BackgroundAs the second most abundant polysaccharide in nature, hemicellulose can be degraded to xylose as the feedstock for bioconversion to fuels and chemicals. To enhance xylose conversion, the engineered Saccharomyces cerevisiae with xylose metabolic pathway is usually adapted with xylose as the carbon source in the laboratory. However, the mechanism under the adaptation phenomena of the engineered strain is still unclear.ResultsIn this study, xylose-utilizing S. cerevisiae was constructed and used for the adaptation study. It was found that xylose consumption rate increased 1.24-fold in the second incubation of the yYST12 strain in synthetic complete-xylose medium compared with the first incubation. The study figured out that it was observed at the single-cell level that the stagnation time for xylose utilization was reduced after adaptation with xylose medium in the microfluidic device. Such transient memory of xylose metabolism after adaptation with xylose medium, named “xylose consumption memory”, was observed in the strains with both xylose isomerase pathway and xylose reductase and xylitol dehydrogenase pathways. In further, the proteomic acetylation of the strains before and after adaptation was investigated, and it was revealed that H4K5 was one of the most differential acetylation sites related to xylose consumption memory of engineered S. cerevisiae. We tested 8 genes encoding acetylase or deacetylase, and it was found that the knockout of the GCN5 and HPA2 encoding acetylases enhanced the xylose consumption memory.ConclusionsThe behavior of xylose consumption memory in engineered S. cerevisiae can be successfully induced with xylose in the adaptation. H4K5Ac and two genes of GCN5 and HPA2 are related to xylose consumption memory of engineered S. cerevisiae during adaptation. This study provides valuable insights into the xylose adaptation of engineered S. cerevisiae.

BackgroundKluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far.ResultsIn this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2–Ire1–Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells.ConclusionsOur results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.

BackgroundDiatoms are well known for high photosynthetic efficiency and rapid growth rate, which are not only important oceanic primary producer, but also ideal feedstock for microalgae industrialization. Their high success is mainly due to the rapid response of photosynthesis to inorganic carbon fluctuations. Thus, an in-depth understanding of the photosynthetic carbon fixation mechanism of diatoms will be of great help to microalgae-based applications. This work directed toward the analysis of whether C4 photosynthetic pathway functions in the model marine diatom Phaeodactylum tricornutum, which possesses biophysical CO2-concentrating mechanism (CCM) as well as metabolic enzymes potentially involved in C4 photosynthetic pathway.ResultsFor P. tricornutum, differential proteome, enzyme activities and transcript abundance of carbon metabolism-related genes especially biophysical and biochemical CCM-related genes in response to different concentrations of CO2 were tracked in this study. The upregulated protein abundance of a carbonic anhydrases and a bicarbonate transporter suggested biophysical CCM activated under low CO2 (LC). The upregulation of a number of key C4-related enzymes in enzymatic activity, transcript and protein abundance under LC indicated the induction of a mitochondria-mediated CCM in P. tricornutum. Moreover, protein abundance of a number of glycolysis, tricarboxylic acid cycle, photorespiration and ornithine–urea cycle related proteins upregulated under LC, while numbers of proteins involved in the Calvin cycle and pentose phosphate pathway were downregulated. Under high CO2 (HC), protein abundance of most central carbon metabolism and photosynthesis-related proteins were upregulated.ConclusionsThe proteomic and biochemical responses to different concentrations of CO2 suggested multiple carbon metabolism strategies exist in P. tricornutum. Namely, LC might induce a mitochondrial-mediated C4-like CCM and the improvement of glycolysis, tricarboxylic acid cycle, photorespiration and ornithine–urea cycle activity contribute to the energy supply and carbon and nitrogen recapture in P. tricornutum to cope with the CO2 limitation, while P. tricornutum responds to the HC environment by improving photosynthesis and central carbon metabolism activity. These findings can not only provide evidences for revealing the global picture of biophysical and biochemical CCM in P. tricornutum, but also provide target genes for further microalgal strain modification to improve carbon fixation and biomass yield in algal-based industry.

BackgroundAs methane is 84 times more potent than carbon dioxide in exacerbating the greenhouse effect, there is an increasing interest in the utilization of methanotrophic bacteria that can convert harmful methane into various value-added compounds. A recently isolated methanotroph, Methylomonas sp. DH-1, is a promising biofactory platform because of its relatively fast growth. However, the lack of genetic engineering tools hampers its wide use in the bioindustry.ResultsThrough three different approaches, we constructed a tunable promoter library comprising 33 promoters that can be used for the metabolic engineering of Methylomonas sp. DH-1. The library had an expression level of 0.24–410% when compared with the strength of the lac promoter. For practical application of the promoter library, we fine-tuned the expressions of cadA and cadB genes, required for cadaverine synthesis and export, respectively. The strain with PrpmB-cadA and PDnaA-cadB produced the highest cadaverine titre (18.12 ± 1.06 mg/L) in Methylomonas sp. DH-1, which was up to 2.8-fold higher than that obtained from a non-optimized strain. In addition, cell growth and lysine (a precursor of cadaverine) production assays suggested that gene expression optimization through transcription tuning can afford a balance between the growth and precursor supply.ConclusionsThe tunable promoter library provides standard and tunable components for gene expression, thereby facilitating the use of methanotrophs, specifically Methylomonas sp. DH-1, as a sustainable cell factory.Graphical

BackgroundThe ability of oxidative enzyme-producing micro-organisms to efficiently valorize organic pollutants is critical in this context. Yeasts are promising enzyme producers with potential applications in waste management, while lipid accumulation offers significant bioenergy production opportunities. The aim of this study was to explore manganese peroxidase-producing oleaginous yeasts inhabiting the guts of wood-feeding termites for azo dye decolorization, tolerating lignocellulose degradation inhibitors, and biodiesel production.ResultsOut of 38 yeast isolates screened from wood-feeding termite gut symbionts, nine isolates exhibited high levels of extracellular manganese peroxidase (MnP) activity ranged between 23 and 27 U/mL after 5 days of incubation in an optimal substrate. Of these MnP-producing yeasts, four strains had lipid accumulation greater than 20% (oleaginous nature), with Meyerozyma caribbica SSA1654 having the highest lipid content (47.25%, w/w). In terms of tolerance to lignocellulose degradation inhibitors, the four MnP-producing oleaginous yeast strains could grow in the presence of furfural, 5-hydroxymethyl furfural, acetic acid, vanillin, and formic acid in the tested range. M. caribbica SSA1654 showed the highest tolerance to furfural (1.0 g/L), 5-hydroxymethyl furfural (2.5 g/L) and vanillin (2.0 g/L). Furthermore, M. caribbica SSA1654 could grow in the presence of 2.5 g/L acetic acid but grew moderately. Furfural and formic acid had a significant inhibitory effect on lipid accumulation by M. caribbica SSA1654, compared to the other lignocellulose degradation inhibitors tested. On the other hand, a new MnP-producing oleaginous yeast consortium designated as NYC-1 was constructed. This consortium demonstrated effective decolorization of all individual azo dyes tested within 24 h, up to a dye concentration of 250 mg/L. The NYC-1 consortium's decolorization performance against Acid Orange 7 (AO7) was investigated under the influence of several parameters, such as temperature, pH, salt concentration, and co-substrates (e.g., carbon, nitrogen, or agricultural wastes). The main physicochemical properties of biodiesel produced by AO7-degraded NYC-1 consortium were estimated and the results were compared to those obtained from international standards.ConclusionThe findings of this study open up a new avenue for using peroxidase-producing oleaginous yeasts inhabiting wood-feeding termite gut symbionts, which hold great promise for the remediation of recalcitrant azo dye wastewater and lignocellulosic biomass for biofuel production.

Rhizopus oryzae (R. oryzae) can effectively produce organic acids, and its pellet formation in seed cultures has been shown to significantly enhance subsequent fermentation processes. Despite advances in strain development, simple and effective methods for inducing pellet morphology and a basic understanding of the mechanisms controlling this process could facilitate substantial increases in efficiency and product output. Here, we report that 1.5% triethanolamine (TEOA) in seed culture medium can activate the growth of R. oryzae spores in compact and uniform pellets which is optimal for fermentation conditions. Analysis of fermentation kinetics showed that the production of fumaric and L-malic acid increases 293% and 177%, respectively. Transcriptomic analysis revealed that exposure of R. oryzae to 1.5% TEOA during the seed culture activated the phosphatidylinositol and mitogen-activated protein kinase signaling pathways. Theses pathways subsequently stimulated the downstream carbohydrate-active synthases and hydrolases that required for cell wall component synthesis and reconstruction. Our results thus provide insight into the regulatory pathways controlling pellet morphology germane to the viability of seed cultures, and provide valuable reference data for subsequent optimization of organic acid fermentation by R. oryzae.

BackgroundCellulolytic enzyme production in filamentous fungi requires a release from carbon catabolite repression (CCR). The protein CRE1/CreA (CRE = catabolite responsive element) is a key transcription factor (TF) that is involved in CCR and represses cellulolytic gene expression. CRE1/CreA represents the functional equivalent of Mig1p, an important Saccharomyces cerevisiae TF in CCR that exerts its repressive effect by recruiting a corepressor complex Tup1p–Cyc8p. Although it is known from S. cerevisiae that CRE1/CreA might repress gene expression via interacting with the corepressor complex Tup1–Cyc8, this mechanism is unconfirmed in other filamentous fungi, since the physical interaction has not yet been verified in these organisms. The precise mechanism on how CRE1/CreA achieves transcriptional repression after DNA binding remains unknown.ResultsThe results from tandem affinity purification and bimolecular fluorescence complementation revealed a direct physical interaction between the TF CRE1/CreA and the complex Tup1–Cyc8 in the nucleus of cellulolytic fungus Trichoderma reesei and Penicillium oxalicum. Both fungi have the ability to secrete a complex arsenal of enzymes to synergistically degrade lignocellulosic materials. In P. oxalicum, the protein PoCyc8, a subunit of complex Tup1–Cyc8, interacts directly with TF PoCreA and histone H3 lysine 36 (H3K36) methyltransferase PoSet2 in the nucleus. The di-methylation level of H3K36 in the promoter of prominent cellulolytic genes (cellobiohydrolase-encoding gene Pocbh1/cel7A and endoglucanase-encoding gene Poegl1/cel7B) is positively correlated with the expression levels of TF PoCreA. Since the methylation of H3K36 was also demonstrated to be a repression marker of cellulolytic gene expression, it appears feasible that the cellulolytic genes are repressed via PoCreA-Tup1–Cyc8-Set2-mediated transcriptional repression.ConclusionThis study verifies the long-standing conjecture that the TF CRE1/CreA represses gene expression by interacting with the corepressor complex Tup1–Cyc8 in filamentous fungi. A reasonable explanation is proposed that PoCreA represses gene expression by recruiting complex PoTup1–Cyc8. Histone methyltransferase Set2, which methylates H3K36, is also involved in the regulatory network by interacting with PoCyc8. The findings contribute to the understanding of CCR mechanism in filamentous fungi and could aid in biotechnologically relevant enzyme production.

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal–methanogen physical associations and fungal cell wall development and remodeling.

BackgroundWax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings, and cosmetics. They can be produced chemically from fossil fuel or plant-derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, tightly controlled and tailor-made WE synthesis in plants would be a sustainable, beneficial, and valuable commodity. Here, we report the expression of ten combinations of WE producing transgenes in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and composition in the transgenic plants.ResultsThe transgenes consisted of different combinations of a FATTY ACYL-COA/ACP REDUCTASE (FAR) and two WAX SYNTHASES/ACYL-COA:DIACYLGLYCEROL O-ACYLTRANSFERASES (WSD), namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access distinct alcohol and acyl substrate pools within A. thaliana cells. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis.ConclusionsA shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE that are mainly derived from mid- and long-chain saturated and monounsaturated substrates.

BackgroundPleurotus ostreatus is an edible mushroom popularly cultivated worldwide. Distilled grain waste (DGW) is a potential substrate for P. ostreatus cultivation. However, components in DGW restrict P. ostreatus mycelial growth. Therefore, a cost-effective approach to facilitate rapid P. ostreatus colonization on DGW substrate will benefit P. ostreatus cultivation and DGW recycling.ResultsFive dominant indigenous bacteria, Sphingobacterium sp. X1, Ureibacillus sp. X2, Pseudoxanthomonas sp. X3, Geobacillus sp. X4, and Aeribacillus sp. X5, were isolated from DGW and selected to develop a consortium-based microbial agent to compost DGW for P. ostreatus cultivation. Microbial agent inoculation led to faster carbohydrate metabolism, a higher temperature (73.2 vs. 71.2 °C), a longer thermophilic phase (5 vs. 3 days), and significant dynamic changes in microbial community composition and diversity in composts than those of the controls. Metagenomic analysis showed the enhanced microbial metabolisms, such as xenobiotic biodegradation and metabolism and terpenoid and polyketide metabolism, during the mesophilic phase after microbial agent inoculation, which may facilitate the fungal colonization on the substrate. In accordance with the bioinformatic analysis, a faster colonization of P. ostreatus was observed in the composts with microbial inoculation than in control after composting for 48 h, as indicated from substantially higher fungal ergosterol content, faster lignocellulose degradation, and higher lignocellulase activities in the former than in the latter. The final mushroom yield shared no significant difference between composts with microbial inoculation and control, with 0.67 ± 0.05 and 0.60 ± 0.04 kg fresh mushroom/kg DGW, respectively (p > 0.05).ConclusionThe consortium-based microbial agent comprised indigenous microorganisms showing application potential in composting DGW for providing substrate for P. ostreatus cultivation and will provide an alternative to facilitate DGW recycling.