Biotechnology for Biofuels最新文献

Filamentous fungi can convert a wide variety of naturally occurring chemical compounds, including organic biomass and waste streams, into a range of products. They have long been used for industrial organic acid production and food preparation. In this review, we will discuss production of products such as organic acids, lipids, small molecules, enzymes, materials, and foods, and highlight advances in metabolic and protein engineering, including CRISPR-Cas9-mediated strain improvements. We discuss to what extent these products are already being made on a commercial scale, as well as what is still required to make certain promising concepts industrially and commercially relevant. Despite significant progress, the systematic application of synthetic biology to filamentous fungi remains in its infancy, with many opportunities for discovery and innovation as new strains and genetic tools are developed. The integration of fungal biotechnology into circular and bio-based economies promises to address critical challenges in waste management, resource sustainability, and the development of new materials for terrestrial and extraterrestrial applications, but requires further developments in genetic engineering and process design.

Background

Intensive agricultural practices are increasing the generation of by-products and wastes, which require appropriate management strategies to prevent environmental pollution and recover valuable resources. Waste-to-energy technologies, such as anaerobic digestion, are gaining attention for integrating local feedstocks to produce biofuels and biofertilisers, contributing to closed nutrient cycles. In the Mediterranean region, olive pomace is very abundant, but its intrinsic characteristics hinder the production of biogas via anaerobic digestion. Concurrently, the direct application of untreated pig manure on agricultural land continues to pose significant environmental risks.

Results

This study assessed the biomethane potential of olive pomace and pig manure, along with microbial population dynamics during the transition from mono- to co-digestion. Mono-digestion of olive pomace led to complete process inhibition, while co-digestion with pig manure increased methane yield more than fivefold (from 53 to 283 mL CH₄ g⁻¹ VS). Co-digestion also enhanced the microbial diversity, improving the ecosystem resilience and metabolic versatility. A notable increase in the relative abundance of methanogenic archaea, particularly Methanosarcina, was observed. An energy assessment indicated that a full-scale plant co-digesting olive pomace and pig manure could not only operate without external energy consumption, but also produce excess electricity (577 MWh y⁻¹) and heat (1074 MWh y⁻¹).

Conclusions

These findings demonstrate that co-digestion can overcome the limitations of olive pomace mono-digestion, enabling the effective treatment of two challenging agro-industrial by-products. This approach aligns with circular bioeconomy principles and supports the decarbonisation of the olive oil and pig farming sectors, contributing to the energy transition. The presented approach serves as a baseline scenario, and further research should focus on recovering high-value bioproducts and advancing towards integrated biorefinery systems in rural areas.

Graphical abstract

Background

Yarrowia lipolytica is a promising host for sustainable microbial oil production from waste-derived carboxylic acids such as acetate and propionate. Nonetheless, the molecular mechanisms underlying the metabolism and assimilation of these substrates, particularly under nitrogen limitation, are still not fully understood.

Results

We conducted a multi-condition transcriptomic analysis of Y. lipolytica strain EXF-17398 under nitrogen-limiting conditions to investigate its transcriptional adaptation to acetate and propionate utilisation. Our results revealed distinct transcriptional responses associated with metabolic adaptation, including the coordinated regulation of Jen and Gpr carboxylate transporter families, suggesting a dual system for carboxylate uptake. JEN5 and GPR1 appear central to propionate and acetate utilisation, respectively. Our data suggest that propionate toxicity is mitigated through its conversion via the methylcitrate cycle and potentially the malonate semialdehyde pathway, preventing accumulation of cytotoxic propionyl-CoA in the cytosol. The upregulation of carnitine acyltransferases suggests active mitochondrial transport of acyl-CoAs, linking detoxification with energy metabolism. Under tested conditions, the de novo lipid synthesis was consistent with carbon overflow from acetyl-CoA and propionyl-CoA, supported by intracellular nitrogen recycling and redox balancing, independent of classical nitrogen regulatory pathways.

Conclusions

These findings illustrate the capacity of Y. lipolytica to coordinate carbon and nitrogen metabolism during carboxylate utilisation, such as acetate and propionate, offering insights to guide the optimisation of microbial oil production from renewable feedstocks.

Natural microbial consortia exhibit characteristics of environmental robustness and the ability to utilize complex substrates. Inspired by them, various synthetic microbial consortia capable of utilizing economical substrates have been constructed. As an abundant renewable biomass resource, lignocellulose is regarded as the most promising raw material for replacing fossil energy to produce biofuels and bioproducts. The hydrolysis process of lignocellulose is a key step limiting its bioconversion. Filamentous fungi, due to their strong ability to secrete and hydrolyze cellulases, are widely used in synthetic microbial consortia to achieve lignocellulosic biorefinery. This paper reviews the latest progresses in the biosynthesis of biofuels and bioproducts from lignocellulose by various microbial consortia integrated with filamentous fungi. In addition, the construction strategies of synthetic microbial consortia integrated with filamentous fungi are also reviewed. Finally, the applications of filamentous fungi in synthetic microbial consortia are prospected.

Background

Efficient enzymatic hydrolysis of lignocellulosic biomass (LCB) is essential for maximizing the recovery of fermentable sugars for diverse biotechnological applications. However, pretreatment by-products including lignin interact with hydrolytic enzymes, blocking their access to substrates and leading to poor monomeric sugar recovery. This study evaluated the effects of all 20 exogenous amino acids (AAs) as additives to block lignin active sites and facilitate enzyme access to polysaccharide substrates for enhanced sugar recovery. The hydrolysates were subsequently tested for microbial lipid production by Rhodotorula toruloides CGMCC 2.1389 as a model application.

Results

The study found that most AAs enhanced enzymatic hydrolysis of 1% (w/v) H₂SO₄- and Na₂CO₃-pretreated corn stover (CS), with l-proline (Pro) increasing total reducing sugar (TRS) recovery by over 20%. Lipid production by R. toruloides on the hydrolysates was verified under single-stage and two-stage culture conditions. The lipid yield reached over 17 g/100 g TRS with some AAs, despite lower titers. Moreover, the dominance of C16 and C18 fatty acids in the lipids suggest no adverse effects of AAs on the yeast's metabolism.

Conclusion

Exogenous AA addition during enzymatic hydrolysis enhanced sugar recovery; however, its impact on R. toruloides lipid production varies with culture conditions, where a two-stage process with nutrient limitation could be more favorable for high lipid production. While this strategy proved to be more effective for enhanced sugar recovery, future studies are expected to uncover the underlying mechanisms that drive this improvement.

Background

Liquid fuels from lignocellulosic feedstocks are required for transition to a sustainable bioeconomy. However, the recalcitrance of carbon-containing feedstock cell walls to deconstruction poses a barrier to cost effective biological conversion of plant biomass to biofuels. One-step consolidated bioprocessing (CBP) in which anaerobic thermophilic bacteria convert lignocellulosic biomass into liquid fuels is a platform for overcoming the recalcitrance of plant biomass.

Results

The amounts of hemicellulosic and pectic polysaccharides, two complex cell wall glycans that contribute to plant biomass recalcitrance and that are partially solubilized during CBP of switchgrass aerial biomass by Clostridium thermocellum were evaluated in the liquor, solid residues and residue washate recovered during a 120-h CBP process. After 120 h, 24% of milled switchgrass was solubilized in the C. thermocellum CBP platform. Higher concentrations of arabinose, xylose, galactose, and glucose accumulated in the CBP-fermentation liquor and washate compared to fermentation controls without C. thermocellum, indicating that C. thermocellum solubilized hemicelluloses, but did not fully metabolize them. After five days of fermentation, the relative amount of rhamnose in the solid residues increased by 16% compared to controls, and CBP solid residues had more than 23% increased reactivity against RG-I reactive monoclonal antibodies, indicating that the pectic polymer rhamnogalacturonan I (RG-I) was not effectively solubilized from switchgrass biomass by C. thermocellum CBP. Similarly, the amount of mannose (Man) in the CBP solid residues increased by 7% and reactivity against galactomannan reactive antibodies increased by greater than 14%, indicating that the hemicellulosic polymer galactomannan was also resistant to degradation by C. thermocellum during CBP fermentation.

Conclusions

These findings show that C. thermocellum is unable to effectively degrade RG-I pectic and galactomannan hemicellulosic components in switchgrass biomass. Targeting these polymers for improved solubilization could enhance the efficiency of conversion of grass biomass to biofuels.

Background

Plant-based materials have the potential to replace some petroleum-based products, offering compostability and biodegradability as critical advantages. Xylan-rich biomass sources are gaining recognition due to their abundance and underutilization in current industrial applications. Research of potential xylan applications has been complicated by the complex and heterogeneous structure that varies for different xylan feedstocks. Acylation is a broadly used reaction in functionalization of polysaccharides at an industrial scale. However, the efficiency of this reaction varies with the xylan source. To optimize xylan valorization, a systematic understanding of structure–reactivity relationships is essential.

Results

This study explores, characterizes, and compares various xylan feedstocks in the acylation process. Xylan feedstocks were analyzed for their chemical composition, degree of polymerization, branching, solubility, and presence of impurities. These features were correlated with xylan glycotypes’ reactivity toward functionalization with succinic anhydride in an optimized DMSO/KOH condition, achieving carboxyl contents of up to 1.46. We used principal component analysis and hierarchical clustering to identify key structural features of xylan that promote its reactivity. Our findings reveal that xylans with higher xylose content and lower degrees of branching exhibit enhanced reactivity, achieving higher carboxyl content and yields. Structural analyses confirmed successful modification, and light scattering analyses showed dramatic changes in the solution properties. Succinylation improves the solubility and film-forming properties of native xylans.

Conclusions

This study shows key structure–reactivity relationships in xylan succinylation, establishing that low branching, high xylose content, and reduced lignin impurity enhance chemical functionalization. The results offer a framework for selecting optimal biomass feedstocks and support future efforts in genetic and synthetic biology to design plants with tunable xylan architectures. These findings advance the hemicellulose valorization for applications in coatings and packaging.

Background

Polyhydroxyalkanoates (PHAs), biodegradable polymers, can be synthesised and degraded by a number of bacteria. With a range of monomer composition and molecular weight, these polymers can be used for packaging to medical applications. However, the production cost, inadequate mechanical properties, and challenging melt processing properties are major impediments.

Understanding and harnessing the regulatory networks underpinning PHA production in a model organism Pseudomonas putida KT2440 is an invaluable tool to increase PHA production and alter polymer properties for specific applications.

Results

The small RNAs CrcY and CrcZ, key components of the carbon catabolite repression (CCR) system, are implicated in PHA metabolism in P. putida KT2440. Their in trans overexpression in P. putida KT2440 shows a 1.3- to 3.5-fold increase in PHA titre (g/L), using glucose or octanoate as feedstocks. This is accompanied by a decrease in the Mw of the synthesised polymer. Among the proteins showing differential expression in response to CrcY and CrcZ overexpression, glutaryl-CoA dehydrogenase GcdH, involved in the catabolism of lysine, hydroxylysine, and tryptophan, and gamma-glutamyl transpeptidase GGT, involved in glutathione metabolism, showed a consistent increase in abundance across different conditions. It also appears that CrcY and CrcZ can compensate for each other, as only when both sRNAs are removed is a 2.5-fold decrease in PHA observed. We also show that these sRNAs require other CCR elements, Hfq and Crc, for their role in PHA metabolism.

Conclusions

One strategy to overcome poor mechanical properties of PHAs is to blend them with a second polymer. Medium chain length (mcl)-PHA acts as a plasticiser when blended with poly-3-hydroxybutyrate (PHB), the most widespread used PHA resin. Here we show a clear effect of the overexpression of CCR elements CrcY and CrcZ in P. putida KT2440 on the amount of the accumulated mcl-PHA and its Mw, making this tool valuable to produce mcl-PHA-based additives.

These findings highlight the complementary regulatory roles of CrcY and CrcZ in modulating CCR to optimise PHA production. This study provides insights into leveraging CCR elements to enhance the efficiency of PHA biosynthesis, contributing to the development of sustainable bioplastic production.

Xylitol, a five-carbon sugar alcohol, is recognized as a desirable sugar alternative due to its low-calorie content and metabolism independent of insulin. Its commercial production generally involves the chemical hydrogenation of D-xylose, an approach that is energy-demanding and environmentally unfriendly. Although fermentation offers a biological alternative, it often suffers from low conversion efficiency and limited yields. However, xylitol is an intermediate metabolite in various microbial species, and its biosynthesis can be enhanced through metabolic engineering. Genetically modifying microbial cell factories—such as bacteria, fungi, and yeast—has shown significant improvements in xylitol production. Furthermore, the precursor xylose, which is utilized by microbes, can be derived from lignocellulosic biomass through hydrolysis, offering a more sustainable and cost-effective production route. This review discusses recent advances in the bioproduction of xylitol and highlights various metabolic engineering strategies employed to enhance xylitol yield in microbial cell factories.

Graphical Abstract

Background

The degradation of agricultural wastes is crucial for sustainable economic and environmental development, necessitating efficient cellulolytic enzymes to enable high-value bioconversion. The filamentous fungus Trichoderma reesei is a widely used cellulase producer for deconstructing agricultural wastes in biomass conversion. However, its enzyme system remains suboptimal and requires further refinement to achieve economical bioconversion of agricultural wastes.

Results

Herein, a hyper-cellulolytic T. reesei mutant strain CU7-4 derived from the industrial strain RUT-C30 was obtained by UV mutagenesis. When degrading the different pretreated corncob residues, CU7-4 exhibited a 20% improvement in saccharification efficiency compared to the parental strain RUT-C30. Furthermore, comparative proteomics was employed to decipher the variation between the secretomes of CU7-4 and RUT-C30. It was found that the discrepancy of the protein proportion between the secretomes may enable the changed saccharification efficiency towards the pretreated corncob residues. Then, three small secreted proteins (SSP1, EPL1, CUT1) and two β-glucosidases (Cel3H, Cel3F) were identified through the significant differences analysis in protein abundance between CU7-4 and RUT-C30, combined with responding to the essential transcriptional regulator Xyr1. Further investigation of these five proteins was conducted. Deletion of SSP1 and EPL1 was certified to facilitate degrading corncob residues and corn stover. Overexpression of Cel3F improved the activities of cellobiohydrolase and β-glucosidase, and the in vitro addition of Cel3F significantly promoted the saccharification efficiency of RUT-C30 toward corncob residues.

Conclusions

This study not only expands the protein functions for deciphering the mechanism of lignocellulose degradation, but also provides valuable protein targets for engineering the robust and powerful lignocellulolytic enzyme system, thereby facilitating the efficient degradation of agricultural wastes.