Biotechnology for Biofuels最新文献

Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the β-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass.

Background

5-Aminolevulinic acid (ALA) recently received much attention due to its potential application in many fields such as medicine, nutrition and agriculture. Metabolic engineering is an efficient strategy to improve microbial production of 5-ALA.

Results

In this study, an ALA production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement. A metabolic strategy to produce ALA directly from glucose in this recombinant E. coli via both C4 and C5 pathways was applied herein. The expression of a modified hemA^RS gene and rational metabolic engineering by gene knockouts significantly improved ALA production from 765.9 to 2056.1 mg/L. Next, we tried to improve ALA production by RGMS-directed evolution of eamA gene. After RGMS, the ALA yield of strain A2-ASK reached 2471.3 mg/L in flask. Then, we aimed to improve the oxidation resistance of cells by overexpressing sodB and katE genes and ALA yield reached 2703.8 mg/L. A final attempt is to replace original promoter of hemB gene in genome with a weaker one to decrease its expression. After 24 h cultivation, a high ALA yield of 19.02 g/L was achieved by 108-ASK in a 5 L fermenter.

Conclusions

These results suggested that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification and optimization of gene expression.

Background

Non-conventional yeasts hold significant potential as biorefinery cell factories for microbial bioproduction. Currently, gene editing systems used for these yeasts rely on antibiotic and auxotrophic selection mechanisms. However, the drawbacks of antibiotics, including high costs, environmental concerns, and the dissemination of resistance genes, make them unsuitable for large-scale industrial fermentation. For auxotrophic selection system, the engineered strains harboring auxotrophic marker genes are typically supplemented with complex nutrient-rich components instead of precisely defined synthetic media in large-scale industrial fermentations, thus lack selection pressure to ensure the stability of heterologous metabolic pathways. Therefore, it is a critical to explore alternative selection systems that can be adapted for large-scale industrial fermentation.

Results

Here, a novel glucose-dependent selection system was developed in a high pullulan-producing non-conventional strain A. melanogenum P16. The system comprised a glucose-deficient chassis cell Δpfk obtained through the knockout of the phosphofructokinase gene (PFK) and a series of chromosomal integration plasmids carrying a selection marker PFK controlled by different strength promoters. Utilizing the green fluorescent protein gene (GFP) as a reporter gene, this system achieved a 100% positive rate of transformation, and the chromosomal integration numbers of GFP showed an inverse relationship with promoter strength, with a customizable copy number ranging from 2 to 54. More importantly, the chromosomal integration numbers of target genes remained stable during successive inoculation and fermentation process, facilitated simply by using glucose as a cost-effective and environmental-friendly selectable molecule to maintain a constant and rigorous screening pressure. Moreover, this glucose-dependent selection system exhibited no significant effect on cell growth and product synthesis, and the glucose-deficient related selectable marker PFK has universal application potential in non-conventional yeasts.

Conclusion

Here, we have developed a novel glucose-dependent selection system to achieve customizable and stable multilocus chromosomal integration of target genes. Therefore, this study presents a promising new tool for genetic manipulation and strain enhancement in non-conventional yeasts, particularly tailored for industrial fermentation applications.

To increase the production of biomass and astaxanthin from Haematococcus pluvialis to meet the high market demand for astaxanthin, this study recruited two typical and negligible phytohormones (namely resveratrol and catechol) for the stepwise treatments of H. pluvialis. It was found that the hybrid and sequential treatments of resveratrol (200 μmol) and catechol (100 μmol) had achieved the maximum astaxanthin content at 33.96 mg/L and 42.99 mg/L, respectively. Compared with the hybrid treatment, the physiological data of H. pluvialis using the sequential strategy revealed that the enhanced photosynthetic performance via the Calvin cycle by RuBisCO improved the biomass accumulation during the macrozooid stage; meanwhile, the excessive ROS production had occurred to enhance astaxanthin production with the help of NADPH overproduction during the hematocyst stage. Overall, this study provides improved knowledge of the impacts of phytohormones in improving biomass and astaxanthin of H. pluvialis, which shed valuable insights for advancing microalgae-based biorefinery.

Background

The need for addition of external electron donors such as ethanol or lactate impairs the economic viability of chain elongation (CE) processes for the production of medium-chain carboxylates (MCC). However, using feedstocks with inherent electron donors such as silages of waste biomass can improve the economics. Moreover, the use of an appropriate inoculum is critical to the overall efficiency of the CE process, as the production of a desired MCC can significantly be influenced by the presence or absence of specific microorganisms and their metabolic interactions. Beyond, it is necessary to generate data that can be used for reactor design, simulation and optimization of a given CE process. Such data can be obtained using appropriate mathematical models to predict the dynamics of the CE process.

Results

In batch experiments using silages of sugar beet leaves, cassava leaves, and Elodea/wheat straw as substrates, caproate was the only MCC produced with maximum yields of 1.97, 3.48, and 0.88 g/kgVS, respectively. The MCC concentrations were accurately predicted with the modified Gompertz model. In a semi-continuous fermentation with ensiled sugar beet leaves as substrate and digestate from a biogas reactor as the sole inoculum, a prolonged lag phase of 7 days was observed for the production of MCC (C6–C8). The lag phase was significantly shortened by at least 4 days when an enriched inoculum was added to the system. With the enriched inoculum, an MCC yield of 93.67 g/kgVS and a productivity of 2.05 gMCC/L/d were achieved. Without the enriched inoculum, MCC yield and productivity were 43.30 g/kgVS and 0.95 gMCC/L/d, respectively. The higher MCC production was accompanied by higher relative abundances of Lachnospiraceae and Eubacteriaceae.

Conclusions

Ensiled waste biomass is a suitable substrate for MCC production using CE. For an enhanced production of MCC from ensiled sugar beet leaves, the use of an enriched inoculum is recommended for a fast process start and high production performance.

Background

Lignin is a highly abundant but strongly underutilized natural resource that could serve as a sustainable feedstock for producing chemicals by microbial cell factories. Because of the heterogeneous nature of the lignin feedstocks, the biological upgrading of lignin relying on the metabolic routes of aerobic bacteria is currently considered as the most promising approach. However, the limited substrate range and the inefficient catabolism of the production hosts hinder the upgrading of lignin-related aromatics. Particularly, the aerobic O-demethylation of the methoxyl groups in aromatic substrates is energy-limited, inhibits growth, and results in carbon loss in the form of CO₂.

Results

In this study, we present a novel approach for carbon-wise utilization of lignin-related aromatics by the integration of anaerobic and aerobic metabolisms. In practice, we employed an acetogenic bacterium Acetobacterium woodii for anaerobic O-demethylation of aromatic compounds, which distinctively differs from the aerobic O-demethylation; in the process, the carbon from the methoxyl groups is fixed together with CO₂ to form acetate, while the aromatic ring remains unchanged. These accessible end-metabolites were then utilized by an aerobic bacterium Acinetobacter baylyi ADP1. By utilizing this cocultivation approach, we demonstrated an upgrading of guaiacol, an abundant but inaccessible substrate to most microbes, into a plastic precursor muconate, with a nearly equimolar yields (0.9 mol/mol in a small-scale cultivation and 1.0 mol/mol in a one-pot bioreactor cultivation). The process required only a minor genetic engineering, namely a single gene knock-out. Noticeably, by employing a metabolic integration of the two bacteria, it was possible to produce biomass and muconate by utilizing only CO₂ and guaiacol as carbon sources.

Conclusions

By the novel approach, we were able to overcome the issues related to aerobic O-demethylation of methoxylated aromatic substrates and demonstrated carbon-wise conversion of lignin-related aromatics to products with yields unattainable by aerobic processes. This study highlights the power of synergistic integration of distinctive metabolic features of bacteria, thus unlocking new opportunities for harnessing microbial cocultures in upgrading challenging feedstocks.

Plastic consumption has increased globally, and environmental issues associated with it have only gotten more severe; as a result, the search for environmentally friendly alternatives has intensified. Polyhydroxyalkanoates (PHA), as biopolymers produced by microalgae, might be an excellent option; however, large-scale production is a relevant barrier that hinders their application. Recently, innovative materials such as carbon dots (CDs) have been explored to enhance PHA production sustainably. This study added green synthesized multi-doped CDs to Scenedesmus sp. microalgae cultures to improve PHA production. Prickly pear was selected as the carbon precursor for the hydrothermally synthesized CDs doped with nitrogen, phosphorous, and nitrogen–phosphorous elements. CDs were characterized by different techniques, such as FTIR, SEM, ζ potential, UV–Vis, and XRD. They exhibited a semi-crystalline structure with high concentrations of carboxylic groups on their surface and other elements, such as copper and phosphorus. A medium without nitrogen and phosphorous was used as a control to compare CDs-enriched mediums. Cultures regarding biomass growth, carbohydrates, lipids, proteins, and PHA content were analyzed. The obtained results demonstrated that CDs-enriched cultures produced higher content of biomass and PHA; CDs-enriched cultures presented an increase of 26.9% in PHA concentration and an increase of 32% in terms of cell growth compared to the standard cultures.

Background

The aim of this study was to increase the accessibility and accelerate the breakdown of lignocellulosic biomass to methane in an anaerobic fermentation system by mechanical cotreatment: milling during fermentation, as an alternative to conventional pretreatment prior to biological deconstruction. Effluent from a mesophilic anaerobic digester running with unpretreated senescent switchgrass as the predominant carbon source was collected and subjected to ball milling for 0.5, 2, 5 and 10 min. Following this, a batch fermentation test was conducted with this material in triplicate for an additional 18 days with unmilled effluent as the ‘status quo’ control.

Results

The results indicate 0.5 – 10 min of cotreatment increased sugar solubilization by 5– 13% when compared to the unmilled control, with greater solubilization correlated with increased milling duration. Biogas concentrations ranged from 44% to 55.5% methane with the balance carbon dioxide. The total biogas production was statistically higher than the unmilled control for all treatments with 2 or more minutes of milling (α = 0.1). Cotreatment also decreased mean particle size. Energy consumption measurements of a lab-scale mill indicate that longer durations of milling offer diminishing benefits with respect to additional methane production.

Conclusions

Cotreatment in anaerobic digestion systems, as demonstrated in this study, provides an alternative approach to conventional pretreatments to increase biogas production from lignocellulosic grassy material.

Background

Schizochytrium sp. is commercially used for production of docosahexaenoic acid (DHA). Schizochytrium sp. utilizes the polyketide synthase complex (PKS) and a single type I fatty acid synthase (FAS) to synthesize polyunsaturated fatty acids and saturated fatty acids, respectively. The acyl carrier protein (ACP) domains of FAS or PKS are used to load acyl groups during fatty acids biosynthesis. Phosphopantetheinyl transferase (PPTase) transfers the pantetheine moiety from Coenzyme A to the conserved serine residue of an inactive ACP domain to produce its active form.

Results

In this study, in order to improve production and content of DHA, we decreased the expression of fas, strengthened the expression of the PKS pathway, and enhanced the supply of active ACP in Schizochytrium sp. ATCC20888. Weakening the expression of fas or disruption of orfA both led to growth defect and reduction of lipid yields in the resulting strains WFAS and DPKSA, indicating that both FAS and PKS were indispensable for growth and lipid accumulation. Although WFAS had a higher DHA content in total fatty acids than the wild-type strain (WT), its growth defect and low DHA yield hinders its use for DHA production. Overexpression of the orfAB, orfC, orfC-DH (truncated orfC), or ppt promoted DHA and lipid production, respectively. The yields and contents of DHA were further increased by combined overexpression of these genes. Highest values of DHA yield (7.2 g/L) and DHA content (40.6%) were achieved in a recombinant OPKSABC-PPT, ⁓56.5% and 15.3% higher than the WT values, respectively.

Conclusions

This study demonstrates that genetic engineering of the fatty acid biosynthetic pathways provides a new strategy to enhance DHA production in Schizochytrium.

Background

Manganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure–function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule.

Results

Two MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD—referred to as Ape-MnP1 and Cst-MnP1, respectively—were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation.

Conclusions

Understanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure–function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.