resulted in a 38% increase in dry mass (3.1 g/L) and improved mass transfer and mixing times by 16.6% and 15.3%, respectively. The optimized cultivation conditions led to a 39.9% enhancement in CO₂ fixation and an 8.7% increase in photosynthetic efficiency (Fv/Fm) compared to traditional systems. The strategic movement of poorly illuminated ORWP to the NB-PBR maximized light absorption and nutrient uptake, significantly boosting overall productivity. These findings highlight the potential of hybrid photobioreactor systems in improving microalgal growth efficiency and advancing sustainable algal cultivation for commercial applications.

Background

Many industrial applications of wood and woody biomass require harsh physicochemical pretreatments to improve the hydrophobicity and durability of the products. Environmentally friendly wood biorefineries necessitate the replacement of chemicals and energy-consuming wood processing. Here, our goal was to increase wood hydrophobicity via the ectopic expression of Jojoba (Simmondsia chinensis) wax ester synthase (ScWS) in poplar (Populus × canescens). We expressed ScWS under a wood-specific promoter (DX15), which naturally controls the expression of FASCICLIN-like ARABINOGALACTAN PROTEIN 15 (FLA15) in the xylem.

Results

In the DX15::ScWS lines, ScWS was highly expressed in wood but not in leaves. The transgenic lines exhibited normal photosynthesis and growth similar to the wild-type poplars. Compared with the wild-type poplars, the DX15::ScWS lines accumulated greater amounts of triacylglycerol in wood and a greater number of lipid droplets in ray parenchyma cells. The composition of the bark cuticle wax esters was unaffected. The wood of the DX15::ScWS lines showed greater water repellency and less swelling than that of the wild-type poplars. Furthermore, the DX15::ScWS lines had an increased expression of FLA15 and increased cell wall deposition in fibers, resulting in increased wood density.

Conclusions

Our results highlight the potential of combining the wood-specific DX15 promoter with ScWS to enhance the technological properties of poplar wood. Reduced wood hydrophilicity represents a significant improvement in wood quality. In addition, our results suggest that the overexpression of the DX15 promoter could be a promising strategy for improving lignocellulose biomass in plants. Since poplars are highly productive species that can be cultivated in short-rotation plantations, our results have high translational potential for advancing sustainable wood utilization for a wider range of applications.

Background

Itaconic acid is a valuable platform chemical with applications in polymer synthesis and other industrial sectors. Microbial fermentation offers a sustainable production route, involving two fungi such as Aspergillus terreus and Ustilago maydis. However, their employment in industrial bioprocesses for itaconic acid production is characterized by several challenges. Yarrowia lipolytica is a non-conventional yeast that shows suitability for industrial production and it is widely employed as heterologous host to obtain relevant metabolites. This study aimed to engineer Y. lipolytica for the selective production of itaconic acid by optimising intracellular metabolic fluxes and transport mechanisms.

Results

A metabolic engineering strategy was developed to prevent the secretion of citric and isocitric acids by blocking their transport at both mitochondrial and plasma membrane levels in Y. lipolytica strains. Specifically, the inactivation of YlYHM2 and YlCEX1 genes reduced secretion of citric and isocitric acid, enabling their accumulation in the mitochondria. Additionally, heterologous transporters from Aspergillus terreus (mttA and mfsA) and Ustilago maydis (mtt1 and itp1) were introduced to enhance the mitochondrial export of cis-aconitate and the extracellular secretion of itaconic acid. For the first time, complete gene set of the itaconate biosynthetic pathways from both fungal species were functionally expressed and compared in a yeast system with a similar genetic background. A synergistic increase in itaconic acid production was observed when both pathways were co-expressed, combined with the inactivation of native citric and isocitric transport. In contrast to previously engineered Y. lipolytica strains for itaconic acid production, the optimised strain obtained in this study does not require complex or nutrient-rich media, while achieving the highest product yield (0.343 mol IA/mol glucose) and productivity (0.256 g/L/h) reported in yeast, with minimal by-product formation.

Conclusions

By integrating transporter engineering and pathway diversification, this study demonstrates an effective strategy to enhance itaconic acid production in Y. lipolytica while minimising by-product formation. The findings provide new insights into organic acid transport in yeast and open avenues for further optimization of microbial cell factories for sustainable biochemical production.

Graphical Abstract

Isoprenoids constitute a large and various number of bio-compounds, with many profitable applications in pharmaceutical, nutraceutical, and industrial fields. The complexity of isoprenoid molecules leads to a challenging, expensive, and environmentally unfriendly chemical synthesis of these metabolites. In addition, the awareness and desire of many consumers for products generated by natural microbial processes has increased recently. Metabolic engineering tools and synthetic biology strategies have been used as a means for the enhancement and optimization of the natural isoprenoid biosynthetic pathways of wild strains. Microalgae as production organisms have been manipulated for the bioproduction of diverse isoprenoids. Particularly when cultivated in unsuitable conditions (such as wastewater, unbalanced nutritional sources, and distinct environmental conditions), microalgae can adjust their metabolic pathways and generate compounds with significant technological potential. Several metabolic engineering approaches have been developed, modifying the metabolic pathways in microalgae to redirect the flow of carbon toward isoprenoid biosynthesis, including pathway engineering, strain improvement, and synthetic biology. In this review, some beneficial features of these high-value metabolites are summarized. Besides, recent advancements in metabolic engineering approaches for the biosynthesis of isoprenoids are discussed in detail. At last, the viewpoints and challenges for the biosynthesis of novel compositions with isoprene units in the microalgae are also included.

Background

Filamentous fungi form a range of macromorphologies during submerged cultivation including dispersed mycelia, loose clumps, and pellets. Macromorphological development is usually heterogenous, whereby mixtures form due to a complex interplay of growth, aggregation, and fragmentation. Submerged macromorphology strongly impacts product titres and rheological performance. Nevertheless, studies that systematically investigate the quantitative effect of cultivation parameters on macromorphology and heterogeneity are lacking.

Results

In this study, we have developed shake flask cultivation conditions which enable reproducible macromorphological control of the multipurpose cell factory Aspergillus niger. Tested culture parameters included various spore titres, concentration of talc microparticles, shaking frequency, and presence/absence of baffles (n = 48 conditions). We quantified macromorphology (e.g., pellet diameter) using high-throughput two-dimensional image analysis and report intra-flask heterogeneity and flask-to-flask variation. These data identified optimal culture conditions which cause minimal macromorphological variation within individual flasks and between technical replicates. We demonstrate that pellet diameter can be reproducibly adjusted between experiments using simple cultivation conditions, and use these parameters to prove larger pellets secrete more protein while consuming less glucose. Linear regression models allowed us to identify spore concentration, shaking frequency, and talc concentration as crucial parameters impacting pellet diameter. Finally, we used a newly developed microtomography (µ-CT) approach to quantify the three-dimensional internal architecture for thousands of pellets at the cellular level. Cultivation conditions drastically impacted internal architecture. For the first time we report distinct types of pellets- those formed from a single (I) or multi-spore (II) core, and additionally pellets formed by agglomeration of mature pellets (III). Remarkably, these data show that a pellet of 2 mm consists of up to about 30 m of total hyphal length and contain approximately 200,000 tips.

Conclusions

This study identifies simple methods for adjusting macromorphology and heterogeneity, which will enable facile testing of different macromorphologies for maximizing product titres. For the first time we have investigated how pellet internal architecture is impacted by numerous culture parameters. We propose a new pellet classification system based on internal spore core architecture, thus broadening our understanding of fungal macromorphological development and opening up new avenues for bioprocess or strain engineering.

Background

A key objective in microbial biorefinery technologies is to identify resilient microorganisms capable of simultaneously synthesizing diverse bioactive metabolites. Among these, Rhodotorula yeasts emerge as promising candidates for converting various waste streams and by-products into high-value chemicals. Their industrial potential stems from their ability to accumulate significant amounts of lipids and carotenoids while also secreting extracellular polymers such as exopolysaccharides, polyol esters of fatty acids, glycolipids, and enzymes—many of which remain to be fully characterized.

Results

Among the five Rhodotorula strains tested, three exhibited substantial exopolysaccharide production. Notably, Rhodotorula graminis CCY 20-2-47 strain was identified, for the first time, to produce two distinct extracellular biopolymers—exopolysaccharides or polyol esters of fatty acids—depending on the growth conditions. It was observed enhanced production of exopolysaccharides up to 7.2 g L⁻¹ and 14.7 g L⁻¹ lipid-rich biomass by Rhodotorula graminis CCY 20-2-47 using lignocellulose hydrolysate and urea by-product. This study, for the first time, reports triggering effect of Mn²⁺ on exopolysaccharide production in Rhodotorula. Glucose-based medium resulted in co-production of polyol esters of fatty acids (3.9 g L⁻¹) and lipid-rich biomass (15 g L⁻¹) for Rhodotorula graminis CCY 20-2-47. Batch bioreactor fermentation for Rhodotorula graminis CCY 20-2-47 resulted in production of 13.1 g L⁻¹ of exopolysaccharides and 50% w/w intracellular lipids when using lignocellulose hydrolysate and urea by-product. In contrast, 7.4 g L⁻¹ of polyol esters of fatty acids and 35% w/w intracellular lipids were produced by the same strain on medium with pure glucose.

Conclusions

In conclusion, Rhodotorula yeasts demonstrate significant potential for microbial biorefineries due to their ability to efficiently convert diverse waste substrates into valuable biomaterials, including lipids and extracellular biopolymers. This study provides new insights into a potential metabolic switch in extracellular polymer biosynthesis, driven by Mn²⁺ availability in the culture medium.

Graphical abstract