Biotechnology for Biofuels最新文献

Glycolipid biosurfactant, sophorolipids (SLs) and rhamnolipids (RLs) can be widely used in agriculture, food and chemical industries. The different physicochemical properties of SLs and RLs, such as hydrophilic lipophilic value (HLB) and critical micelle concentration (CMC), determine they have different application focus. Researchers are still hoping to obtain new glycolipid surfactants with unique surface activities. In this study, we successfully transformed two rhamnosyltransferase genes rhlA and rhlB from Pseudomonas aeruginosa to the sophorolipid-producing Starmerella bombicola CGMGG 1576 to obtain a recombinant strain was Sb_rhlAB. Two novel components with molecular weight of 554 (C₂₆H₅₀O₁₂) and 536 (C₂₆H₄₈O₁₁) were identified with the ASB C₁₈ column from the fermentation broth of Sb_rhlAB, the former was a non-acetylated acidic C14:0 glycolipid containing one glucose and one rhamnose, and the latter was an acidic C14:1 glycolipid containing two rhamnoses. With the Venusil MP C₁₈ column, one new glycolipid component was identified as an acidic C18:3 glycolipid with one rhamnose (C₂₄H₄₀O₇), which has not been reported before. Our present study demonstrated that novel glycolipids can be synthesized in vivo by reasonable genetic engineering. The results will be helpful to engineer sophorolipid-producing yeast to produce some specific SLs or their derivatives in more rational and controllable way.

Background

Escherichia coli JU15 is a metabolically engineered strain capable to metabolize C5 and C6 sugars with a high yield of d-lactic acid production at its optimal growth temperature (37 °C). The simultaneous saccharification and fermentation process allow to use lignocellulosic biomass as a cost-effective and high-yield strategy. However, this process requires microorganisms capable of growth at a temperature close to 50 °C, at which the activity of cellulolytic enzymes works efficiently.

Results

The thermotolerant strain GT48 was generated by adaptive laboratory evolution in batch and chemostat cultures under temperature increments until 48 °C. The strain GT48 was able to grow and ferment glucose to d-lactate at 47 °C. It was found that a pH of 6.3 conciliated with GT48 growth and cellulase activity of a commercial cocktail. Hence, this pH was used for the SSF of a diluted acid-pretreated corn stover (DAPCS) at a solid load of 15% (w/w), 15 FPU/g-_DAPCS, and 47 °C. Under such conditions, the strain GT48 exhibited remarkable performance, producing d-lactate at a level of 1.41, 1.42, and 1.48-fold higher in titer, productivity, and yield, respectively, compared to parental strain at 45 °C.

Conclusions

In general, our results show for the first time that a thermal-adapted strain of E. coli is capable of being used in the simultaneous saccharification and fermentation process without pre-saccharification stage at high temperatures.

Background

Hydroxy fatty acids represent an emerging class of compounds with promising applications in the chemical, medicinal and functional food sectors. The challenges associated with their chemical synthesis have spurred exploration of biological synthesis as an alternative route, particularly through the use of fatty acid hydratases. Fatty acid hydratases catalyse the regioselective addition of a hydrogen atom and a hydroxyl group from a water molecule to the carbon–carbon cis-double bond of unsaturated fatty acids to form hydroxy fatty acids. Despite having been discovered in the early 1960s, previous research has primarily focused on characterizing single fatty acid hydratase variants with a limited range of substrates. Comprehensive studies that systematically examine and compare the characteristics of multiple variants of fatty acid hydratases are still lacking.

Results

In this study, we employed an integrated bioinformatics workflow to identify 23 fatty acid hydratases and characterized their activities against nine unsaturated fatty acid substrates using whole-cell biotransformation assays. Additionally, we tested a dual-protein system involving two fatty acid hydratases of distinct regioselectivity and demonstrated its suitability in enhancing the biosynthesis of di-hydroxy fatty acids.

Conclusions

Our study demonstrates that fatty acid hydratases can be classified into three subtypes based on their regioselectivity and provides insights into their preferred substrate structures. These understandings pave ways for the design of optimal fatty acid hydratase variants and bioprocesses for the cost-efficient biosynthesis of hydroxy fatty acids.

Tryptophol (IET) is a metabolite derived from L-tryptophan that can be isolated from plants, bacteria, and fungi and has a wide range of biological activities in living systems. Despite the fact that IET biosynthesis pathways exist naturally in living organisms, industrial-scale production of IET and its derivatives is solely based on environmentally unfriendly chemical conversion. With diminishing petroleum reserves and a significant increase in global demand in all major commercial segments, it becomes essential to develop new technologies to produce chemicals from renewable resources and under mild conditions, such as microbial fermentation. Here we characterized and engineered the less-studied L-tryptophan pathway and IET biosynthesis in the baker’s yeast Saccharomyces cerevisiae, with the goal of investigating microbial fermentation as an alternative/green strategy to produce IET. In detail, we divided the aromatic amino acids (AAAs) metabolism related to IET synthesis into the shikimate pathway, the L-tryptophan pathway, the competing L-tyrosine/L-phenylalanine pathways, and the Ehrlich pathway based on a modular engineering concept. Through stepwise engineering of these modules, we obtained a yeast mutant capable of producing IET up to 1.04 g/L through fed-batch fermentation, a ~ 650-fold improvement over the wild-type strain. Besides, our engineering process also revealed many insights about the regulation of AAAs metabolism in S. cerevisiae. Finally, during our engineering process, we also discovered yeast mutants that accumulate anthranilate and L-tryptophan, both of which are precursors of various valuable secondary metabolites from fungi and plants. These strains could be developed to the chassis for natural product biosynthesis upon introducing heterologous pathways.

Background

Lignin is an aromatic polymer deposited in secondary cell walls of higher plants to provide strength, rigidity, and hydrophobicity to vascular tissues. Due to its interconnections with cell wall polysaccharides, lignin plays important roles during plant growth and defense, but also has a negative impact on industrial processes aimed at obtaining monosaccharides from plant biomass. Engineering lignin offers a solution to this issue. For example, previous work showed that heterologous expression of a coliphage S-adenosylmethionine hydrolase (AdoMetase) was an effective approach to reduce lignin in the model plant Arabidopsis. The efficacy of this engineering strategy remains to be evaluated in bioenergy crops.

Results

We studied the impact of expressing AdoMetase on lignin synthesis in sorghum (Sorghum bicolor L. Moench). Lignin content, monomer composition, and size, as well as biomass saccharification efficiency were determined in transgenic sorghum lines. The transcriptome and metabolome were analyzed in stems at three developmental stages. Plant growth and biomass composition was further evaluated under field conditions. Results evidenced that lignin was reduced by 18% in the best transgenic line, presumably due to reduced activity of the S-adenosylmethionine-dependent O-methyltransferases involved in lignin synthesis. The modified sorghum features altered lignin monomer composition and increased lignin molecular weights. The degree of methylation of glucuronic acid on xylan was reduced. These changes enabled a ~20% increase in glucose yield after biomass pretreatment and saccharification compared to wild type. RNA-seq and untargeted metabolomic analyses evidenced some pleiotropic effects associated with AdoMetase expression. The transgenic sorghum showed developmental delay and reduced biomass yields at harvest, especially under field growing conditions.

Conclusions

The expression of AdoMetase represents an effective lignin engineering approach in sorghum. However, considering that this strategy potentially impacts multiple S-adenosylmethionine-dependent methyltransferases, adequate promoters for fine-tuning AdoMetase expression will be needed to mitigate yield penalty.

Biofuel production has emerged as a leading contender in the quest for renewable energy solutions, offering a promising path toward a greener future. This comprehensive state-of-the-art review delves into the current landscape of biofuel production, exploring its potential as a viable alternative to conventional fossil fuels. This study extensively examines various feedstock options, encompassing diverse sources such as plants, algae, and agricultural waste, and investigates the technological advancements driving biofuel production processes. This review highlights the environmental benefits of biofuels, emphasizing their capacity to significantly reduce greenhouse gas emissions compared to those of fossil fuels. Additionally, this study elucidates the role of biofuels in enhancing energy security by decreasing reliance on finite fossil fuel reserves, thereby mitigating vulnerabilities to geopolitical tensions and price fluctuations. The economic prospects associated with biofuel production are also elucidated, encompassing job creation, rural development, and the potential for additional revenue streams for farmers and landowners engaged in biofuel feedstock cultivation. While highlighting the promise of biofuels, the review also addresses the challenges and considerations surrounding their production. Potential issues such as land use competition, resource availability, and sustainability implications are critically evaluated. Responsible implementation, including proper land-use planning, resource management, and adherence to sustainability criteria, is emphasized as critical for the long-term viability of biofuel production. Moreover, the review underscores the importance of ongoing research and development efforts aimed at enhancing biofuel production efficiency, feedstock productivity, and conversion processes. Technological advancements hold the key to increasing biofuel yields, reducing production costs, and improving overall sustainability. This review uniquely synthesizes the latest advancements across the entire spectrum of biofuel production, from feedstock selection to end-use applications. It addresses critical research gaps by providing a comprehensive analysis of emerging technologies, sustainability metrics, and economic viability of various biofuel pathways. Unlike previous reviews, this work offers an integrated perspective on the interplay between technological innovation, environmental impact, and socio-economic factors in biofuel development, thereby providing a holistic framework for future research and policy directions in renewable energy.

Background

The efficient use of softwood in biorefineries is hampered by its recalcitrance to enzymatic saccharification. In the present study, the fungus Thermothielavioides terrestris LPH172 was cultivated on three steam-pretreated spruce materials (STEX_180°C/auto, STEX_210°C/auto, and STEX_210°C/H2SO4), characterized by different hemicellulose content and structure, as well as on untreated biomass. The aim of the study was to map substrate-induced changes in the secretome of T. terrestris grown on differently treated spruce materials and to evaluate the hydrolytic efficiency of the secretome as supplement for a commercial enzyme mixture.

Results

The cultivation of T. terrestris was monitored by endo-cellulase, endo-xylanase, endo-mannanase, laccase, and peroxidase activity measurements. Proteomic analysis was performed on the secretomes induced by the spruce materials to map the differences in enzyme production. Growth of T. terrestris on STEX_180°C/auto and STEX_210°C/auto induced higher expression level of mannanases and mannosidases of the GH5_7 CAZy family compared to cultivation on the other materials. Cultivation on untreated biomass led to overexpression of GH47, GH76, and several hemicellulose debranching enzymes compared to the cultivation on the pretreated materials. T. terrestris grown on untreated, STEX_180°C/auto and STEX_210°C/auto induced three arabinofuranosidases of the GH43 and GH62 families; while growth on STEX_210°C/H2SO4 induced a GH51 arabinofuranosidase and a GH115 glucuronidase. All secretomes contained five lytic polysaccharide monooxygenases of the AA9 family. Supplementation of Celluclast® + Novozym188 with the secretome obtained by growing the fungus grown on STEX_180°C/auto achieved a twofold higher release of mannose from spruce steam-pretreated with acetic acid as catalyst, compared to the commercial enzyme cocktail alone.

Conclusions

Minor changes in the structure and composition of spruce affect the composition of fungal secretomes, with differences in some classes explaining an increased hydrolytic efficiency. As demonstrated here, saccharification of spruce biomass with commercial enzyme cocktails can be further enhanced by supplementation with tailor-made secretomes.

Switchgrass (Panicum virgatum L.) is a perennial C4 warm-season grass known for its high-biomass yield and wide environmental adaptability, making it an ideal bioenergy crop. Despite its potential, switchgrass seedlings grow slowly, often losing out to weeds in field conditions and producing limited biomass in the first year of planting. Furthermore, during the reproductive growth stage, the above-ground biomass rapidly increases in lignin content, creating a significant saccharification barrier. Previous studies have identified rhythm-related genes TOC1 and LHY as crucial to the slow seedling development in switchgrass, yet the precise regulatory functions of these genes remain largely unexplored. In this study, the genes TOC1 and LHY were characterized within the tetraploid genome of switchgrass. Gene expression analysis revealed that PvTOC1 and PvLHY exhibit circadian patterns under normal growth conditions, with opposing expression levels over time. PvTOC1 genes were predominantly expressed in florets, vascular bundles, and seeds, while PvLHY genes showed higher expression in stems, leaf sheaths, and nodes. Overexpression of PvTOC1 from the N chromosome group (PvTOC1-N) or PvLHY from the K chromosome group (PvLHY-K) in Arabidopsis thaliana led to alterations in circadian rhythm and hormone metabolism, resulting in shorter roots, delayed flowering, and decreased resistance to oxidative stress. These transgenic lines exhibited reduced sensitivity to hormones and hormone inhibitors, and displayed altered gene expression in the biosynthesis and signal transduction pathways of abscisic acid (ABA), gibberellin (GA), 3-indoleacetic acid (IAA), and strigolactone (SL). These findings highlight roles of PvTOC1-N and PvLHY-K in plant development and offer a theoretical foundation for genetic improvements in switchgrass and other crops.

Background

Ionizing radiation has been used for mutagenesis or material modification. The potential to use microalgae as a platform for antimicrobial production has been reported, but little work has been done to advance it beyond characterization to biotechnology. This study explored two different applications of ionizing radiation as a metabolic remodeler and a molecular modifier to enhance the antimicrobial activity of total protein and solvent extracts of Chlamydomonas reinhardtii cells.

Results

First, highly efficient transgenic C. reinhardtii strains expressing the plant-derived antimicrobial peptides, AtPR1 or AtTHI2.1, were developed using the radiation-inducible promoter, CrRPA70Ap. Low transgene expression was significantly improved through X-irradiation (12–50 Gy), with peak activity observed within 2 h. Protein extracts from these strains after X-irradiation showed enhanced antimicrobial activity against the prokaryotic bacterium, Pseudomonas syringae, and the eukaryotic fungus, Cryptococcus neoformans. In addition, X-irradiation (12 Gy) increased the growth and biomass of the transgenic strains. Second, C. reinhardtii cell extracts in ethanol were γ-irradiated (5–20 kGy), leading to molecular modifications and increased antimicrobial activity against the phytopathogenic bacteria, P. syringae and Burkholderia glumae, in a dose-dependent manner. These changes were associated with alterations in fatty acid composition. When both transgenic expression of antimicrobial peptides and molecular modification of bioactive substances were applied, the antimicrobial activity of C. reinhardtii cell extracts was further enhanced to some extent.

Conclusion

Overall, these findings suggest that ionizing radiation can significantly enhance the antimicrobial potential of C. reinhardtii through efficient transgene expression and molecular modification of bioactive substances, making it a valuable source of natural antimicrobial agents. Ionizing radiation can act not only as a metabolic remodeler of transgene expression in microalgae but also as a molecular modifier of the bioactive substances.

Background

Production of cheese whey in the EU exceeded 55 million tons in 2022, resulting in lactose-rich effluents that pose significant environmental challenges. To address this issue, the present study investigated cheese-whey treatment via membrane filtration and the utilization of its components as fermentation feedstock. A simulation model was developed for an industrial-scale facility located in Italy’s Apulia region, designed to process 539 m³/day of untreated cheese-whey. The model integrated experimental data from ethanolic fermentation using a selected strain of Kluyveromyces marxianus in lactose-supplemented media, along with relevant published data.

Results

The simulation was divided into three different sections. The first section focused on cheese-whey pretreatment through membrane filtration, enabling the recovery of 56%_w/w whey protein concentrate, process water recirculation, and lactose concentration. In the second section, the recovered lactose was directed towards fermentation and downstream anhydrous ethanol production. The third section encompassed anaerobic digestion of organic residue, sludge handling, and combined heat and power production. Moreover, three different scenarios were produced based on ethanol yield on lactose (Y_E/L), biomass yield on lactose, and final lactose concentration in the medium. A techno-economic assessment based on the collected data was performed as well as a sensitivity analysis focused on economic parameters, encompassing considerations on cheese-whey by assessing its economical impact as a credit for the simulated facility, dictated by a gate fee, or as a cost by considering it a raw material. The techno-economic analysis revealed different minimum ethanol selling prices across the three scenarios. The best performance was obtained in the scenario presenting a Y_E/L = 0.45 g/g, with a minimum selling price of 1.43 €/kg. Finally, sensitivity analysis highlighted the model’s dependence on the price or credit associated with cheese-whey handling.

Conclusions

This work highlighted the importance of policy implementation in this kind of study, demonstrating how a gate fee approach applied to cheese-whey procurement positively impacted the final minimum selling price for ethanol across all scenarios. Additionally, considerations should be made about the implementation of the simulated process as a plug-in addition in to existing processes dealing with dairy products or handling multiple biomasses to produce ethanol.