Biotechnology for Biofuels最新文献

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.

Background

Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.

Results

Arabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes.

Conclusions

Simultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Succinic acid (SA) is one of the top platform chemicals with huge applications in diverse sectors. The presence of two carboxylic acid groups on the terminal carbon atoms makes SA a highly functional molecule that can be derivatized into a wide range of products. The biological route for SA production is a cleaner, greener, and promising technological option with huge potential to sequester the potent greenhouse gas, carbon dioxide. The recycling of renewable carbon of biomass (an indirect form of CO₂), along with fixing CO₂ in the form of SA, offers a carbon-negative SA manufacturing route to reduce atmospheric CO₂ load. These attractive attributes compel a paradigm shift from fossil-based to microbial SA manufacturing, as evidenced by several commercial-scale bio-SA production in the last decade. The current review article scrutinizes the existing knowledge and covers SA production by the most efficient SA producers, including several bacteria and yeast strains. The review starts with the biochemistry of the major pathways accumulating SA as an end product. It discusses the SA production from a variety of pure and crude renewable sources by native as well as engineered strains with details of pathway/metabolic, evolutionary, and process engineering approaches for enhancing TYP (titer, yield, and productivity) metrics. The review is then extended to recent progress on separation technologies to recover SA from fermentation broth. Thereafter, SA derivatization opportunities via chemo-catalysis are discussed for various high-value products, which are only a few steps away. The last two sections are devoted to the current scenario of industrial production of bio-SA and associated challenges, along with the author's perspective.

Background

Aspergillus tubingensis is a citric acid-producing fungus that can utilize sugars in hydrolysate of lignocellulosic biomass such as sugarcane bagasse and, unlike A. niger, does not produce mycotoxins. To date, no attempt has been made to model its metabolism at genome scale.

Results

Here, we utilized the whole-genome sequence (34.96 Mb length) and the measured biomass composition to reconstruct a genome-scale metabolic model (GSMM) of A. tubingensis DJU120 strain. The model, named iMK1652, consists of 1652 genes, 1657 metabolites and 2039 reactions distributed over four cellular compartments. The model has been extensively curated manually. This included removal of dead-end metabolites and generic reactions, addition of secondary metabolite pathways and several transporters. Several mycotoxin synthesis pathways were either absent or incomplete in the genome, providing a genomic basis for the non-toxinogenic nature of this species. The model was further refined based on the experimental phenotypic microarray (Biolog) data. The model closely captured DJU120 fermentative data on glucose, xylose, and phosphate consumption, as well as citric acid and biomass production, showing its applicability to capture citric acid fermentation of lignocellulosic biomass hydrolysate.

Conclusions

The model offers a framework to conduct metabolic systems biology investigations and can act as a scaffold for integrative modelling of A. tubingensis.

Background

The red oleaginous yeast Rhodotorula toruloides is a promising cell factory to produce microbial oils and carotenoids from lignocellulosic hydrolysates (LCH). A multi-stress tolerant strain towards four major inhibitory compounds present in LCH and methanol, was derived in our laboratory from strain IST536 (PYCC 5615) through adaptive laboratory evolution (ALE) under methanol and high glycerol selective pressure.

Results

Comparative genomic analysis suggested the reduction of the original strain ploidy from triploid to diploid, the occurrence of 21,489 mutations, and 242 genes displaying copy number variants in the evolved strain. Transcriptomic analysis identified 634 genes with altered transcript levels (465 up, 178 down) in the multi-stress tolerant strain. Genes associated with cell surface biogenesis, integrity, and remodelling and involved in stress-responsive pathways exhibit the most substantial alterations at the genome and transcriptome levels. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid-induced stresses.

Conclusions

The comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance and a list of promising genes, pathways, and regulatory networks, as targets for synthetic biology approaches applied to promising cell factories, toward more robust and superior industrial strains. This study lays the foundations for understanding the mechanisms underlying tolerance to multiple stresses in R. toruloides, underscoring the potential of ALE for enhancing the robustness of industrial yeast strains.

Background

Bacterial nanocellulose (BNC), a natural polymer material, gained significant popularity among researchers and industry. It has great potential in areas, such as textile manufacturing, fiber-based paper, and packaging products, food industry, biomedical materials, and advanced functional bionanocomposites. The main current fermentation methods for BNC involved static culture, as the agitated culture methods had lower raw material conversion rates and resulted in non-uniform product formation. Currently, studies have shown that the production of BNC can be enhanced by incorporating specific additives into the culture medium. These additives included organic acids or polysaccharides. γ-Polyglutamic acid (γ-PGA), known for its high polymerization, excellent biodegradability, and environmental friendliness, has found extensive application in various industries including daily chemicals, medicine, food, and agriculture.

Results

In this particular study, 0.15 g/L of γ-PGA was incorporated as a medium additive to cultivate BNC under agitated culture conditions of 120 rpm and 30 ℃. The BNC production increased remarkably by 209% in the medium with 0.15 g/L γ-PGA and initial pH of 5.0 compared to that in the standard medium, and BNC production increased by 7.3% in the medium with 0.06 g/L γ-PGA. The addition of γ-PGA as a medium additive resulted in significant improvements in BNC production. Similarly, at initial pH levels of 4.0 and 6.0, the BNC production also increased by 39.3% and 102.3%, respectively. To assess the characteristics of the BNC products, scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis were used. The average diameter of BNC fibers, which was prepared from the medium adding 0.15 g/L γ-PGA, was twice thicker than that of BNC fibers prepared from the control culture medium. That might be because that polyglutamic acid relieved the BNC synthesis from the shear stress from the agitation.

Conclusions

This experiment held great significance as it explored the use of a novel medium additive, γ-PGA, to improve the production and the glucose conversion rate in BNC fermentation. And the BNC fibers became thicker, with better thermal stability, higher crystallinity, and higher degree of polymerization (DPv). These findings lay a solid foundation for future large-scale fermentation production of BNC using bioreactors.

Background

MicroRNA396 (miR396) plays an important role in the regulation of plant growth and development by repressing the expression level of its target growth-regulating factor (GRF) family genes. In our previous study, we found that overexpression of miR396 negatively regulated both tillering and biomass yield in switchgrass (Panicum virgatum L.). We, therefore, speculated that blocking the expression of miR396 could enhance switchgrass tillering and biomass yield. Here, we produced transgenic switchgrass plants overexpressing a target mimicry form of miR396 (MIM396) in wild type (WT) and Os-MIR319b overexpressing switchgrass plant (with higher enzymatic hydrolysis efficiency, but reduced tillering), in which the expression of miR396 was blocked. The phenotype and biological yields of these plants were analyzed.

Results

Blocking miR396 to improve its target PvGRFs expression in switchgrass improved the tiller number and dry weight of transgenic plants. Further morphological analysis revealed that MIM396 plants increased the number of aerial branches and basal tillers compared to those of wild-type plants. The enzymatic efficiency of MIM396 plants was reduced; however, the total sugar production per plant was still significantly higher than that of wild-type plants due to the increase in biomass. In addition, blocking miR396 in a transgenic switchgrass plant overexpressing Os-MIR319b (TG21-Ms) significantly increased the PvGRF1/3/5 expression level and tiller number and biomass yield. The miR156-target gene PvSPL4, playing a negative role in aerial and basal buds outgrowth, showed significant downregulated in MIM396 and TG21-Ms. Those results indicate that miR396-PvGRFs, through disrupting the PvSPL4 expression, are involved in miR319-PvPCFs in regulating tiller number, at least partly.

Conclusions

MIM396 could be used as a molecular tool to improving tiller number and biomass yield in switchgrass wild type and miR319b transgenic plants. This finding may be applied to other graminaceous plants to regulate plant biological yield.

Biotransformation with enzymes and de novo syntheses with whole-cell biocatalysts each have specific advantages. These can be combined to achieve processes with optimal performance. A recent approach is to perform bioconversion processes and enzymatic catalysis simultaneously in one-pot. This is a well-established process in the biorefinery, where starchy or cellulosic material is degraded enzymatically and simultaneously used as substrate for microbial cultivations. This procedure leads to a number of advantages like saving in time but also in the needed equipment (e.g., reaction vessels). In addition, the inhibition or side-reaction of high sugar concentrations can be overcome by combining the processes. These benefits of coupling microbial conversion and enzymatic biotransformation can also be transferred to other processes for example in the sector of biofuel production or in the food industry. However, finding a compromise between the different requirements of the two processes is challenging in some cases. This article summarises the latest developments and process variations.

Graphical Abstract

Background

Power-to-gas is the pivotal link between electricity and gas infrastructure, enabling the broader integration of renewable energy. Yet, enhancements are necessary for its full potential. In the biomethanation process, transferring H₂ into the liquid phase is a rate-limiting step. To address this, we developed a novel tubular foam-bed reactor (TFBR) and investigated its performance at laboratory scale.

Results

A non-ionic polymeric surfactant (Pluronic^® F-68) at 1.5% w/v was added to the TFBR’s culture medium to generate a stabilized liquid foam structure. This increased both the gas–liquid surface area and the bubble retention time. Within the tubing, cells predominantly traveled evenly suspended in the liquid phase or were entrapped in the thin liquid film of bubbles flowing inside the tube. Phase (I) of the experiment focused primarily on mesophilic (40 °C) operation of the tubular reactor, followed by phase (II), when Pluronic^® F-68 was added. In phase (II), the TFBR exhibited 6.5-fold increase in biomethane production rate (MPR) to 15.1 (({text{L}}_{{text{CH}}_{4}}text{/}{text{L}}_{text{R}}text{/d)}), with a CH₄ concentration exceeding 90% (grid quality), suggesting improved H₂ transfer. Transitioning to phase (III) with continuous operation at 55 °C, the MPR reached 29.7 ({text{L}}_{{text{CH}}_{4}}text{/}{text{L}}_{text{R}}text{/d}) while maintaining the grid quality CH₄. Despite, reduced gas–liquid solubility and gas–liquid mass transfer at higher temperatures, the twofold increase in MPR compared to phase (II) might be attributed to other factors, i.e., higher metabolic activity of the methanogenic archaea.

To assess process robustness for phase (II) conditions, a partial H₂ feeding regime (12 h 100% and 12 h 10% of the nominal feeding rate) was implemented. Results demonstrated a resilient MPR of approximately 14.8 ({text{L}}_{{text{CH}}_{4}}text{/}{text{L}}_{text{R}}text{/d}) even with intermittent, low H₂ concentration.

Conclusions

Overall, the TFBR’s performance plant sets the course for an accelerated introduction of biomethanation technology for the storage of volatile renewable energy. Robust process performance, even under H₂ starvation, underscores its reliability. Further steps towards an optimum operation regime and scale-up should be initiated. Additionally, the use of TFBR systems should be considered for biotechnological processes in which gas–liquid mass transfer is a limiting factor for achieving higher reaction rates.