Biotechnology for Biofuels and Bioproducts最新文献

Straw biorefinery offers economical and sustainable production of chemicals. The merits of cell immobilization technology have become the key technology to meet D-lactic acid production from non- detoxified corn stover. In this paper, Low acyl gellan gum (LA-GAGR) was employed first time for Lactobacillus bulgaricus T15 immobilization and applied in D-lactic acid (D-LA) production from non-detoxified corn stover hydrolysate. Compared with the conventional calcium alginate (E404), LA-GAGR has a hencky stress of 82.09 kPa and excellent tolerance to 5-hydroxymethylfurfural (5-HMF), ferulic acid (FA), and vanillin. These features make LA-GAGR immobilized T15 work for 50 days via cell-recycle fermentation with D-LA yield of 2.77 ± 0.27 g/L h, while E404 immobilized T15 can only work for 30 days. The production of D-LA from non-detoxified corn stover hydrolysate with LA-GAGR immobilized T15 was also higher than that of free T15 fermentation and E404 immobilized T15 fermentation. In conclusion, LA-GAGR is an excellent cell immobilization material with great potential for industrial application in straw biorefinery industry.

Background: Lipid formation from glycerol was previously found to be activated in Rhodotorula toruloides when the yeast was cultivated in a mixture of crude glycerol (CG) and hemicellulose hydrolysate (CGHH) compared to CG as the only carbon source. RNA samples from R. toruloides CBS14 cell cultures grown on either CG or CGHH were collected at different timepoints of cultivation, and a differential gene expression analysis was performed between cells grown at a similar physiological situation.

Results: We observed enhanced transcription of genes involved in oxidative phosphorylation and enzymes localized in mitochondria in CGHH compared to CG. Genes involved in protein turnover, including those encoding ribosomal proteins, translation elongation factors, and genes involved in building the proteasome also showed an enhanced transcription in CGHH compared to CG. At 10 h cultivation, another group of activated genes in CGHH was involved in β-oxidation, handling oxidative stress and degradation of xylose and aromatic compounds. Potential bypasses of the standard GUT1 and GUT2-glycerol assimilation pathway were also expressed and upregulated in CGHH 10 h. When the additional carbon sources from HH were completely consumed, at CGHH 36 h, their transcription decreased and NAD⁺-dependent glycerol-3-phosphate dehydrogenase was upregulated compared to CG 60 h, generating NADH instead of NADPH with glycerol catabolism. TPI1 was upregulated in CGHH compared to cells grown on CG in all physiological situations, potentially channeling the DHAP formed through glycerol catabolism into glycolysis. The highest number of upregulated genes encoding glycolytic enzymes was found after 36 h in CGHH, when all additional carbon sources were already consumed.

Conclusions: We suspect that the physiological reason for the accelerated glycerol assimilation and faster lipid production, was primarily the activation of enzymes that provide energy.

Background: High-throughput metabolomics analytical methodology is needed for population-scale studies of bioenergy-relevant feedstocks such as poplar (Populus sp.). Here, the authors report the relative abundance of extractable aromatic metabolites in Populus trichocarpa leaves rapidly estimated using pyrolysis-molecular beam mass spectrometry (py-MBMS). Poplar leaves were analyzed in conjunction with and validated by GC/MS analysis of extracts to determine key spectral features used to build PLS models to predict the relative composition of extractable aromatic metabolites in whole poplar leaves.

Results: The Pearson correlation coefficient for the relative abundance of extractable aromatic metabolites based on ranking between GC/MS analysis and py-MBMS analysis of the Boardman leaf set was 0.86 with R² = 0.76 using a simplified prediction approach from select ions in MBMS spectra. Metabolites most influential to py-MBMS spectral features in the Clatskanie set included the following compounds: catechol, salicortin, salicyloyl-coumaroyl-glucoside conjugates, α-salicyloylsalicin, tremulacin, as well as other salicylates, trichocarpin, salicylic acid, and various tremuloidin conjugates. Ions in py-MBMS spectra with the highest correlation to the abundance of extractable aromatic metabolites as determined by GC/MS analysis of extracts, included m/z 68, 71, 77, 91, 94, 105, 107, 108, and 122, and were used to develop the simplified prediction approach without PLS models or a priori measurements.

Conclusions: The simplified py-MBMS method is capable of rapidly screening leaf tissue for relative abundance of extractable aromatic secondary metabolites to enable prioritization of samples in large populations requiring comprehensive metabolomics that will ultimately inform plant systems biology models and advance the development of optimized biomass feedstocks for renewable fuels and chemicals.

Background: Glycoside hydrolase (GH) family 30 xylanases are a distinct group of xylanases, most of which have a highly specific catalytic activity for glucuronoxylan. Since GH30 xylanases do not normally carry carbohydrate-binding modules (CBMs), our knowledge of the function of their CBMs is lacking.

Results: In this work, the CBM functions of CrXyl30 were investigated. CrXyl30 was a GH30 glucuronoxylanase containing tandem CBM13 (CrCBM13) and CBM2 (CrCBM2) at its C terminus, which was identified in a lignocellulolytic bacterial consortium previously. Both CBMs could bind insoluble and soluble xylan, with CrCBM13 having binding specificity for the xylan with L-arabinosyl substitutions, whereas CrCBM2 targeted L-arabinosyl side chains themselves. Such binding abilities of these two CBMs were completely different from other CBMs in their respective families. Phylogenetic analysis also suggested that both CrCBM13 and CrCBM2 belong to novel branches. Inspection of the simulated structure of CrCBM13 identified a pocket that just accommodates the side chain of 3(2)-alpha-L-arabinofuranosyl-xylotriose, which forms hydrogen bonds with three of the five amino acid residues involved in ligand interaction. The truncation of either CrCBM13 or CrCBM2 did not alter the substrate specificity and optimal reaction conditions of CrXyl30, whereas truncation of CrCBM2 decreased the k_cat/K_m value by 83% (± 0%). Moreover, the absence of CrCBM2 and CrCBM13 resulted in a 5% (± 1%) and a 7% (± 0%) decrease, respectively, in the amount of reducing sugar released by the synergistic hydrolysis of delignified corncob whose hemicellulose is arabinoglucuronoxylan, respectively. In addition, fusion of CrCBM2 with a GH10 xylanase enhanced its catalytic activity against the branched xylan and improved the synergistic hydrolysis efficiency by more than fivefold when delignified corncob was used as substrate. Such a strong stimulation of hydrolysis resulted from the enhancement of hemicellulose hydrolysis on the one hand, and the cellulose hydrolysis is also improved according to the lignocellulose conversion rate measured by HPLC.

Conclusions: This study identifies the functions of two novel CBMs in CrXyl30 and shows the good potential of such CBMs specific for branched ligands in the development of efficient enzyme preparations.

Humic substances (HS) are originated from naturally decaying biomass. The main products of HS are humic acids, fulvic acids, and humins. HS are extracted from natural origins (e.g., coals, lignite, forest, and river sediments). However, the production of HS from these resources is not environmentally friendly, potentially impacting ecological systems. Earlier theories claimed that the HS might be transformed from lignin by enzymatic or aerobic oxidation. On the other hand, lignin is a by-product of pulp and paper production processes and is available commercially. However, it is still under-utilized. To address the challenges of producing environmentally friendly HS and accommodating lignin in valorized processes, the production of lignin-derived HS has attracted attention. Currently, several chemical modification pathways can be followed to convert lignin into HS-like materials, such as alkaline aerobic oxidation, alkaline oxidative digestion, and oxidative ammonolysis of lignin. This review paper discusses the fundamental aspects of lignin transformation to HS comprehensively. The applications of natural HS and lignin-derived HS in various fields, such as soil enrichment, fertilizers, wastewater treatment, water decontamination, and medicines, were comprehensively discussed. Furthermore, the current challenges associated with the production and use of HS from lignin were described.

Background: Owing to the Crabtree effect, Saccharomyces cerevisiae produces a large amount of ethanol in the presence of oxygen and excess glucose, leading to a loss of carbon for the biosynthesis of non-ethanol chemicals. In the present study, the potential of a newly constructed Crabtree negative S. cerevisiae, as a chassis cell, was explored for the biosynthesis of various non-ethanol compounds.

Results: To understand the metabolic characteristics of Crabtree negative S. cerevisiae sZJD-28, its transcriptional profile was compared with that of Crabtree positive S. cerevisiae CEN.PK113-11C. The reporter GO term analysis showed that, in sZJD-28, genes associated with translational processes were down-regulated, while those related to carbon metabolism were significantly up-regulated. To verify a potential increase in carbon metabolism for the Crabtree negative strain, the production of non-ethanol chemicals, derived from different metabolic nodes, was then undertaken for both sZJD-28 and CEN.PK113-11C. At the pyruvate node, production of 2,3-butanediol and lactate in sZJD-28-based strains was remarkably higher than that of CEN.PK113-11C-based ones, representing 16.8- and 1.65-fold increase in titer, as well as 4.5-fold and 0.65-fold increase in specific titer (mg/L/OD), respectively. Similarly, for shikimate derived p-coumaric acid, the titer of sZJD-28-based strain was 0.68-fold higher than for CEN.PK113-11C-based one, with a 0.98-fold increase in specific titer. While farnesene and lycopene, two acetoacetyl-CoA derivatives, showed 0.21- and 1.88-fold increases in titer, respectively. From malonyl-CoA, the titer of 3-hydroxypropionate and fatty acids in sZJD-28-based strains were 0.19- and 0.76-fold higher than that of CEN.PK113-11C-based ones, respectively. In fact, yields of products also improved by the same fold due to the absence of residual glucose. Fed-batch fermentation further showed that the titer of free fatty acids in sZJD-28-based strain 28-FFA-E reached 6295.6 mg/L with a highest reported specific titer of 247.7 mg/L/OD in S. cerevisiae.

Conclusions: Compared with CEN.PK113-11C, the Crabtree negative sZJD-28 strain displayed a significantly different transcriptional profile and obvious advantages in the biosynthesis of non-ethanol chemicals due to redirected carbon and energy sources towards metabolite biosynthesis. The findings, therefore, suggest that a Crabtree negative S. cerevisiae strain could be a promising chassis cell for the biosynthesis of various chemicals.

Background: The dense structure of cellulose lowers its reactivity and hinders its applications. Concentrated sulfuric acid is an ideal solvent to dissolve cellulose and thus has been used widely to treat cellulose. However, the changes of cellulose after reaction with concentrated sulfuric acid at near-limit S/L ratio and its effect on enzymatic saccharification still need further investigation.

Results: In this study, the interactions between cellulose (Avicel) and 72% sulfuric acid at very low acid loading conditions of 1:2 to 1:3 (S/L ratio) were studied for the enhanced production of glucose. The Avicel gradually transformed from cellulose I structure to cellulose II structure during the sulfuric acid treatment. Other physicochemical characteristics of Avicel also changed dramatically, such as the degree of polymerization, particle size, crystallinity index, and surface morphology. After acid treatment, both the yield and productivity of glucose from cellulose increased significantly under a very low enzyme loading of 5 FPU/g-cellulose. The glucose yields for raw cellulose and acid-treated (30 min) were 57% and 85%, respectively.

Conclusion: Low loadings of concentrated sulfuric acid were proven to be effective to break the recalcitrance of cellulose for enzymatic saccharification. A positive correlation between cellulose CrI and glucose yield was found for concentrated sulfuric acid-treated cellulose, which was opposite to previous reports. Cellulose II content was found to be an important factor that affects the conversion of cellulose to glucose.

Background: Terrequinone A is a bis-indolylquinone natural product with antitumor activity. Due to its unique asymmetric quinone core structure and multiple functional groups, biosynthesis is more efficient and environmentally friendly than traditional chemical synthesis. Currently, most bis-indolylquinones are obtained by direct extraction from fungi or by chemical synthesis. By focusing on the biosynthesis of terrequinone A, we hope to explore the way to synthesize bis-indolylquinones de novo using Escherichia coli as a cell factory.

Results: In this study, a terrequinone A synthesis pathway containing the tdiA-tdiE genes was constructed into Escherichia coli and activated by a phosphopantetheinyl transferase gene sfp, enabling the strain to synthesize 1.54 mg/L of terrequinone A. Subsequently, a two-step isopentenol utilization pathway was introduced to enhance the supply of endogenous dimethylallyl diphosphate (DMAPP) in E. coli, increasing the level of terrequinone A to 20.1 mg/L. By adjusting the L-tryptophan (L-Trp)/prenol ratio, the major product could be changed from ochrindole D to terrequinone A, and the content of terrequinone A reached the highest 106.3 mg/L under the optimized culture conditions. Metabolic analysis of L-Trp indicated that the conversion of large amounts of L-Trp to indole was an important factor preventing the further improvement of terrequinone A yield.

Conclusions: A comprehensive approach was adopted and terrequinone A was successfully synthesized from low-cost L-Trp and prenol in E. coli. This study provides a metabolic engineering strategy for the efficient synthesis of terrequinone A and other similar bis-indolylquinones with asymmetric quinone cores. In addition, this is the first report on the de novo biosyhthesis of terrequinone A in an engineered strain.

Background: Switchgrass (Panicum virgatum L.) is an important biofuel crop that may contribute to replacing petroleum fuels. However, slow seedling growth and soil salinization affect the growth and development of switchgrass. An increasing number of studies have shown that beneficial microorganisms promote plant growth and increase tolerance to salinity stress. However, the feasibility of inoculating switchgrass with Azorhizobium caulinodans ORS571 to enhance the growth and salt tolerance of its seedlings is unclear. Our previous study showed that A. caulinodans ORS571 could colonize wheat (Triticum aestivum L.) and thereby promote its growth and development and regulate the gene expression levels of microRNAs (miRNAs).

Results: In this study, we systematically studied the impact of A. caulinodans ORS571 on switchgrass growth and development and the response to salinity stress; we also studied the underlying mechanisms during these biological processes. Inoculation with A. caulinodans ORS571 significantly alleviated the effect of salt stress on seedling growth. Under normal conditions, A. caulinodans ORS571 significantly increased fresh plant weight, chlorophyll a content, protein content, and peroxidase (POD) activity in switchgrass seedlings. Under salt stress, the fresh weight, dry weight, shoot and root lengths, and chlorophyll contents were all significantly increased, and some of these parameters even recovered to normal levels after inoculation with A. caulinodans ORS571. Soluble sugar and protein contents and POD and superoxide dismutase (SOD) activities were also significantly increased, contrary to the results for proline. Additionally, A. caulinodans ORS571 may alleviate salt stress by regulating miRNAs. Twelve selected miRNAs were all upregulated to different degrees under salt stress in switchgrass seedlings. However, the levels of miR169, miR171, miR319, miR393, miR535, and miR854 were decreased significantly after inoculation with A. caulinodans ORS571 under salt stress, in contrast to the expression level of miR399.

Conclusion: This study revealed that A. caulinodans ORS571 increased the salt tolerance of switchgrass seedlings by increasing their water content, photosynthetic efficiency, osmotic pressure maintenance, and reactive oxygen species (ROS) scavenging abilities and regulating miRNA expression. This work provides a new, creative idea for improving the salt tolerance of switchgrass seedlings.