Biotechnology for Biofuels and Bioproducts

Napier grass biomass can be hydrolyzed mainly containing glucose and xylose after alkaline pretreatment and enzymatic hydrolysis. This biomass can be fermented using Actinobacillus succinogenes to produce succinic acid. The yield of succinic acid was 0.58 g/g. Because metabolizing xylose could produce more acetic acid, this yield of succinic acid was lower than that achieved using glucose as the sole carbon source.

The addition of glycerol as a fermentation substrate to Napier grass hydrolysate increased the reducing power of the hydrolysate, which not only increased the production of succinic acid but also reduced the formation of undesirable acetic acid in bacterial cells. At a hydrolysate:glycerol ratio of 10:1, the succinic acid yield reached 0.65 g/g. The succinic acid yield increased to 0.88 g/g when a 1:1 ratio of hydrolysate:glycerol was used. For the recovery of succinic acid from the fermentation broth, an outside-in module of an ultrafiltration membrane was used to remove bacterial cells. Air sparging at the feed side with a flow rate of 3 L/min increased the filtration rate. When the air flow rate was increased from 0 to 3 L/min, the average filtration rate increased from 25.0 to 45.7 mL/min, which corresponds to an increase of 82.8%. The clarified fermentation broth was then electrodialized to separate succinate from other contaminated ions. After electrodialysis, the acid products were concentrated through water removal, decolorized through treatment with activated carbon, and precipitated to obtain a purified product.

The yield of succinic acid was increased by adding glycerol to the hydrolysate of Napier grass. The downstream processing consisting of ultrafiltration membrane separation and single-stage electrodialysis was effective for product separation and purification. An overall recovery yield of 74.7% ± 4.5% and a purity of 99.4% ± 0.1% were achieved for succinic acid.

Rapeseed (Brassica napus) is the second largest oil crop worldwide. It is widely used in food, energy production and the chemical industry, as well as being an ornamental. Consequently, it has a large economic value and developmental potential. Waterlogging is an important abiotic stress that restricts plant growth and development. However, little is known about the molecular mechanisms underlying waterlogging tolerance in B. napus.

In the present study, the physiological changes and transcriptomes of germination-stage rapeseed in response to waterlogging stress were investigated in the B. napus cultivar ‘Zhongshuang 11’ (ZS11) and its anthocyanin-more (am) mutant, which was identified in our previous study. The mutant showed stronger waterlogging tolerance compared with ZS11, and waterlogging stress significantly increased anthocyanin, soluble sugar and malondialdehyde contents and decreased chlorophyll contents in the mutant after 12 days of waterlogging. An RNA-seq analysis identified 1370 and 2336 differently expressed genes (DEGs) responding to waterlogging stress in ZS11 and am, respectively. An enrichment analysis revealed that the DEGs in ZS11 were predominately involved in carbohydrate metabolism, whereas those in the am mutant were particularly enriched in plant hormone signal transduction and response to endogenous stimulation. In total, 299 DEGs were identified as anthocyanin biosynthesis-related structural genes (24) and regulatory genes encoding transcription factors (275), which may explain the increased anthocyanin content in the am mutant. A total of 110 genes clustered in the plant hormone signal transduction pathway were also identified as DEGs, including 70 involved in auxin and ethylene signal transduction that were significantly changed in the mutant. Furthermore, the expression levels of 16 DEGs with putative roles in anthocyanin accumulation and biotic/abiotic stress responses were validated by quantitative real-time PCR as being consistent with the transcriptome profiles.

This study provides new insights into the molecular mechanisms of increased anthocyanin contents in rapeseed in response to waterlogging stress, which should be useful for reducing the damage caused by waterlogging stress and for further breeding new rapeseed varieties with high waterlogging tolerance.

Vernonia galamensis native to Africa is an annual oleaginous plant of Asteraceae family. As a newly established industrial oil crop, this plant produces high level (> 70%) of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid), which is an unusual epoxy fatty acid (EFA) with multiple industrial applications. Here, transcriptome analysis and fatty acid profiling from developing V. galamensis seeds were integrated to uncover the critical metabolic pathways responsible for high EFA accumulation, aiming to identify the target genes that could be used in the biotechnological production of high-value oils.

Based on oil accumulation dynamics of V. galamensis seeds, we harvested seed samples from three stages (17, 38, and 45 days after pollination, DAP) representing the initial, fast and final EFA accumulation phases, and one mixed sample from different tissues for RNA-sequencing, with three biological replicates for each sample. Using Illumina platform, we have generated a total of 265 million raw cDNA reads. After filtering process, de novo assembly of clean reads yielded 67,114 unigenes with an N50 length of 1316 nt. Functional annotation resulted in the identification of almost all genes involved in diverse lipid-metabolic pathways, including the novel fatty acid desaturase/epoxygenase, diacylglycerol acyltransferases, and phospholipid:diacylglycerol acyltransferases. Expression profiling revealed that various genes associated with acyl editing, fatty acid β-oxidation, triacylglycerol assembly and oil-body formation had greater expression levels at middle developmental stage (38 DAP), which were consistent with the fast accumulation of EFA in V. galamensis developing seed, these genes were detected to play fundamental roles in EFA production. In addition, we isolated some transcription factors (such as WRI1, FUS3 and ABI4), which putatively regulated the production of V. galamensis seed oils. The transient expression of the selected genes resulted in a synergistic increase of EFA-enriched TAG accumulation in tobacco leaves. Transcriptome data were further confirmed by quantitative real-time PCR for twelve key genes in EFA biosynthesis. Finally, a comprehensive network for high EFA accumulation in V. galamensis seed was established.

Our findings provide new insights into molecular mechanisms underlying the natural epoxy oil production in V. galamensis. A set of genes identified here could be used as the targets to develop other oilseeds highly accumulating valued epoxy oils for commercial production.

The production and processing of animal-based products generates many collagen-rich by-products, which have received attention both for exploitation to increase their added value and to reduce their negative environmental impact. The collagen-rich by-products can be hydrolyzed by collagenases for further utilization. Therefore, collagenases are of benefit for efficient collagen materials processing. An alternative and safe way to produce secreted collagenases is needed.

Two collagenases from Hathewaya histolytica, ColG and ColH, were successfully secreted by the yeast Saccharomyces cerevisiae. Compared with the native signal peptide of collagenase, the α-factor leader is more efficient in guiding collagenase secretion. Collagenase secretion was significantly increased in YPD medium by supplementing with calcium and zinc ions. Recombinant collagenase titers reached 68 U/mL and 55 U/mL for ColG and ColH, respectively. Collagenase expression imposed metabolic perturbations on yeast cells; substrate consumption, metabolites production and intracellular cofactor levels changed in engineered strains. Both recombinant collagenases from yeast could hydrolyze soluble and insoluble collagen materials. Recombinant ColG and ColH showed a synergistic effect on efficient collagen digestion.

Sufficient calcium and zinc ions are essential for active collagenase production by yeast. Collagenase secretion was increased by optimization of expression cassettes. Collagenase expression imposed metabolic burden and cofactor perturbations on yeast cells, which could be improved through metabolic engineering. Our work provides a useful way to produce collagenases for collagen resource utilization.

Microalgae can absorb CO₂ during photosynthesis, which causes the aquatic environmental pH to rise. However, the pH is reduced when microalga Euglena gracilis (EG) is cultivated under photoautotrophic conditions. The mechanism behind this unique phenomenon is not yet elucidated.

The present study evaluated the growth of EG, compared to Chlorella vulgaris (CV), as the control group; analyzed the dissolved organic matter (DOM) in the aquatic environment; finally revealed the mechanism of the decrease in the aquatic environmental pH via comparative metabolomics analysis. Although the CV cell density was 28.3-fold that of EG, the secreted-DOM content from EG cell was 49.8-fold that of CV (p-value < 0.001). The main component of EG’s DOM was rich in humic acids, which contained more DOM composed of chemical bonds such as N–H, O–H, C–H, C=O, C–O–C, and C–OH than that of CV. Essentially, the 24 candidate biomarkers metabolites secreted by EG into the aquatic environment were acidic substances, mainly lipids and lipid-like molecules, organoheterocyclic compounds, organic acids, and derivatives. Moreover, six potential critical secreted-metabolic pathways were identified.

This study demonstrated that EG secreted acidic metabolites, resulting in decreased aquatic environmental pH. This study provides novel insights into a new understanding of the ecological niche of EG and the rule of pH change in the microalgae aquatic environment.

A butyric acid recovery process using octyl acetate is proposed, and the design details of the extraction and subsequent distillation processes were investigated. Ternary equilibrium data for the extractor design were derived from molecular simulations and experimental measurements.

A new procedure for estimating the thermodynamic parameters was introduced to determine the effect of the parameters on extractor design by comparison with previously reported parameters. Using the proposed recovery process with the newly estimated thermodynamic model, 99.8% butyric acid was recovered from the fermentation broth at a recovery rate of 99%. The energy demand for the proposed process was found to be lower than the average demand for several reported butyric acid recovery processes.

The investment cost is projected to be lower than that of other butyric acid processes due to the high efficiency of extraction solvent. The recovery cost of butyric acid was comparable to its selling price.

The thermotolerant yeast is beneficial in terms of efficiency improvement of processes and reduction of costs, while Saccharomyces cerevisiae does not efficiently grow and ferment at high-temperature conditions. The sterol composition alteration from ergosterol to fecosterol in the cell membrane of S. cerevisiae affects the thermotolerant capability.

In this study, S. cerevisiae ERG5, ERG4, and ERG3 were knocked out using the CRISPR–Cas9 approach to impact the gene expression involved in ergosterol synthesis. The highest thermotolerant strain was S. cerevisiae ERG5ΔERG4ΔERG3Δ, which produced 22.1 g/L ethanol at 37 °C using the initial glucose concentration of 50 g/L with an increase by 9.4% compared with the wild type (20.2 g/L). The ethanol concentration of 9.4 g/L was produced at 42 ℃, which was 2.85-fold of the wild-type strain (3.3 g/L). The molecular mechanism of engineered S. cerevisiae at the RNA level was analyzed using the transcriptomics method. The simultaneous deletion of S. cerevisiae ERG5, ERG4, and ERG3 caused 278 up-regulated genes and 1892 down-regulated genes in comparison with the wild-type strain. KEGG pathway analysis indicated that the up-regulated genes relevant to ergosterol metabolism were ERG1, ERG11, and ERG5, while the down-regulated genes were ERG9 and ERG26. S. cerevisiae ERG5ΔERG4ΔERG3Δ produced 41.6 g/L of ethanol at 37 °C with 107.7 g/L of corn liquefied glucose as carbon source.

Simultaneous deletion of ERG5, ERG4, and ERG3 resulted in the thermotolerance improvement of S. cerevisiae ERG5ΔERG4ΔERG3Δ with cell viability improvement by 1.19-fold at 42 °C via modification of steroid metabolic pathway. S. cerevisiae ERG5ΔERG4ΔERG3Δ could effectively produce ethanol at 37 °C using corn liquefied glucose as carbon source. Therefore, S. cerevisiae ERG5ΔERG4ΔERG3Δ had potential in ethanol production at a large scale under supra-optimal temperature.

The budding yeast Komagataella phaffii (Pichia pastoris) is widely employed to secrete proteins of academic and industrial interest. For secretory proteins, signal peptides are the sorting signal to direct proteins from cytosol to extracellular matrix, and their secretion efficiency directly impacts the yields of the targeted proteins in fermentation broth. Although the α-mating factor (MF) secretion signal from S. cerevisiae, the most common and widely used signal sequence for protein secretion, works in most cases, limitation exists as some proteins cannot be secreted efficiently. As the optimal choice of secretion signals is often protein specific, more secretion signals need to be developed to augment protein expression levels in K. phaffii.

In this study, the secretion efficiency of 40 α-MF secretion signals from various yeast species and 32 endogenous signal peptides from K. phaffii were investigated using enhanced green fluorescent protein (EGFP) as the model protein. All of the evaluated α-MF secretion signals successfully directed EGFP secretion except for the secretion signals of the yeast D. hansenii CBS767 and H. opuntiae. The secretion efficiency of α-MF secretion signal from Wickerhamomyces ciferrii was higher than that from S. cerevisiae. 24 out of 32 endogenous signal peptides successfully mediated EGFP secretion. The signal peptides of chr3_1145 and FragB_0048 had similar efficiency to S. cerevisiae α-MF secretion signal for EGFP secretion and expression.

The screened α-MF secretion signals and endogenous signal peptides in this study confer an abundance of signal peptide selection for efficient secretion and expression of heterologous proteins in K. phaffii.