Biotechnology for Biofuels最新文献

This study is the first to apply dilute acid pretreatment (DAP) under different severity conditions to poplar wood genetically modified for the cinnamyl alcohol dehydrogenase (CAD1) gene, which is involved in the lignin biosynthesis pathway. The carefully selected pretreatment conditions resulted in glucose yields that were 15 points higher for the hpCAD poplar line than for the wild-type (WT) wood after 48 h of enzymatic hydrolysis. To explain this higher saccharification rate, the chemical, spectral and structural changes in WT and hpCAD wood were analyzed in relation to the severity of the pretreatment process. Although few differences were found at the chemical level, variations in autofluorescence and cell deformation were more significant: at high severity, the cells of hpCAD wood observed by nanotomography were more easily deformed, but their middle lamella was more resistant than those of WT wood. All these differences are possibly explained by changes in the molecular structure of lignin in hpCAD wood, leading to the formation of more hydrophobic shorter monomer chains with fewer lignin‒carbohydrate interactions.

Graphical Abstract

Background

Tyrosol is an important drug precursor, and Saccharomyces cerevisiae is one of the main microorganisms that produces tyrosol. Although excessive metabolic modification increases the production of tyrosol, it also causes a decrease in the growth rate of yeast. Therefore, this study attempted to restore the growth of S. cerevisiae through adaptive evolution and further improve tyrosol production.

Results

After the adaptive laboratory evolution of S. cerevisiae S26, three evolutionary strains were obtained. The biomass of strain S26-AE2 reached 17.82 g DCW/L in the presence of 100 g/L glucose, which was 15.33% higher than that of S26, and its tyrosol production reached 817.83 mg/L. The transcriptome analysis revealed that, upon exposure to 100 g/L glucose, the S26-AE2 strain may reduce the transcriptional regulation of glucose repression through decreased HXK2 expression. The expression of genes related to pyruvate synthesis was increased in strain S26-AE2. Meanwhile, the expression levels of most tricarboxylic acid cycle-related genes in S26-AE2 were increased when cultured with 20 g/L glucose. Furthermore, the amount of tyrosol produced by strain S26 with the SNZ3^Val125Ile mutation increased by 17.01% compared with that of the control strain S26 following exposure to 100 g/L glucose.

Conclusions

In this study, a strain, S26-AE2, with good growth and tyrosol production performance was obtained by adaptive evolution. The transcriptome analysis revealed that the differences in the expression of genes involved in metabolic pathways in adaptive evolutionary strains may be related to yeast growth and tyrosol production. Further reverse engineering verified that the mutation of SNZ3 promoted tyrosol synthesis in S. cerevisiae in glucose-rich medium. This study provides a theoretical basis for the metabolic engineering of S. cerevisiae to synthesise tyrosol and its derivatives.

Graphical Abstract

Background

Astaxanthin is a red pigment required by feed, nutraceutical, and cosmetic industries for its pigmentation and antioxidant properties. This carotenoid is one of the main high-value products that can nowadays be derived from microalgae cultivation, raising important industrial interest. However, state-of-the-art astaxanthin production is the cultivation of the green alga Haematococcus pluvialis (or lacustris), which faces high costs and low production yield. Hence, alternative and efficient sources for astaxanthin need to be developed, and novel biotechnological solutions must be found. The recently discovered cyanobacterium, Synechococcus sp. PCC 11901 is a promising photosynthetic platform for the large-scale production of high-value products, but its potential has yet to be thoroughly tested.

Results

In this study, the cyanobacterium Synechococcus sp. PCC 11901 was engineered for the first time to our knowledge to produce astaxanthin, a high-value ketocarotenoid, by expressing recombinant β-ketolase (bKT) and a β-hydroxylase enzymes (CtrZ). During photoautotrophic growth, the bKT-CtrZ transformed strain (called BC) accumulated astaxanthin to above 80% of the total carotenoid. Moreover, BC cells grew faster than wild-type (WT) cells in high light and continuous bubbling with CO₂-enriched air. The engineered strain reached stationary phase after only 4 days of growth in an airlift 80-mL photobioreactor, producing 7 g/L of dry biomass, and accumulated ~ 10 mg/L/day of astaxanthin, which is more than other CO₂-consuming multi-engineered systems. In addition, BC cells were cultivated in a 330-L photobioreactor to link lab-scale experiments to the industrial scale-up.

Conclusions

The astaxanthin volumetric productivity achieved, 10 mg/L/day, exceeds that previously reported for Haematococcus pluvialis, the standard microalgal species nowadays used at the industrial level for astaxanthin production, or for other microalgal strains engineered to produce ketocarotenoids. Overall, this work identifies a new route to produce astaxanthin on an industrial scale.

Background

Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment is lacking. In this study, small-angle neutron scattering (SANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from Eucalyptus and Spruce wood samples.

Results

Whereas the SANS analysis of Spruce holocellulose revealed an increase in inter-microfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in the ordered arrangement of microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi β-barrel (DPBB) domain surrounded by three α-helices—the largest nestled against the DPBB core and the other two part of loops extending from the core.

Conclusions

The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing regular order in the microfibril arrangement to increasing inter-microfibril spacing. The most impactful PcaLOOL, PcaLOOL12, was previously observed to be the most highly expressed loosenin in P. carnosa. Its structural characterization herein reveals its stabilization through two disulfide linkages, and an extended N-terminal region distal to a negatively charged and surface accessible polysaccharide binding groove.

Background

Saccharomyces cerevisiae has been extensively employed as a host for the production of various biochemicals and recombinant proteins. The expression systems employed in S. cerevisiae typically rely on constitutive or galactose-regulated promoters, and the limited repertoire of gene expression regulations imposes constraints on the productivity of microbial cell factories based on budding yeast.

Results

In this study, we designed and characterized a series of allantoin-inducible expression systems based on the endogenous allantoin catabolic system (DAL-related genes) in S. cerevisiae. We first characterized the expression profile of a set of DAL promoters induced by allantoin, and further combined with the galactose-inducible (GAL) system to create a highly responsive genetic switch that efficiently amplifies the output signals. The resulting allantoin–GAL system could give a ON/OFF ratio of 68.6, with 6.8-fold higher signal output over that of direct P_DAL2-controlled gene expression. Additionally, when a centromeric plasmid was used for EGFP expression, the ON/OFF ratio was increased to > 67.2, surpassing the EGFP expression levels driven by the DAL2 promoter. Subsequently, we successfully demonstrated that allantoin–GAL system can be used to effectively regulate carotenoid production and cell flocculation in S. cerevisiae.

Conclusions

In summary, we characterized several allantoin-inducible DAL promoters from budding yeast and further developed a layered allantoin–GAL system that utilizes the DAL2 promoter to regulate the galactose regulon in budding yeast. The resulting allantoin–GAL system could give an impressive ON/OFF ratio that surpassed the traditional P_DAL2-controlled gene expression. It is anticipated that utilizing our allantoin-inducible system in budding yeast with allantoin as the alternative nitrogen source might favor the low-cost production of biochemicals and pharmaceuticals.

Background

Anthraquinones in the emodin family are produced by bacteria, fungi, and plants. They display various biological activities exploited, e.g., for crop protection, and may also be utilized as sustainable, bio-based colorants for the textile, paints, electronics, and cosmetic industries. Anthraquinone pigments from Cortinarius mushrooms have been used for artisan dyeing because they are stable, colorfast, and compatible with various dyeing methods. However, their chemical synthesis is complex and uneconomical, and harvesting wild mushrooms from forests in commercial quantities is not feasible.

Results

Here, we use genomics, transcriptomics, and synthetic biology to uncover the biosynthesis of the anthraquinone scaffold compounds emodin and endocrocin, and their methylation to the yellow pigments physcion and dermolutein in Cortinarius semisanguineus and C. sp. KIS-3. Both the nonreducing polyketide synthases (nrPKSs), and the regiospecific, fastidious O-methyltransferases (OMTs) are non-orthologous to their Ascomycete counterparts, suggesting a parallel evolutionary origin for the pathway in Basidiomycetes. The genes for the nrPKS and the OMTs are not all clustered in Cortinarius, revealing metabolic crosstalk among paralogous nrPKS biosynthetic gene clusters.

Conclusions

Heterologous biosynthesis of physcion and dermolutein in Saccharomyces cerevisiae opens the way to produce specific Cortinarius anthraquinones, and to modify these scaffolds to tune their chemistry towards their various applications.

Background

Mucoromycota fungi are promising for the production of second-generation biofuel from single-cell oils (SCOs) using lignocellulose biomass. Despite the lack of enzymatic capability for efficiently degrading lignocellulose in Mucoromycota fungi, simultaneous saccharification and fermentation (SSF) offers an attractive solution by combining enzymatic hydrolysis and fermentation in the same procedure. This study explored specific traits of various Mucoromycota species to evaluate their suitability for SSF, due to the frequent and significant gap between the microorganism and enzyme optimal conditions.

Results

The suitability of nine oleaginous fungal strains from the Mucoromycota phylum for use in lignocellulose-based simultaneous saccharification and fermentation was evaluated. Several traits, such as thermal tolerance, biochemical composition changes in response to incubation temperature, cellobiose and cellulose response and induction of β-glucosidase and endoglucanase, were evaluated. Lichtheimia corymbifera was the most suitable species for SSF due to its ability to grow up to 45 °C, with a consequent decrease in lipid unsaturation, and good uptake of cellobiose with induction of β-glucosidase and endoglucanase expression. The Cunninghamella blackesleeana and Mucor circinelloides strains were also considered good candidates; despite the cultivation should not exceed 35 °C, their good uptake of cellobiose and the expression of extracellular β-glucosidase induced by cellobiose indicated that they could increase the enzymatic hydrolysis efficiency. C. blakesleeana outperformed all the other tested strains in terms of β-glucosidase activity expression. In addition, both endoglucanase and β-glucosidase activities of Rhizopus stolonifer and M. circinelloides were induced by cellobiose. Mortierella alpina and Mortierella hyalina were not considered suitable for simultaneous saccharification and fermentation due to their reduced tolerance to high temperatures and poor response to cellobiose utilization.

Conclusions

This study identified beneficial traits of Mucoromycota species for simultaneous saccharification and fermentation using lignocellulose, contributing to an optimal selection for producing lipid-derived second-generation biofuels.

Background

Soybean is a major oil crop and a primary protein source for livestock, and soybean oil is the most common input for biodiesel. Identifying genes that enhance soybean yield and oil content is crucial for breeding programs. Phosphatidic acid (PA) phosphohydrolase (PAH), which dephosphorylates PA to diacylglycerol (DAG), plays a critical role in lipid synthesis, and yet their potential in improving agronomic traits of oil crops remains unexplored.

Results

This study shows that seed-specific expression of AtPAH1/2 enhances PA turnover into DAG and triacylglycerol (TAG) accumulation in soybean seeds. PAH overexpression upregulated the expression of DAG acyltransferase (DGAT) but suppressed phospholipid: DAG acyltransferase (PDAT). In addition, seed-specific expression of AtPAH1/2 increases soybean seed size and weight. Furthermore, analysis of the variation of the soybean PAHs in 4414 soybean accessions indicated that the advantageous effects of GmPAHs on oil content and seed weight were selected during domestication.

Conclusion

These findings suggest that targeting PAHs represents a promising strategy for enhancing soybean seed oil content and yield in current cultivars and landraces soybeans.

Utilizing microalgae as “photosynthetic cell factories” for compound production holds significant potential for sustainable carbon neutrality. However, the inherent inefficiency of algal photosynthesis, a limiting factor for productivity, represents a critical area for enhancement. Among the key regulatory mechanisms, the Target of Rapamycin (TOR), essential for cell growth regulation and known for its conserved structure across eukaryotes, remains underexplored in Nannochloropsis gaditana. In this study, we identified conserved component of the TOR complex in N. gaditana. Rapamycin (RAP) effectively inhibited photosynthetic growth and enhanced lipid accumulation in N. gaditana, as demonstrated by sensitivity tests. Transcriptomic analysis revealed that NgTOR modulates multiple intracellular metabolic and signaling pathways. Specifically, genes associated with photosynthesis and chlorophyll synthesis were significantly down-regulated following NgTOR inhibition. Additionally, genes involved in carbon metabolism, the TCA cycle, and amino acid biosynthesis were markedly reduced, while those related to lipid metabolism were up-regulated, resulting in stunted cell growth and increased lipid accumulation. Furthermore, blocking photosynthesis with DCMU significantly reduced the transcriptional activity of TOR-related complexes, highlighting a bidirectional regulatory interaction. These findings underscore the pivotal role of the TOR signaling pathway in regulating photosynthesis, carbon metabolism, and lipid metabolism in N. gaditana, setting the stage for further studies on photosynthetic autotrophy and lipid metabolic pathways in this species.

Background

Lignocellulose is the most abundant renewable bioresource on earth, and its biodegradation and utilization would contribute to the sustainable development of the global environment. Ruminiclostridium papyrosolvens, an anaerobic, mesophilic, and cellulolytic bacterium, produces an enzymatic complex known as the cellulosome. As one of the most highly evolved species among Ruminiclostridium-type species, R. papyrosolvens is particularly relevant for understanding how cellulolytic clostridia modulate their biomass degradation mechanisms in response to diverse carbon sources.

Results

Our study investigates the transcriptional responses of Ruminiclostridium papyrosolvens to different carbon sources to understand its lignocellulose utilization. Using RNA-seq, we analyzed gene expression under glucose, cellobiose, xylan, cellulose, and corn stover, identifying distinct metabolic preferences and regulatory responses. We found significant gene expression changes under corn stover compared to other carbon sources, with enrichment in ABC transporters and cell growth pathways. CAZyme gene expression was regulated by TCSs, affecting sugar transporter systems. Metabolic profiling showed R. papyrosolvens produced more complex metabolites during corn stover fermentation, revealing its adaptability to various carbon sources and implications for metabolic engineering.

Conclusion

This study not only uncovers the intricate response mechanisms of R. papyrosolvens to lignocellulose and its hydrolysates, but it also outlines the strategy for using R. papyrosolvens as a cellulolytic chassis in genetic engineering.